Immunostimulatory herpes virus

Abstract

Provided is an oncolytic herpes simplex virus having a gene encoding an immune checkpoint inhibitor. This herpes simplex virus preferably has the following features (a)-(c). (a) The ICP6 gene is deleted or inactivated, (b) the γ34.5 gene is deleted or inactivated, and (c) the α47 gene is deleted or inactivated. Since the expressed immune checkpoint inhibitor remains at the tumor locus and does not flow out into the blood, systemic side effects are not generated.

Description

Immunostimulatory herpesviruses

[0001] The present invention relates to an oncolytic herpesvirus, and more particularly to a functionalized oncolytic herpesvirus that enhances the therapeutic effect of virotherapy.

[0002] Research is currently being conducted into viral therapies to treat cancer, in which the virus infects cancer cells and destroys them as the virus replicates. Unlike existing therapies, this method not only has a direct cytocidal effect associated with viral replication, but also elicits cancer-cell-specific immunity during the process of destroying cancer cells, resulting in a systemic therapeutic effect via the immune system.

[0003] The present inventors have invented a third-generation oncolytic herpes simplex virus type 1 (HSV-1) G47Δ with a triple mutation, which they have subsequently developed clinically and successfully commercialized (Patent Document 1, Non-Patent Document 1). The present inventors are also developing next-generation oncolytic HSV-1s, such as functionalized G47Δ viruses incorporating various foreign genes into the G47Δ backbone, including a swelling-suppressing oncolytic virus incorporating a gene encoding a vascular endothelial growth factor (VEGF) antagonist (Patent Document 2). They have also reported an oncolytic virus incorporating a gene expressing interleukin-12 (IL-12) as a fusion polypeptide in which the light and heavy chains are linked by an elastin motif (Non-Patent Document 2).

[0004] Many cancer cells utilize the immunosuppressive function of immune checkpoint molecules to avoid attack by the immune system. Immune checkpoint inhibitor therapy aims to reverse the suppression of immune cell activation, including T cells, by inhibiting the transmission of immunosuppressive signals by immune checkpoint molecules, thereby enhancing the patient's immune activity and achieving anti-cancer effects. Immune checkpoint inhibitors, such as nivolumab (trade name Opdivo), a PD-1 (Programmed Death-1) inhibitor antibody, atezolizumab (trade name Tecentriq), a PD-L1 (Programmed cell Death 1-Ligand 1) inhibitor antibody, and tremelimumab and ipilimumab, CTLA-4 (Cytotoxic T-lymphocyte-associated antigen 4) inhibitor antibodies, have been used to treat certain cancers.

[0005] Patent No. 4212897 Publication WO2019 / 189643

[0006] Todo, T. et al., Nat Commun 13, 4119 (2022), https: / / doi.org / 10.1038 / s41467-022-31262-y105, 841-846.Fukuhara, H. et al., Commun Med (Lond) 3, 40 (2023), https: / / www.nature.com / articles / s43856-023-00270-4

[0007] Immune checkpoint inhibitor antibodies can shift the entire immune system to attack, but they are not effective unless the immune system recognizes cancer as non-self. Furthermore, systemic administration of immune checkpoint inhibitor antibodies is known to frequently cause severe side effects such as autoimmune diseases.

[0008] In this study, the inventors developed a functionalized, third-generation oncolytic HSV-1 that expresses immune checkpoint inhibitory antibodies by incorporating immune checkpoint antibodies into the G47Δ backbone. Virotherapy using HSV-1 is characterized by inducing the immune system to recognize cancer cells as non-self, thereby eliciting anti-cancer immunity. Therefore, combining HSV-1 with immune checkpoint inhibitory antibodies is expected to significantly improve therapeutic efficacy.

[0009] Furthermore, the oncolytic HSV-1 of this invention induces the expression of immune checkpoint inhibitory antibodies each time the virus replicates and infects tumors, which allows immune checkpoint inhibitory antibodies to be expressed and accumulated only locally in tumors, potentially enhancing the therapeutic effects of viral therapy without causing systemic side effects.

[0010] The present invention provides the following: [1] An oncolytic herpes simplex virus having a gene encoding an immune checkpoint inhibitor. [2] The virus according to 1, wherein the herpes simplex virus has the following characteristics (a) to (c): (a) the ICP6 gene is deleted or inactivated, (b) the γ34.5 gene is deleted or inactivated, and (c) the α47 gene is deleted or inactivated. [3] The virus according to 1 or 2, wherein a gene encoding an immune checkpoint inhibitor has been inserted into the site where the IPC6 gene has been deleted or inactivated. [4] The virus according to any one of 1 to 3, wherein the immune checkpoint inhibitor is an immune checkpoint inhibitor antibody or a fragment thereof. [5] The virus according to any one of 1 to 4, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody, or a fragment thereof. [6] A pharmaceutical composition comprising the virus according to any one of 1 to 5. [7] The pharmaceutical composition according to 6, which is for local injection. [8] The pharmaceutical composition according to 6 or 7, which suppresses blood levels of an immune checkpoint inhibitor. [9] A combination of the virus according to any one of 1 to 5 and at least one oncolytic herpes simplex virus having a gene encoding an immune checkpoint inhibitor different from the virus according to 1 to 5.

[10] A combination of the virus according to any one of 1 to 5 and at least one oncolytic herpes simplex virus having a gene encoding a soluble B7-1 gene and an oncolytic herpes simplex virus having a gene encoding IL-12.

[0011] The oncolytic herpes simplex virus carrying a gene encoding an immune checkpoint inhibitor provided by the present application can express immune checkpoint inhibitor antibodies in conjunction with viral replication when administered to tumor cells. Furthermore, the following effects can be achieved: The expressed antibodies have the same functions as commercially available systemically administered immune checkpoint inhibitor antibodies. Administration of a single virus alone can achieve the combined effects of systemically administered immune checkpoint inhibitor antibodies and virotherapy. Compared to a virus expressing no immune checkpoint inhibitor, the virus exhibits enhanced therapeutic effects in both administered and distant unadministered tumors. The expressed immune checkpoint inhibitor antibodies remain localized in the tumor and are barely released into the bloodstream, eliminating systemic side effects. The therapeutic effect can be further enhanced by administering multiple viruses expressing different immune checkpoint inhibitor antibodies in combination (mixed administration or sequential administration). The therapeutic effect can be further enhanced by combining the virus with a soluble B7-1-expressing virus and / or an IL-12-expressing virus.

[0012] Amino acid sequence (derived from J43) for expression of anti-mouse PD1 antibody. As with nivolumab, the heavy chain constant region was derived from human IgG4. The light chain constant region was derived from human lambda chain. The underlined parts are hypervariable regions predicted by IMGT / DomainGapAlign. Amino acid sequence (derived from J43) for expression of anti-mouse PD-1 antibody. The heavy chain constant region was derived from Armenian Ham IgG, and the light chain constant region was derived from Armenian Ham Ig kappa chain. Comparison of anti-PD-1 antibody expression levels. HEK293T cells were infected with T-J43 and T-01 at an MOI of 1. 48 hours later, the supernatant was collected and concentrated by ultrafiltration (Amicon Ultra-30K). A) Human Fc ELISA (Human Fc antibody immobilized, detected with HRP-Human Fc antibody) and B) PD-1 binding ELISA (mouse PD-1 immobilized, detected with HRP-Human Fc antibody) were performed. Comparison of anti-mouse PD-1 antibody expression levels between cell lines. PD-1 binding ELISA (mouse PD-1 immobilized, detected with HRP-Human Fc antibody) was performed. J43 / SV01 was used as the standard (quantified with human IgG). PD-1 / PD-L1 inhibitory activity measurement results. A) Culture supernatant from transfections of J43 / SV-01 and SV-01 (empty vector). B) The IC50 value, calculated by converting the concentration of J43 / SV-01 used in the experiment in A) into Human Fc, was 6.81 ng / ml. C) Measurement of the inhibitory activity of commercially available anti-mouse PD-1 antibodies (RMP1-14, J43) and isotype control antibody (2A3) against PD-1 / PD-L1. Cytotoxicity Assay. Replication Assay. Infection efficiency of the prepared virus (X-gal staining). Structural confirmation of the prepared virus by Southern blotting. Structural confirmation of the prepared virus by Southern blotting. Comparison of the antitumor effects of anti-mouse PD-1 antibody-expressing HSV-1 in a unilateral subcutaneous tumor model in Neuro2a / A / J mice. *; P<0.05 (Two-way ANOVA and Bonferroni comparison test).On day 13, there was a significant difference between mock and T-J43 (***; P<0.001) and between T-01 and T-J43 (*; P<0.05) (One-way ANOVA and Tukey's comparison test). Bar = SEM. On day 16, 200 mm. 3 The number of mice with tumor sizes below were considered to have an inhibitory effect. Comparison of the antitumor effect of anti-mouse PD-1 antibody-expressing HSV-1 in a bilateral subcutaneous tumor model in Neuro2a / A / J mice. *: P<0.05 (two-way ANOVA and Tukey's comparison test). On the non-treated side, there was a significant difference only between mock and T-J43 on day 10 (one-way ANOVA and Tukey's comparison test). Bar = SEM. On day 14, 200 mm 3 The number of mice is shown when the following tumor sizes were considered to have an inhibitory effect. Antitumor effect in clone M3 / DBA / 2 unilateral subcutaneous tumor model. *: P<0.05, **: P<0.01 (Two-way ANOVA and Bonferroni comparison test). Treatment side: On day 13, there was a significant difference only between T-01 and T-J43 (t-test). Bar = SEM. On day 27, 200 mm 3 The percentage of tumor disappearance is shown below. Antitumor effect in a clone M3 / DBA / 2 mouse bilateral subcutaneous tumor model. *: P<0.05, ***: P<0.001, ****: P<0.0001 (Two-way ANOVA and Bonferroni comparison test). At day 13, 200 mm 3The percentage of patients with tumor disappearance is shown below. Anti-tumor effect in a bilateral subcutaneous tumor model in cloneM3 / DBA / 2 mice. Survival curve when observation was terminated (death) at death or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 38. T-01 vs T-J43; P=0.0069, Log-rank test. Comparison of the anti-tumor effects of anti-mouse PD-1 antibody (T-J43) and anti-mouse CTLA-4 antibody (T-4F10HA)-expressing HSV-1 in a unilateral subcutaneous tumor model in Renca / Balb / c mice. Bars = SEM. The percentage of patients with tumor disappearance (50 mm3 or less) at day 80 is shown. Kaplan-Meier survival curve. Survival curve when observation was terminated (death) at death or when the maximum tumor diameter reached 24 mm. Observation ended on day 80. *; P<0.05, **; P<0.01, ***; P<0.001 log-rank test. Comparison of the antitumor effects of the combination of T-01 and J43 antibody and T-J43 alone in a Neuro2a / A / J mouse bilateral subcutaneous tumor model. P<0.05, T-01+J43 vs T-J43+J43 (2-way ANOVA, Bonferroni's multiple comparison test). Comparison of the antitumor effects of the combination of T-01 and J43 antibody and T-J43 alone in a Neuro2a / A / J mouse bilateral subcutaneous tumor model (treatment side). Tumor loss (50 mm) on day 48 was observed. 3 Comparison of the antitumor effects of the combination of T-01 and J43 antibody with T-J43 alone in a bilateral subcutaneous tumor model in Neuro2a / A / J mice (non-treated side). Tumor loss (50 mm) was observed on day 48. 3The percentage of patients who died (below) is shown. Kaplan-Meier survival curve. Survival curve when observation was terminated (death) when death occurred or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 48. Comparison of blood and intratumoral antibody concentrations following intraperitoneal administration of J43 and intratumoral administration of T-J43HA in a unilateral Neuro2a / A / J subcutaneous tumor model. Amino acid sequences for expression of anti-human and mouse PD-L1 antibodies (derived from Atezolizumab). The heavy chain constant region was derived from human IgG1. The light chain constant region was derived from human κ chain. The underlined parts are hypervariable sites predicted by IMGT / DomainGapAlign. Comparison of anti-PD-L1 antibody expression levels. HEK293T cells were infected with T-ATZ and T-01 at an MOI of 1. 48 hours later, the supernatant was collected and concentrated by ultrafiltration (Amicon Ultra-30K). A) Human Fc ELISA (Human Fc antibody immobilized, detected with HRP-Human Fc antibody) and B) PD-L1 binding ELISA (Mouse PD-L1 immobilized, detected with HRP-Human Fc antibody) were performed. Comparison of anti-mouse PD-L1 antibody expression levels among each cell line. PD-L1 binding ELISA (Mouse PD-1 immobilized, detected with HRP-Human Fc antibody) was performed. Atezolizumab (ATZ) was used as the standard. Comparison of anti-human PD-L1 antibody expression levels among each cell line. PD-L1 binding ELISA (Human PD-1 immobilized, detected with HRP-Human Fc antibody) was performed. ATZ was used as the standard. Measurement of PD-1 / PD-L1 inhibitory activity. A) Culture supernatants transfected with ATZ / SV-01 and SV-01 (empty vector). B) The IC50 value was calculated by converting the concentration of ATZ / SV-01 used in experiment A into the human Fc concentration, which was 19.14 ng / ml. Measurement of PD-1 / PD-L1 inhibitory activity. A) Culture supernatants transfected with ATZ / SV-01 and SV-01 (empty vector). B) The IC50 value was calculated by converting the concentration of ATZ / SV-01 used in experiment A into the human Fc concentration, which was 15.37 ng / ml.Mouse PD-1 / PD-L1 inhibitory activity measurement results Human PD-1 / PD-L1 inhibitory activity measurement results Cytotoxicity Assay Replication Assay Infection efficiency of the prepared virus (X-gal staining) Structure confirmation of the prepared virus by Southern blotting Comparison of the antitumor effect of anti-mouse PD-L1 antibody-expressing HSV-1 in a unilateral subcutaneous tumor model in Neuro2a / A / J mice. *; P<0.05, **; P<0.01 (Two-way ANOVA and Bonferroni comparison test), 200mm at day 16. 3 The number of mice is shown when the following tumor sizes were considered to have an inhibitory effect. Anti-tumor effect on the Neuro2a / A / J bilateral subcutaneous tumor model. *: P<0.05 (two-way ANOVA and Bonferroni comparison test). Anti-tumor effect on the cloneM3 / DBA / 2 unilateral subcutaneous tumor model. *: P<0.05, ***: P<0.001 (two-way ANOVA and Bonferroni comparison test). Anti-tumor effect on the cloneM3 / DBA / 2 bilateral subcutaneous tumor model. Treatment side: ***; P<0.001, ****; P<0.0001 (Two-way ANOVA and Tukey's comparison test); Non-treatment side: *; P<0.05. On day 11, there was a significant difference only between mock and T-J43 (One-way ANOVA and Tukey's comparison test). Bar=SEM. Comparison of the antitumor effect of anti-mouse PD-L1 antibody (T-ATZ)-expressing HSV-1 in a unilateral subcutaneous tumor model in Renca / Balb / c mice. The results were almost equivalent to those of T-01. Bar=SEM. Comparison of the antitumor effect of the combination of T-01 and ATZ antibody and T-ATZ alone in a bilateral subcutaneous tumor model in Neuro2a / A / J mice. Comparison of the antitumor effect of the combination of T-01 and ATZ antibody and T-ATZ alone in a bilateral subcutaneous tumor model in Neuro2a / A / J mice (treatment side). Tumor loss (50 mm) on day 87. 3Comparison of the antitumor effects of the combination of T-01 and ATZ antibody with T-ATZ alone in a bilateral subcutaneous tumor model in Neuro2a / A / J mice (non-treated side). Tumor loss (50 mm) was observed on day 87. 3The percentage of patients who died (or died after 1 year of age) is shown. Kaplan-Meier survival curve. This survival curve is based on the assumption that observation was terminated (death) when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 135. *; P<0.05, log-rank test. Comparison of blood and intratumoral antibody concentrations following intraperitoneal administration of ATZ and intratumoral administration of T-ATZ in a unilateral Neuro2a / A / J subcutaneous tumor model. For the 200 μg ATZ group, blood concentrations exceeded the measurement range even at a 1 / 1000 dilution, so this was designated ND. For intratumoral administration of T-ATZ, the line in the figure is below the detection limit, but it was calculated using the calibration curve formula and is included for reference. Amino acid sequence for expression of anti-mouse CTLA4 antibody (derived from 9H10). The heavy chain constant region was derived from human IgG1, and the light chain constant region was derived from human κ chain. The underlined red text indicates the hypervariable region predicted by IMGT / DomainGapAlign. Amino acid sequence for expression of anti-mouse CTLA4 antibody (derived from 4F10). The heavy chain constant region was derived from human IgG1, and the light chain constant region was derived from human κ chain. The underlined and red letters indicate the hypervariable regions predicted by IMGT / DomainGapAlign. Amino acid sequence for expression of anti-mouse CTLA4 antibody (derived from 4F10). The heavy chain constant region was derived from Armenian ham IgG, and the light chain constant region was derived from Armenian ham Ig κ chain. Comparison of anti-CTLA-4 antibody expression levels Comparison of anti-mouse CTLA-4 antibody expression levels between each cell line CTLA-4 binding ELISA was performed (mouse CTLA4 was immobilized and detected with HRP-labeled anti-Armenian hamster IgG antibody). 4F10 was used as the standard. Measurement results of inhibitory activity of CTLA-4 / CD80 bindingCytotoxicity AssayReplication AssayInfection efficiency of the prepared virus (X-gal staining)Infection efficiency of the prepared virus (X-gal staining)Confirmation of the structure of the prepared virus by Southern blottingAnti-tumor effect on a Neuro2a / A / J unilateral subcutaneous tumor model. **; P<0.01 (Two-way ANOVA and Bonferroni comparison test)Anti-tumor effect on a Neuro2a / A / J bilateral subcutaneous tumor model.Treatment side: **; P<0.01 (two-way ANOVA and Bonferroni comparison test). Non-treatment side: *; P<0.05 on day 7, there was a significant difference only between mock and T-9H10 (one-way ANOVA and Tukey comparison test). Bar=SEM. Anti-tumor effect on the cloneM3 / DBA / 2 unilateral subcutaneous tumor model. **; P<0.01 (two-way ANOVA and Bonferroni comparison test). Anti-tumor effect on the cloneM3 / DBA / 2 bilateral subcutaneous tumor model. Treatment side: *; P<0.05, **; P<0.01, ****; P<0.0001 (two-way ANOVA and Bonferroni comparison test). Untreated side: *; P<0.05. Significant difference only between T-01 and T-9H10 on days 23 and 26 (t-test). Bar=SEM. Comparison of the antitumor effects of the combination of T-01 and 4F10 antibody and T-4F10HA alone in a Neuro2a / A / J mouse bilateral subcutaneous tumor model. Comparison of the antitumor effects of the combination of T-01 and 4F10 antibody and T-4F10HA alone in a Neuro2a / A / J mouse bilateral subcutaneous tumor model (treated side). The percentage of mice that achieved tumor-free status at day 42 is shown on the left, and the percentage of mice that achieved tumor-free status at day 87 is shown on the right. Comparison of the antitumor effects of the combination of T-01 and 4F10 antibody and T-4F10HA alone in a Neuro2a / A / J mouse bilateral subcutaneous tumor model (untreated side). The percentage of mice that achieved tumor-free status at day 42 is shown on the left, and the percentage of mice that achieved tumor-free status at day 87 is shown on the right. Kaplan-Meier survival curve. Survival curve when observation was terminated (death) when the animal died or when the maximum tumor diameter reached 20 mm. Observation ended on day 38. **; P<0.01, ***; P<0.001, log-rank test (only major significant differences are shown). Comparison of blood and intratumoral antibody concentrations following intraperitoneal administration of 4F10 and intratumoral administration of T-4F10HA in a Neuro2a / A / J unilateral subcutaneous tumor model. Effect of a combination of anti-CTLA-4 antibody (T-9H10) and anti-PD-1 antibody (T-J43)-expressing HSV-1 in a Neuro2a / A / J mouse bilateral subcutaneous tumor model. Tumor disappeared on day 48 (50 mm). 3The percentage of patients who underwent treatment with T-4F10 or the mix is ​​shown. Untreated side: There was a significant difference between T-4F10 and the mix (*; P<0.05, two-way ANOVA and Bonferroni comparison test). Kaplan-Meier survival curve. This is a survival curve where observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 38. **; P<0.01, log-rank test. Effect of the combination (mix) of anti-CTLA-4 antibody (T-9H10) and anti-PD-L1 antibody (T-ATZ) expressing HSV-1 in a bilateral subcutaneous tumor model in Neuro2a / A / J mice. Tumors disappeared on day 62 (50 mm 3 The percentage of patients who died (below) is shown. Kaplan-Meier survival curve. This is a survival curve assuming that observation ended (death) when death occurred or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 62. Effect of a combination (mix) of anti-CTLA-4 antibody (T-9H10) and anti-PD-1 antibody (T-J43) expressing HSV-1 in a bilateral subcutaneous tumor model in clone M3 / DBA / 2 mice. Effect of a combination (mix) of anti-CTLA-4 antibody (T-9H10) and anti-PD-L1 antibody (T-ATZ) expressing HSV-1 in a bilateral subcutaneous tumor model in clone M3 / DBA / 2 mice. The therapeutic effect of a cocktail (mix) of two types of HSV-1 expressing anti-CTLA-4 antibody (T-4F10HA) and anti-PD-1 antibody (T-J43) in a bilateral subcutaneous tumor model in Neuro2a / A / J mice. Tumor disappearance (50 mm) was observed on day 48. 3 The percentage of patients who died or reached a maximum tumor diameter of 20 mm is shown. Kaplan-Meier survival curve. Survival curve (during observation) when observation was terminated (death) when death occurred or when the maximum tumor diameter reached 20 mm. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, log-rank test. The therapeutic effect of a cocktail (mix) of two types of HSV-1 expressing anti-CTLA-4 antibody (T-4F10HA) and anti-PD-L1 antibody (T-ATZ) in a bilateral subcutaneous tumor model in Neuro2a / A / J mice, followed by sequential administration. Tumors disappeared on day 48 (50 mm). 3The percentage of patients who died or reached a maximum tumor diameter of 20 mm is shown. Kaplan-Meier survival curve. Survival curve (during observation) when observation was terminated (death) when the patient died or when the maximum tumor diameter reached 20 mm. *: P<0.05, **: P<0.01, log-rank. Therapeutic effect of a cocktail (mix) of two types of HSV-1 expressing anti-CTLA-4 antibody (T-4F10HA) and anti-PD-1 antibody (T-J43) in a bilateral subcutaneous tumor model in testclone M3 / DBA / 2 mice, with sequential administration. Tumor disappearance at 50 days (50 mm 3 The percentage of patients who died or reached a maximum tumor diameter of 20 mm is shown. Kaplan-Meier survival curve. Survival curve (during observation) when observation was terminated (death) when the patient died or the maximum tumor diameter reached 20 mm. *: P<0.05, **: P<0.01, ***: P<0.001, log-rank. Therapeutic effect of a cocktail (mix) of anti-CTLA-4 antibody expressing (T-4F10HA) and anti-PD-L1 antibody expressing (T-ATZ) HSV-1 in a bilateral subcutaneous tumor model in testclone M3 / DBA / 2 mice, followed by sequential administration. Tumors disappeared on day 49 (50 mm 3The percentage of patients who died (or died as defined below) is shown. Kaplan-Meier survival curve. Survival curve (during observation) when observation was terminated (death) upon death or when the maximum tumor diameter reached 20 mm. *: P<0.05, **: P<0.01, log-rank test. Amino acid sequence for expression of anti-human PD-1 antibody (derived from Nivolmab). The heavy chain constant region was derived from human IgG4. The light chain constant region was derived from human κ chain. The underlined parts indicate a comparison of the expression levels of anti-PD-1 antibody at hypervariable sites predicted by IMGT / DomainGapAlign. HEK293T cells were infected with T-Niv and T-01 at an MOI of 0.1. 48 hours later, the supernatant was collected and concentrated by ultrafiltration (Amicon Ultra-30K). A) Human Fc ELISA (Human Fc antibody immobilized, detected with HRP-Human Fc antibody) and B) PD-1 binding ELISA (Human PD-1 immobilized, detected with HRP-Human Fc antibody) were performed. Comparison of anti-human PD-1 antibody expression levels between cell lines. PD-1 binding ELISA (Human PD-1 immobilized, detected with HRP-conjugated anti-Human IgG Fc antibody) was performed. Nivolumab was used as the standard. PD-1 / PD-L1 binding inhibitory activity measurement results. A) Culture supernatants transfected with Niv / SV-01 and SV-01 (empty vector). B) The IC50 value was calculated by converting the concentration of Niv / SV-01 used in experiment A to the Human Fc concentration, and was found to be 23.64 ng / ml. PD-1 / PD-L1 binding inhibitory activity measurement results. Cytotoxicity Assay. Replication Assay. Infection efficiency of the prepared virus (X-gal staining). Structural confirmation of the prepared virus by Southern blotting. Amino acid sequence for expression of anti-human CTLA4 antibody (derived from ipilimumab). The H-chain constant region was derived from human IgG1, and the L-chain constant region was derived from human κ chain. The underlined and red characters indicate the hypervariable sites predicted by IMGT / DomainGapAlign. Amino acid sequence for expression of anti-human CTLA4 antibody (derived from ticilimumab). The H-chain constant region was derived from human IgG2, and the L-chain constant region was derived from human κ chain. The underlined and red characters indicate the hypervariable sites predicted by IMGT / DomainGapAlign. Comparison of anti-CTLA4 antibody expression levels.A) Ipi / SV-01 and Tic / SV-01 plasmids were transiently expressed in HEK293T cells, and the culture supernatants were concentrated to serve as samples. A negative control sample was also prepared by treating the SV-01 plasmid in the same manner. ELISA for human Fc (human Fc antibody immobilized and detected with HRP-human Fc antibody) was performed. B) HEK293T cells were infected with T-Ipi at an MOI of 1. After 48 hours, the supernatant was collected and concentrated by ultrafiltration (Amicon Ultra-30K). Then, CTLA-4 binding ELISA (human CTLA-4 immobilized and detected with HRP-human Fc antibody; ipilimumab was used as the standard) was performed. C) HEK293T cells were infected with T-Tic at an MOI of 1. 48 hours later, the supernatant was collected and concentrated by ultrafiltration (Amicon Ultra-30K). CTLA-4 binding ELISA was performed (human CTLA-4 was immobilized, detected with HRP-Human Fc antibody, and the standard was ticilimumab). Comparison of anti-human CTLA-4 antibody expression levels among cell lines. A) T-Ipi and B) T-Tic expression. Ipilimumab (A) and ticilimumab (B) were used as standards, respectively. CTLA-4 / CD80 (B7-1) inhibitory activity was measured. A) Culture supernatants transfected with Ipi / SV-01 and SV-01 (empty vector). B) The IC50 value, calculated by converting the concentration of Ipi / SV-01 used in experiment A) into Human Fc, was 8.24 ng / ml. Measurement results of CTLA-4 / CD80 (B7-1) inhibitory activity. A) Culture supernatant of Tic / SV-01 and SV-01 (empty vector) transfection. B) The IC50 value was calculated by converting the concentration of Tic / SV-01 used in the experiment in A) into Human Fc, and was found to be 25.43 ng / ml. Measurement results of CTLA-4 / CD80 binding inhibitory activity. Cytotoxicity Assay. Replication Assay. Infection efficiency of the prepared virus (X-gal staining). Structural confirmation of the prepared virus by Southern blotting. Insertion site of the gene encoding the immune checkpoint inhibitor and the base sequence surrounding it. γ34.5 gene and the base sequence surrounding it (TR). LWithin the region). The α47 gene and the base sequence before and after it. Effect of combined (mix) and sequential administration of anti-CTLA-4 antibody (T-4F10HA) and soluble B7-1 (T-mB7.1) expressing HSV-1 in a Neuro2a mouse bilateral subcutaneous tumor model. Top: Significant differences between the treatment side and mock. Significant differences between the non-treatment side and mock. Middle: Tumor disappeared on day 58 (50mm 3 The percentage of mice that achieved survival (50 mm or less) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the mouse died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 58. ***; p<0.001, Log-rank test. Effect of combined (mix) and sequential administration of anti-CTLA-4 antibody (T-4F10HA) and IL12 (T-mfIL12)-expressing HSV-1 in a Neuro2a mouse bilateral subcutaneous tumor model. Top: Significant differences from the treatment side and Mock. Non-treatment side, **; p<0.01, two-way ANOVA and Bonferroni comparison test. Middle: Tumor disappeared on day 59 (50 mm or less). 3 The percentage of patients who achieved survival (50 mm or less) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 70. ***; p<0.001, Log-rank test. Effect of combined (mix) and sequential administration of anti-PD-1 antibody (T-J43) and soluble B7-1 (T-mB7.1)-expressing HSV-1 in a Neuro2a mouse bilateral subcutaneous tumor model. Top: Significant differences from the treatment side and Mock. Non-treatment side, *; p<0.05, two-way ANOVA and Bonferroni comparison test. Middle: Tumor disappeared on day 49 (50 mm or less). 3The percentage of patients who achieved survival (50 mm or less) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 49. ***; p<0.001, Log-rank test. Effect of combined (mix) and sequential administration of anti-PD-1 antibody (T-J43) and IL12 (T-mfIL12)-expressing HSV-1 in a Neuro2a mouse bilateral subcutaneous tumor model. Top: All significant differences from the treatment side and mock. Non-treatment side, *; p<0.05, ***; p<0.001, two-way ANOVA and Bonferroni comparison test. Middle: Tumor disappeared on day 59 (50 mm or less). 3 The percentage of patients who achieved 50% or more of the following (50mm or less) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20mm. Observation ended on day 70. ***; p<0.001, Log-rank test Effect of combined (mix) and sequential administration of anti-PD-L1 antibody (T-ATZ) and soluble B7-1 (T-mB7.1)-expressing HSV-1 in a Neuro2a mouse bilateral subcutaneous tumor model. Top: Significant differences between the treatment side and Mock. Significant differences between the non-treatment side and Mock. Middle: Tumor disappeared on day 58 (50mm or less) 3 The percentage of patients who achieved 50% or more of the above (below) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20mm. Observation ended on day 62. **; p<0.01, ***; p<0.001, Log-rank test. Effect of combined (mix) and sequential administration of anti-PD-L1 antibody (T-ATZ) and IL12 (T-mfIL12)-expressing HSV-1 in a Neuro2a mouse bilateral subcutaneous tumor model 1. Top: All significant differences from the treatment side and mock. Non-treatment side, **; p<0.01, two-way ANOVA and Bonferroni comparison test. Middle: Tumor disappearance on day 59 (50mm 3The percentage of mice that achieved survival (50 mm or less) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the mouse died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 58. *; p<0.05, **; p<0.01, ***; p<0.001, Log-rank Testclone M3 mice. Effects of combined (mix) and sequential administration of anti-CTLA-4 antibody (T-4F10HA) and soluble B7-1 (T-mB.7)-expressing HSV-1 in a bilateral subcutaneous tumor model. Top: Significant differences between the treated side and Mock. Significant differences between the untreated side and Mock. Middle: Tumor disappeared on day 59 (50 mm or less). 3 The percentage of mice that achieved survival (50 mm or less) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the mouse died or when the maximum tumor diameter on either side reached 20 mm. Observation was terminated on day 87. ***; p<0.001, Log-rank testclone M3 mice. Effects of combined (mix) and sequential administration of anti-CTLA4 antibody (T-4F10HA) and IL12 (T-mfIL12)-expressing HSV-1 in a bilateral subcutaneous tumor model. Top: All significant differences from the treated side and mock. Non-treated side, *; p<0.05, **; p<0.01, two-way ANOVA and Bonferroni comparison test. Middle: Tumor disappeared on day 58 (50 mm or less). 3 The percentage of patients who achieved 50% or more of the above (below) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 79. ***; p<0.001, Log-rank Testclone M3 mice bilateral subcutaneous tumor model. Effect of combined (mix) and sequential administration of anti-PD-1 antibody (T-J43) and soluble B7-1 (T-mB7.1)-expressing HSV-1 in a bilateral subcutaneous tumor model. Top: Significant differences between the treatment side and Mock. Significant differences between the non-treatment side and Mock. Middle: Tumor disappeared on day 58 (50 mm 3The percentage of mice that achieved survival (50 mm or less) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the mouse died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 87. ***; p<0.001, Log-rank test. Effect of combined (mix) and sequential administration of anti-PD-1 antibody (T-J43) and IL12 (T-mfIL12)-expressing HSV-1 in a bilateral subcutaneous tumor model in cloneM3 mice. Top: All significant differences from the treatment side and mock. Non-treatment side, *; p<0.05, two-way ANOVA and Bonferroni comparison test. Middle: Tumor disappeared on day 58 (50 mm 3 The percentage of patients who achieved 50% or more of the above (below) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20mm. Observation ended on day 79. *; p<0.05, **; p<0.01, ***; p<0.001, Log-rank Testclone M3 mice: Effect of combined (mix) and sequential administration of anti-PD-L1 antibody (T-ATZ) and soluble B7-1 (T-mB7.1)-expressing HSV-1 in a bilateral subcutaneous tumor model. Top: Significant differences between the treatment side and Mock. Significant differences between the non-treatment side and Mock. Middle: Tumor disappeared on day 59 (50mm 3 The percentage of patients who achieved survival (50 mm or less) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 58. ***; p<0.001, Log-rank testcloneM3 mice. Effect of combined (mix) and sequential administration of anti-PD-L1 antibody (T-ATZ) and IL12 (T-mfIL12)-expressing HSV-1 in a bilateral subcutaneous tumor model. Top: All significant differences from the treatment side and mock. Non-treatment side, *; p<0.05, ***; p<0.001, two-way ANOVA and Bonferroni comparison test. Middle: Tumor disappearance (50 mm or less) on day 59. 3The percentage of patients who achieved 50% or more of the above (below) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20 mm. Observation was terminated on day 69. *; p<0.05, ***; p<0.001, Log-rank test. Effect of combined (mix) and sequential administration of anti-CTLA-4 antibody (T-4F10HA) and soluble B7-1 (T-mB7.1)-expressing HSV-1 in a bilateral subcutaneous tumor model in Renca / Balb / c mice. Top: Significant differences between the treatment side and mock. Non-treatment side, *; p<0.05, **; p<0.01, two-way ANOVA and Bonferroni comparison test. Middle: Tumor disappearance on day 59 (50 mm 3 The percentage of mice that died (below) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 73. *; p<0.05, ***; p<0.001, Log-rank test. Effect of combined (mix) and sequential administration of anti-CTLA-4 antibody (T-4F10HA) and IL12 (T-mfIL12) expressing HSV-1 in a bilateral subcutaneous tumor model in Renca / Balb / c mice. Top: There were significant differences between the treatment side and mock, except for 12→4F. ***; p<0.001, two-way ANOVA and Bonferroni comparison test. There were no significant differences between the non-treatment side and non-treatment side. Middle: There was tumor disappearance (50 mm) on day 58. 3 The percentage of patients who achieved tumor disappearance (50mm or less) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20mm. Observation ended on day 79. **;p<0.01, ***;p<0.001, Log-rank test. Effect of combined (mix) and sequential administration of anti-PD-1 antibody (T-J43) and soluble 7-1 (T-mB7.1)-expressing HSV-1 in a bilateral subcutaneous tumor model in Renca / Balb / c mice. Top: *;p<0.05, **;p<0.01, ***;p<0.001, two-way ANOVA and Bonferroni comparison test. Middle: Tumor disappearance (50mm or less) on day 59. 3The percentage of patients who achieved a tumor size of 50 mm or less is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 73. **; p<0.01, ***; p<0.001, Log-rank test. Effect of combined (mix) and sequential administration of anti-PD-1 antibody (T-J43) and IL12 (T-mfIL12) expressing HSV-1 in a bilateral subcutaneous tumor model in Renca / Balb / c mice. Top: There were significant differences between the treatment side and Mock except for 12→J. **; p<0.01, two-way ANOVA and Bonferroni comparison test. There were no significant differences between the non-treatment side and the non-treatment side. Middle: Tumor disappeared on day 58 (50 mm 3 The percentage of patients who achieved survival (50 mm or less) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 79. *; p<0.05, **; p<0.001, Log-rank test. Effect of combined (mix) and sequential administration of anti-PD-L1 antibody (T-ATZ) and soluble B7-1 (T-mB7.1)-expressing HSV-1 in a bilateral subcutaneous tumor model in Renca / Balb / c mice. Top: All significant differences from the treatment side and mock. Non-treatment side, **; p<0.01, two-way ANOVA and Bonferroni comparison test. Middle: Tumor disappeared on day 58 (50 mm or less). 3 The percentage of patients who achieved 50% or more of the above (below) is shown in a box. Bottom: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20mm. Observation ended on day 65. No significant difference, Log-rank test. Effect of combined (mix) and sequential administration of anti-PD-L1 antibody (T-ATZ) and IL12 (T-mfIL12) expressing HSV-1 in a bilateral subcutaneous tumor model in Renca / Balb / c mice. Top: Significant differences between the treated side and Mock. No significant differences between the untreated side. Middle: Tumor disappeared on day 58 (50mm 3The percentage of mice that died (death) is indicated by a box. Bottom: Survival curve when observation was terminated (death) when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 65. *; p<0.05, Log-rank test. Effect of the 3-type HSV-1 cocktail in a Neuro2a mouse bilateral subcutaneous tumor model. Top left: Significant differences from the treated side and mock. No significant differences from the untreated side. Bottom left: Survival curve when observation was terminated (death) when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 62. *; p<0.05, **; p<0.01, ***; p<0.001, Log-rank test. Right: Tumor disappeared on day 58 (50 mm 3 The percentage of mice with tumors that disappeared (below) is shown in a box. Effect of the three-type HSV-1 cocktail in a bilateral subcutaneous tumor model in cloneM3 mice. Upper left: All treatments were significantly different from the treated side and Mock. There were significant differences from the untreated side and Mock except for T-01 and 12+B+4. *; p<0.05, **; p<0.01, ***; p<0.001, two-way ANOVA and Bonferroni comparison test. Lower left: Survival curve when observation was terminated (death) when the tumor died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 84. *; p<0.05, ***; p<0.001, Log-rank test. Right: Tumor disappeared on day 60 (50 mm 3 The percentage of patients who received the following treatments is indicated by a box. 12 = T-mfIL12 (IL12), B = T-mB7.1 (soluble B7-1), 4 = T-4F10HA (anti-CTLA4 antibody), J = T-J43 (anti-PD-L1 antibody), A = T-ATZ (anti-PD-1 antibody). Effect of the three-type HSV-1 cocktail in a Renca mouse bilateral subcutaneous tumor model. Upper left: All significant differences from the treated and mock groups. All significant differences except T-01 from the untreated and mock groups. ***; p<0.001, two-way ANOVA and Bonferroni comparison test. Lower left: Survival curve when observation was terminated (death) when the patient died or when the maximum tumor diameter on either side reached 20 mm. Observation ended on day 70. *; p<0.05, ***; p<0.001, Log-rank test. Right: Tumor disappeared on day 60 (50mm 3(below) is expressed by enclosing the percentage in a square. 12=T-mfIL12 (IL12), B=T-mB7.1 (soluble B7-1), 4=T-4F10HA (anti-CTLA4 antibody), J=T-J43 (anti-PD-L1 antibody), A=T-ATZ (anti-PD-1 antibody).

[0013] The present invention relates to an oncolytic herpes simplex virus (HSV) carrying a gene encoding an immune checkpoint inhibitor. Hereinafter, the present invention will be described in detail based on an embodiment of the present invention (also referred to as the "present embodiment").

[0014] (Gene Encoding Immune Checkpoint Inhibitor) This embodiment relates to HSV having a gene encoding an immune checkpoint inhibitor.

[0015] In one aspect, the immune checkpoint inhibitor binds with sufficient affinity and specificity to an immune checkpoint protein (also referred to as an immune checkpoint modulator) or a protein (e.g., a receptor) that interacts with it, and is thereby able to inhibit the activation-suppressing signal from the immune checkpoint protein to an immune cell. The immune checkpoint inhibitor is preferably an antibody or a fragment thereof that binds to the immune checkpoint protein or a protein that interacts with it.

[0016] In one embodiment, the immune checkpoint inhibitor is an antibody or fragment thereof against PD-1 (programmed death 1), CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4), TIM-3 (T-cell immunoglobulin and mucin-containing protein-3), LAG-3 (lymphocyte activation gene-3), B7-H3, B7-H4, B7-H5 (VISTA, V-domain Ig Suppressor of T cell Activation), TIGIT (T cell immunoreceptor with Ig and ITIM domain), PD-L1 (B7-H1), or PD-L2 (B7-H2). It is preferably any one selected from PD-1, PD-L1, and CTLA-4, or a fragment thereof.

[0017] In one embodiment, the immune checkpoint inhibitor antibody or fragment thereof is an anti-PD-1 antibody, such as nivolumab (Opdivo), pembrolizumab (Keytruda), spartalizumab, or cemiplimab; an anti-PD-L1 antibody, such as atezolizumab (Tecentriq), avelumab (Havencio), or durvalumab (Imfinzi); an anti-CTLA-4 antibody, such as ticilimumab (Tremelimumab), ipilimumab (Yervoy), or tremelimumab; an anti-PD-1 antibody, such as spartalizumab or cemiplimab; or a fragment thereof.

[0018] In one embodiment, the type of antibody is not particularly limited and may be any antibody, such as a human antibody, a humanized antibody, a chimeric antibody, or an antibody derived from another animal (e.g., a mouse antibody, a rat antibody, a rabbit antibody, a sheep antibody, a camel antibody, or a chicken antibody), but is preferably an antibody from the same organism as the tumor to be lysed by the oncolytic HSV carrying the gene encoding the antibody. For example, when the oncolytic HSV is to be used in humans, a human antibody or a humanized antibody against a human immune checkpoint protein or a protein that interacts with the human immune checkpoint protein is preferred.

[0019] In one embodiment, the immune checkpoint inhibitor may be an antibody fragment. The antibody fragment refers to either an antibody fragment itself or an antibody fragment bound to any molecule, which recognizes the same epitope as the original antibody. There are no particular limitations on the fragment as long as it contains the complementarity-determining region (CDR) of the original antibody. Specifically, the V L , V H , C L and C H Fab fragments consisting of the Fab region; Fab' fragments, which also contain the hinge region; F(ab')2 fragments, in which two Fabs are linked by a disulfide bond at the hinge region; V L and V H Fv consisting of V L and V H In addition to scFv, which is a single-chain antibody in which two variable regions constituting the antigen-binding site of an antibody are linked by an artificial polypeptide linker, examples of such antibodies include sdFv, diabody, and sc(Fv)2. H ) and the light chain variable region (V L ) is not particularly limited.

[0020] In one embodiment, the scFv is H and V L can be prepared by linking these with a known linker (GS linker (SEQ ID NO: 11) etc.).

[0021] In one aspect, the antibody or fragment thereof preferably has a long half-life in tumor cells. For example, compared to single-chain antibodies, double-chain antibodies have a longer half-life due to the presence of the Fc portion, and antibody-dependent immune responses can be expected.

[0022] In one embodiment, the HSV carrying a gene encoding an immune checkpoint inhibitor is V HA gene encoding any of the following polypeptides (i) to (iii), which are regions: (i) a polypeptide having the sequence of SEQ ID NO: 3, 11, 15, 18, 20, 22, 25, or 27, preferably 11, 22, 25, or 27; (ii) a polypeptide consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the polypeptide having the sequence of SEQ ID NO: 3, 11, 15, 18, 20, 22, 25, or 27, preferably 11, 22, 25, or 27, and having a desired binding ability; (iii) a polypeptide having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the polypeptide of SEQ ID NO: 3, 11, 15, 18, 20, 22, 25, or 27, preferably 11, 22, 25, or 2, and having a desired binding ability; and L The virus has a gene encoding any of the following polypeptides (iv) to (vi), which are regions of the polypeptide sequence: (iv) the polypeptide of SEQ ID NO: 7, 13, 17, 19, 21, 24, 26, or 29, preferably 13, 24, 26, or 29; (v) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the polypeptide of SEQ ID NO: 7, 13, 17, 19, 21, 24, 26, or 29, preferably 13, 24, 26, or 29, and which has a desired binding ability; or (vi) a polypeptide which has 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the polypeptide of SEQ ID NO: 7, 13, 17, 19, 21, 24, 26, or 29, preferably 13, 24, 26, or 29, and has a desired binding ability; and expresses these polypeptides.

[0023] In one aspect, the genetically modified HSV of this embodiment is C HA gene encoding any one of the following polypeptides (vii) to (ix) as a region: (vii) a polypeptide of SEQ ID NO: 4, 9, 12, 16, 23, or 28, preferably 4, 12, 16, 23, or 28; (viii) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the polypeptide of SEQ ID NO: 30, and having a desired binding ability; (ix) a polypeptide having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the polypeptide of SEQ ID NO: 30, and having V H The polypeptide may have a polypeptide that has the desired binding ability when used together with any of the polypeptides of the regions (i) to (iii).

[0024] In one aspect, the genetically modified HSV of this embodiment is C L A gene encoding any one of the following polypeptides (x) to (xii) as a region: (x) a polypeptide of SEQ ID NO: 8, 10, or 14, preferably 8 or 14; (xi) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the polypeptide of SEQ ID NO: 31, and having a desired binding ability; (xii) a polypeptide having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more and 90% or more homology with the polypeptide of SEQ ID NO: 31, and having V L The polypeptide may have a polypeptide that has the desired binding ability when used together with any one of the polypeptides (iv) to (vi).

[0025] In one embodiment, the genetically modified HSV preferably has a gene further encoding a Furin recognition sequence (SEQ ID NO: 5) and a polypeptide of FMDV-2A (SEQ ID NO: 6), and may have a gene encoding the polypeptides of SEQ ID NOs: 5 and 6 between the gene encoding any one of the polypeptides (i) to (iii) and any one of the polypeptides (vii) to (ix) above and the gene encoding any one of the polypeptides (iv) to (vi) and any one of the polypeptides (x) to (xii) above.

[0026] When referring to an amino acid sequence in which one or several amino acids have been deleted, substituted, or added, the number of deleted, substituted, or added amino acids is not particularly limited as long as a polypeptide consisting of that amino acid sequence has the desired binding ability, and can be, for example, 1 to 5, 1 to 3, or 1 to 2. The deletion, substitution, or replacement may occur at either the terminal or middle of the peptide, and at one or more positions. Similarly, when referring to a nucleotide sequence in which one or several bases have been deleted, substituted, or added, the number of deleted, substituted, or added bases is not particularly limited as long as a polypeptide encoded by that nucleotide sequence has the desired binding ability, and can be, for example, 1 to 5, 1 to 3, or 1 to 2. The deletion, substitution, or replacement may occur at either the terminal or middle of the polynucleotide, and at one or more positions. Whether a polypeptide has the desired binding ability can be determined using techniques known to those skilled in the art. For example, this can be determined using the ELISA described in the Examples below. The amino acids may be natural amino acids, derivatives thereof, artificial amino acids, or unnatural amino acids. In one aspect, the amino acids of this embodiment are preferably natural amino acids.

[0027] Known techniques can be used to introduce a gene encoding an immune checkpoint inhibitor into an oncolytic HSV. For example, DNA encoding the immune checkpoint inhibitor can be inserted into an expression vector by a known method (such as a method using a restriction enzyme), and the expression vector can be transfected into an oncolytic HSV. The expression vector can further include a promoter that regulates the expression of the target gene, a replication origin, a selection marker gene, and the like. The promoter and replication origin can be selected appropriately depending on the type of oncolytic HSV and vector into which the gene is to be introduced.

[0028] Specifically, for example, the amino acid sequence or nucleotide sequence of the antibody or fragment thereof to be expressed can be obtained, and the region of interest can be synthesized by known techniques. For example, sequences encoding polypeptides corresponding to the heavy and light chains of an antibody can be designed so that equal amounts of the heavy and light chains are expressed from cDNA, enabling efficient antibody production. Preferably, the virus can be modified by designing the sequences to incorporate both an amino acid sequence encoding a foot-and-mouth disease virus (FMDV)-2A sequence with autocleaving activity and a furin cleavage site. Furthermore, the polypeptides corresponding to the heavy and light chains may each have a signal sequence for secretion at their N-terminus.

[0029] The site at which the gene is introduced is not particularly limited as long as the oncolytic HSV maintains its oncolytic activity. For example, a gene encoding an immune checkpoint inhibitor can be introduced into a site where a non-essential viral gene has been deleted. When the virus is HSV-1, the non-essential genes shown in Table 1 below can be deleted and a gene encoding an immune checkpoint inhibitor can be introduced. Expression of the introduced gene can be confirmed using known techniques, as shown in the Examples below.

[0030] Genetic recombination of viruses with large genome sizes, such as HSV, can be carried out using, for example, the IL-12 expression T-BAC system described in Non-Patent Document 2 or a system similar thereto.

[0031] (Oncolytic HSV) This embodiment relates to oncolytic HSV. Oncolytic refers to a genetic modification that allows for use in tumor treatment.

[0032] In this embodiment, oncolytic HSV is a virus that can be used as an active ingredient in pharmaceutical compositions for virotherapy or cancer treatment. It is not particularly limited as long as it has the ability to infect tumor cells, selectively replicate in tumor cells, destroy tumor cells during viral replication, and infect and further replicate in surrounding tumor cells. Known viruses can be used. Many of these viruses are genetically modified variants of naturally occurring viruses to enhance tumor selectivity. If necessary, they may be attenuated and modified to enhance antitumor activity, such as by incorporating a gene encoding a VEGF antagonist (Patent Document 2) or by incorporating a gene expressing a fusion polypeptide in which the light and heavy chains of IL-12 are linked via an elastin motif (Non-Patent Document 2).

[0033] For example, oncolytic HSV that can be used in this embodiment includes mutants of HSV-1 or HSV-2.

[0034] In one embodiment, the oncolytic HSV is an HSV-1 variant. HSV-1 is classified as an enveloped, double-stranded DNA virus and possesses the following advantageous characteristics for cancer therapy: 1) it can infect all types of human cells; 2) its viral life cycle and genome sequence have been elucidated; 3) the functions of most viral genes are known, making it amenable to genetic manipulation; 4) its large viral genome (approximately 152 kb) allows for the incorporation of large or multiple genes. Furthermore, HSV-1 possesses the following advantages suitable for clinical applications: 5) it can kill all cells at a relatively low multiplicity of infection (MOI); 6) antiviral drugs that inhibit its proliferation are available; 7) circulating anti-HSV-1 antibodies do not affect the cell-to-cell spread of the virus, allowing repeated administration; 8) mice and monkeys susceptible to HSV-1 exist, allowing preclinical evaluation of safety and efficacy in animals; and 9) the viral DNA is not integrated into the host cell genome but remains extrachromosomal.

[0035] The HSV-1 genome consists of 82% long unique regions (U LIt consists of two unique sequence regions, the long region (L) and the short unique region (Us) of 12%, and the terminal repeat (TR) and inverted repeat (IR) sequences located at both ends of each region (Table 1, Todo, T. (2008). A journal and virtual library 13, 2060-2064). The L and S regions can each take two directions independently, so the HSV-1 genome DNA consists of four isomers. This genome contains the TR L RL1, RL2, U above L UL1 to UL56, TR above S Above RS1, U S A total of 84 genes are unidirectionally encoded in US1 to US12, about half of which are unnecessary for viral replication. Deleting these nonessential genes can reduce pathogenicity and enable gene transfer (Carson, J. et al. (2010). Drugs of the future 35, 183-195).

[0036]

[0037] When an oncolytic HSV is an HSV-1 mutant, it may have one or more of the following characteristics: - Acquisition of tumor cell-specific replication ability by inactivating enzymes involved in viral DNA synthesis, such as thymidine kinase (TK), ribonucleotide reductase (RR), and uracil-N-glycosylase (UNG or UDG). - Acquisition of tumor cell-specific replication ability by deleting the γ34.5 gene, which encodes the ICP34.5 protein involved in the pathogenicity of HSV-1. - Acquisition of antitumor effect by deleting α47. - Deletion or inactivation of genes to prevent reversion to the wild type and increase safety (for example, deletion or inactivation of the endogenous γ34.5 gene, α47 gene (ICP47 gene), α0 gene (ICP0 gene), U L 41 genes (vhs genes), U LDeletion or inactivation of 56 genes). - Enhancement of antitumor immunity and prolongation of survival by expressing immune stimulatory genes (IL-4, IL-10, GM-CSF, IL-12, soluble B7-1, etc.). - Enhancement of antiangiogenic and antitumor effects by expressing genes expressing antiangiogenic factors such as platelet factor 4, thrombospondin, endostatin, dominant-negative FGF, and angiostatin. - Enhancement of antitumor effects by expressing metalloproteinase inhibitors involved in local tumor invasion. - Promotion of viral infection spread by overexpression of metalloproteinase. - Enhancement of tumor cell-specific viral replication by controlling viral genes with tumor- or tissue-specific promoters (calponin promoter, E2F-responsive cell cycle-dependent promoter B-myb, Nestin promoter, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), MUC-1, Musashi enhancer / promoter, etc.). - Suppression of swelling by expressing vascular endothelial growth factor (VEGF) antagonists (Patent Document 2).

[0038] In one aspect, the oncolytic HSV of this embodiment is preferably HSV-1 having any one or more of the following characteristics (a) to (c), more preferably all of the following characteristics (a) to (c). HSV-1 mutants having these characteristics can be prepared with reference to WO2011 / 101912, Patent Document 2, etc. (a) The ICP6 gene (UL39 gene) is deleted or inactivated, or is expressed under the control of a tumor-specific promoter or tissue-specific promoter; (b) The γ34.5 gene is deleted or inactivated; and (c) The α47 gene is deleted or inactivated.

[0039] Regarding the above characteristics (a) and (b), by deleting ICP6, the large subunit of RR, an enzyme important for nucleotide metabolism and viral DNA synthesis in non-dividing cells, and / or γ34.5, which antagonizes the function of phosphorylated PKR generated during viral infection, the virus cannot grow in normal cells but can grow only in tumor cells where RR activity is increased due to active division and PKR phosphorylation is suppressed. Regarding the above characteristic (c), the protein encoded by the α47 gene inhibits TAP and suppresses MHC class I expression on the host cell surface, thereby evading host immune surveillance. Therefore, α47 gene-deficient HSV-1 is expected to maintain MHC class I expression on host cells, thereby enhancing immune cell stimulation. Furthermore, in HSV-1 with both characteristics (b) and (c) above, the deletion of α47 simultaneously results in the deletion of the US11 late promoter, which overlaps with α47 in the genome. This puts the expression of the US11 gene under the control of the ICP47 immediate-early promoter, accelerating its expression and restoring the viral replication ability attenuated by the deletion of γ34.5 exclusively to tumor cells.

[0040] Deletion or inactivation of a gene refers to suppressing the expression of the gene by deleting all or part of the gene, or by substituting or modifying some bases, or by inserting unnecessary sequences, and can be carried out by known methods.

[0041] A tumor-specific promoter or tissue-specific promoter refers to a promoter that enables the expression of a gene under its control specifically in a desired tumor cell or tissue, and any known promoter can be used depending on the gene, such as the telomerase reverse transcriptase promoter (hTERT promoter) or the E2F promoter.

[0042] Examples of HSVs containing deletions or inactivations of the γ34.5 gene, ICP6 gene, and / or α47 gene described in (a) to (c) above include G207, which lacks both copies of the γ34.5 gene and inactivates the ICP6 gene, and G47Δ, which lacks both copies of the γ34.5 gene, inactivates the ICP6 gene, and deletes the α47 gene. Therefore, the oncolytic HSV of this embodiment can also be produced by further modifying G207 or G47Δ. In particular, G47Δ is an oncolytic HSV-1 that significantly improves tumor cell-selective viral replication and elicits anti-tumor immunity while improving safety. Its broad therapeutic range allows for safe administration of high doses into the human brain. In June 2021, it was approved in Japan as a regenerative medicine product (generic name: Tecelpatulev, product name: Delitact Injection) for the treatment of malignant glioma.

[0043] With regard to feature (a) above, it is preferable that a region encoding an immune checkpoint inhibitor be inserted into the site of the deleted or inactivated IPC6 gene. Note that when a region encodes a polypeptide, this includes cases where the nucleotide sequence of that region encodes the amino acid sequence of the polypeptide, and cases where the complementary sequence of the nucleotide sequence of that region encodes the amino acid sequence of the polypeptide.

[0044] "A region encoding an immune checkpoint inhibitor has been inserted into the site where the IPC6 gene has been deleted or inactivated" means, for example, that the virus has a sequence that is highly identical to the portion of the nucleotide sequence of SEQ ID NO: 30 in the Sequence Listing excluding Location 5349..9170, and in which a sequence encoding an immunogenic polypeptide or a complementary sequence thereto has been inserted in place of Location 5349..9170. "High identity" here refers to 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity.

[0045] SEQ ID NO: 30 and Figure 8 show the insertion site of the region encoding the immune checkpoint inhibitor in the virus prepared in the experiment described in the Examples section and the nucleotide sequence surrounding it. The insertion site of the region encoding the immune checkpoint inhibitor is Location 5349..9170 in SEQ ID NO: 30, and from the double-underlined TCA to the double-underlined CAT in Figure 8 (the double-underlined TCA is the complementary sequence of the stop codon, and the double-underlined CAT is the complementary sequence of the start codon).

[0046] The virus having the characteristic (b) above means, for example, that in the base sequence of SEQ ID NO: 31 in the Sequence Listing, all or part of Location 88..834 is deleted, or a part of the bases is substituted, modified, or an unnecessary sequence is inserted, thereby suppressing the expression of the γ34.5 gene. L "(b)" refers to a virus having a sequence in which all or part of the nucleotide sequence in either γ34.5 gene has been deleted, or some nucleotides have been substituted or modified, or unnecessary sequences have been inserted, thereby suppressing expression of the γ34.5 gene. Preferably, all or part of the γ34.5 gene sequence in either γ34.5 gene has been deleted, or some nucleotides have been substituted or modified, or unnecessary sequences have been inserted, thereby suppressing expression of both γ34.5 genes. Furthermore, a virus having the above characteristic (b) refers, for example, to a virus having a sequence highly identical to the sequence obtained by excluding locations 177..1128 from the nucleotide sequence of SEQ ID NO: 31 in the Sequence Listing.

[0047] SEQ ID NO: 31 and FIG. 9 show the γ34.5 gene of the strain from which G47Δ was derived and the base sequence surrounding it (TR L In the sequence of SEQ ID NO: 6, Location 88..834, which is shaded in Figure 9, is the coding region of the γ34.5 gene from the initiation codon to the termination codon. The deleted site in the basic backbone of G47Δ (base numbers 576 to 1527 of GU734771.1) is Location 177..1128 in SEQ ID NO: 31, which is the underlined portion in Figure 9. TR L The above sequence and IR LIt completely matches the complementary sequence around the coding region of the γ34.5 gene (nucleotide numbers 124349 to 125798 of GU734771.1).

[0048] A virus having the above characteristic (c) means, for example, that it has a sequence in which all or part of locations 218 to 484 in the nucleotide sequence of SEQ ID NO: 32 in the Sequence Listing have been deleted, or a portion of the nucleotides have been substituted or modified, or an unnecessary sequence has been inserted, thereby suppressing expression of the α47 gene.Furthermore, a virus having the above characteristic (c) means, for example, that it has a sequence highly identical to the sequence obtained by excluding locations 230 to 540 from the nucleotide sequence of SEQ ID NO: 32 in the Sequence Listing.

[0049] SEQ ID NO: 32 and Figure 10 show the α47 gene and the nucleotide sequence surrounding it of the strain from which G47Δ is derived. The α47 gene is encoded on the complementary strand. In the sequence of SEQ ID NO: 32, locations 218..484, which are shaded in Figure 10, represent the coding region of the γ34.5 gene from the initiation codon to the termination codon. The sequence of the site deleted in the basic backbone of G47Δ (nucleotides 576 to 1527 of GU734771.1) is located at locations 177..1128 in the sequence of SEQ ID NO: 32, and is the underlined portion in Figure 10.

[0050] Oncolytic HSV can be produced by referring to, for example, Patent Documents 1 and 2, Non-Patent Document 1, and International Publication WO2005 / 103237.

[0051] (Pharmaceutical Composition) This embodiment relates to a pharmaceutical composition comprising an oncolytic HSV that has a gene encoding the immune checkpoint inhibitor of this embodiment and has been genetically modified for use in tumor treatment. In the pharmaceutical composition of this embodiment, the oncolytic HSV grows specifically in tumor cells, and each time the virus replicates within the tumor, it expresses an immune checkpoint inhibitor (e.g., an immune checkpoint inhibitor antibody) locally in the tumor.

[0052] Administration of the pharmaceutical composition of this embodiment is expected to significantly improve the therapeutic efficacy of viral therapy. Conventional immune checkpoint inhibitor antibodies can shift the entire immune system to attack, but they are not effective unless the immune system recognizes cancer as non-self. Oncolytic HSV, however, can induce the immune system to recognize cancer cells as non-self, thereby eliciting anti-cancer immunity.

[0053] In one embodiment, the pharmaceutical composition suppresses the blood concentration of an immune checkpoint inhibitor. Conventional immune checkpoint inhibitor antibodies are known to cause severe side effects such as autoimmune diseases at a relatively high frequency when administered systemically. In one embodiment, the immune checkpoint inhibitor antibody expressed by administration of the pharmaceutical composition remains localized in the tumor.

[0054] Tumors to which the pharmaceutical composition of the present embodiment is expected to be applied include, but are not limited to, brain tumors, gliomas, eye tumors, neuroblastoma, retinoblastoma, primary central nervous system malignant lymphoma, meningioma, pituitary adenoma, schwannoma, craniopharyngioma, intraocular tumors, retinoblastoma, choroidal malignant melanoma, intraocular malignant lymphoma, ocular adnexal tumors, eyelid tumors, lacrimal gland cancer, ocular adnexal lymphoma, orbital sarcoma, conjunctival tumors, optic nerve tumors, glioma, oral cancer, tongue cancer, pharyngeal cancer, thyroid cancer, auditory cancer, adenoid cystic carcinoma, olfactory neuroblastoma, head and neck sarcoma, lung cancer, breast cancer, thymoma, thymic carcinoma, malignant pleural mesothelioma, trunk sarcoma, cardiac sarcoma, lung neuroendocrine tumor, and thoracic SMARC. A4-deficient tumors, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, colorectal cancer (colon cancer and rectal cancer), small intestinal cancer (duodenal cancer, jejunal cancer, ileal cancer), GIST (gastrointestinal stromal tumor), pancreatic and gastrointestinal neuroendocrine tumors, anal cancer, gastrointestinal neuroendocrine tumors, hepatocellular carcinoma, biliary tract cancer (bile duct cancer [including intrahepatic bile duct cancer], gallbladder cancer, and duodenal ampulla cancer), pancreatic cancer, pancreatic and gastrointestinal neuroendocrine tumors, liver tumors, pancreatic neuroendocrine tumors, renal cell carcinoma, renal pelvis and ureter cancer, bladder cancer, urachal cancer, kidney tumors, prostate cancer, breast cancer, cervical cancer, uterine cancer (endometrial cancer), uterine sarcoma, ovarian cancer, fallopian tube cancer, vaginal cancer, Vulvar cancer, malignant melanoma (skin), basal cell carcinoma, squamous cell carcinoma, lymphoma of the skin, soft tissue sarcoma (adult), desmoid tumor, osteosarcoma (pediatric), Ewing's sarcoma (pediatric), soft tissue sarcoma (pediatric), rhabdomyosarcoma (pediatric), classification of sarcomas (sarcomas), undifferentiated / unclassifiable sarcoma, osteosarcoma, chondrosarcoma, chordoma, Ewing's sarcoma, liposarcoma, fibrosarcoma, myxofibrosarcoma, undifferentiated pleomorphic sarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, epithelioid sarcoma, alveolar soft part sarcoma, clear cell sarcoma, extraskeletal Ewing's sarcoma, acute myeloid Leukemia, acute lymphocytic leukemia / lymphoblastic lymphoma, chronic myeloid leukemia, myelodysplastic syndrome, malignant lymphoma, B-cell lymphoma, T / NK-cell lymphoma, Hodgkin's lymphoma, lymphoma of the skin, chronic lymphocytic leukemia / small lymphocytic lymphoma, adult T-cell leukemia / lymphoma, multiple myeloma, acute promyelocytic leukemia, follicular lymphoma, MALT lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, peripheral T-cell lymphoma, Burkitt's lymphoma, extranodal NK / T-cell lymphoma, cancer of unknown primary origin,Hereditary tumors / familial tumors, neuroendocrine cancers, germ cell tumors, paragangliomas, germ cell tumors, testicular germ cell tumors, ovarian germ cell tumors, extragonadal germ cell tumors, neuroendocrine tumors.

[0055] From another perspective, tumors for which the pharmaceutical composition of this embodiment is effective include tumors for which the application of immune checkpoint inhibitors has been approved or is being considered, such as malignant melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, gastric cancer, malignant pleural mesothelioma, esophageal cancer, MSI-High colorectal cancer, gastroesophageal junction cancer and esophageal cancer, small cell lung cancer, hepatocellular carcinoma, glioblastoma, urothelial cancer, ovarian cancer, bladder cancer, prostate cancer, cervical cancer, uterine cancer, soft tissue sarcoma, primary central nervous system lymphoma, primary testicular lymphoma, pancreatic cancer, biliary tract cancer, etc. Furthermore, tumors for which the pharmaceutical composition of this embodiment is effective include the following, which are currently considered to be diseases for which immune checkpoint inhibitors are used in Japan. Unresectable malignant melanoma, unresectable advanced or recurrent non-small cell lung cancer, maintenance therapy after definitive chemoradiotherapy for unresectable locally advanced non-small cell lung cancer, unresectable or metastatic renal cell carcinoma, unresectable or metastatic renal cell carcinoma, relapsed or refractory classical Hodgkin lymphoma, recurrent or distant metastatic head and neck cancer, unresectable advanced or recurrent gastric cancer that has progressed after cancer chemotherapy, unresectable advanced or recurrent malignant pleural mesothelioma that has progressed after cancer chemotherapy, and cancer that has progressed after cancer chemotherapy. Unresectable, advanced or recurrent microsatellite instability-high (MSI-High) colorectal cancer that has progressed after chemotherapy, unresectable, advanced or recurrent esophageal cancer that has progressed after chemotherapy, unresectable urothelial carcinoma that has progressed after chemotherapy, advanced or recurrent microsatellite instability-high (MSI-High) solid tumors that have progressed after chemotherapy (only when standard treatment is difficult to treat), unresectable or metastatic renal cell carcinoma, extensive-stage small cell lung cancer, PD-L1-positive, hormone receptor-negative and HER2-negative inoperable or recurrent breast cancer, and unresectable Merkel cell carcinoma.

[0056] From another perspective, tumors for which the pharmaceutical composition of this embodiment is effective include those for which viral therapy is considered to be particularly effective, such as nervous system tumors (e.g., astrocytoma, anaplastic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, meningioma, neurofibroma, glioblastoma, diffuse midline glioma, ependymoma, schwannoma, neurofibrosarcoma, and neuroblastoma), pituitary tumors (e.g., pituitary adenoma), medulloblastoma, melanoma, and brain tumors. Examples of cancers that may be present include prostate cancer, head and neck cancer, esophageal cancer, kidney cancer, renal cell carcinoma, pancreatic cancer, breast cancer, lung cancer, colon cancer, colorectal cancer, gastric cancer, skin cancer, ovarian cancer, bladder cancer, sarcoma (e.g., osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma), squamous cell carcinoma, neuroectodermal carcinoma, thyroid tumor, lymphoma, hepatocellular carcinoma, mesothelioma, epidermoid carcinoma, and benign tumors (e.g., benign thyroid tumor or benign prostatic hyperplasia).

[0057] In relation to the present invention and the present embodiments, unless otherwise specified, reference to treatment in relation to a disease or condition includes prevention, reduction of the risk of onset, treatment, inhibition of progression, and prevention of recurrence.

[0058] The method of administration of the pharmaceutical composition of this embodiment is not particularly limited, and may be, for example, intravenous, intraarterial, intraventricular, intraperitoneal, intrathoracic, intraspinal, subcutaneous, intradermal, intraepidermal, intramuscular, or into a mucosal surface (e.g., eye, intranasal, lung, oral cavity, intestine, rectum, vagina, or urinary tract surface). Preferably, it is administered locally directly into tumor tissue by injection, endoscopic, or surgical method. By local administration, the immune checkpoint inhibitor is expressed locally in the tumor, thereby reducing the risk of side effects compared to systemic administration of the immune checkpoint inhibitor. In a preferred aspect, the pharmaceutical composition is for intratumoral administration or local injection.

[0059] The pharmaceutical composition of this embodiment can be formulated by known formulation methods for introducing viruses into the bodies of mammals, including humans. For example, it may contain an adjuvant or any carrier, or it may be simply diluted with a physiologically acceptable solution such as sterile saline or sterile buffered saline without the addition of an adjuvant or carrier. It may also be in the form of a frozen formulation, dried formulation, lyophilized formulation, or the like, suitable for long-term storage.

[0060] The pharmaceutical composition of this embodiment contains a therapeutically effective amount of the oncolytic HSV of this embodiment. As used herein, the term "therapeutically effective amount" refers to the amount of agent that alleviates to some extent one or more symptoms of the tumor being treated, more specifically, an amount that results in at least one of the following: a reduction in tumor size, inhibition (delay or cessation) of tumor metastasis, inhibition (delay or cessation) of tumor growth, and alleviation of one or more symptoms associated with the tumor. The specific dosage can be determined appropriately by those skilled in the art based on the severity of the symptoms, the age, sex, weight, and susceptibility of the subject, the method, timing, and interval of administration, the properties of the formulation, and the strength of the promoter.

[0061] The pharmaceutical composition of this embodiment can be administered in an amount of, for example, about 10 1 ~about 10 12 Plaque-forming units (pfu), preferably about 10 7 ~about 10 10 pfu, more preferably about 10 8 pfu ~ approx. 5×10 9 can be administered by injection once or in divided doses. The number of doses of the pharmaceutical composition of this embodiment can be determined appropriately depending on the target patient and the target disease. For example, the first two doses can be administered within two weeks, and then administered at intervals of 3 to 5 weeks.

[0062] The pharmaceutical composition of this embodiment may further contain other active ingredients, or may be used in combination with pharmaceutical compositions containing other active ingredients. The pharmaceutical composition of this embodiment may also be used in combination with other tumor treatments, such as surgery (tumor resection, etc.), chemotherapy, radiation therapy, immunotherapy, hormone therapy, and combinations thereof. Because viral therapy using the pharmaceutical composition of this embodiment relies on a different mechanism from existing tumor treatments, the tumor treatment effect may be enhanced by combining it with other treatments, such as radiation therapy and chemotherapy.

[0063] The pharmaceutical composition of this embodiment can be administered as a combination of multiple viruses, each expressing a different immune checkpoint inhibitory antibody. It can also be administered in combination with at least one of a soluble B7-11-expressing virus and an IL-12-expressing virus. When administered in combination, the viruses may be mixed and administered simultaneously or sequentially. The order of administration is not particularly limited.

[0064] This embodiment also relates to a method for treating tumors using an oncolytic HSV functionalized to express an immune checkpoint inhibitor. This embodiment also relates to the use of the functionalized oncolytic HSV in the manufacture of a medicament for treating tumors. The method and use can be carried out with reference to the description of the pharmaceutical composition above.

[0065] A) Construction of anti-mouse PD-1 antibody-expressing virus (T-J43) Both viruses were constructed using G47Δ as the base backbone. The T-BAC system was used to construct the viruses. 1) Amino acid sequence of anti-mouse PD-1 antibody The amino acid sequence for the expression of anti-mouse PD-1 antibody (J43) was designed from the sequence information of the J43 clone (derived from Armecian hamster) (Patent No. US 7,858,746 B2) (Figure 1-1-1). From this amino acid sequence, a cDNA sequence with codon optimization for the mouse expression system was artificially synthesized.

[0066] 1-2) Anti-mouse PD-1 antibody expressing virus (T-J43HA) The amino acid sequence for expressing the anti-mouse PD-1 antibody (J43) was designed from the sequence information of the J43 clone (derived from Armecian Hamster) (Patent No. US 7,858,746 B2) (Figure 1-1-2). From this amino acid sequence, a codon-optimized cDNA sequence was artificially synthesized for a mouse expression system. In this case, the constant regions of the heavy and light chains were derived from Armenian Ham IgG, and the virus was named J43HA.

[0067] 2) Confirmation of anti-mouse PD-1 antibody expression. HEK293T cells were infected with T-J43 and T-01 at an MOI of 1. 48 hours later, the supernatant was collected and concentrated by ultrafiltration (Amicon Ultra-30K). Then, ELISA for human Fc (human Fc antibody immobilized, detected with HRP-human Fc antibody) and PD-1 binding ELISA (mouse PD-1 immobilized, detected with HRP-human Fc antibody) were performed. Note that T-01 is a control virus in which only the cytomegalovirus (CMV) promoter and an empty polyA cassette were inserted into the deletion site of the ICP6 gene, with no foreign gene other than LacZ inserted. The results are shown in Figure 1-2-1.

[0068] 2-2) Confirmation of anti-mouse PD-1 antibody expression The expression of the anti-mouse PD-1 antibody loaded onto T-J43 was confirmed in mouse cell lines. The mouse neuroblastoma cell line neuro2a, mouse melanoma cell line cloneM3, mouse kidney cancer cell line Renca, and the African green monkey kidney epithelial cell line Vero as a control cell were infected at an MOI of 2, and the culture supernatant was collected 18 hours later and concentrated by ultrafiltration, after which the expression of the loaded anti-PD-1 antibody was confirmed by ELISA. The results are shown in Figure 1-2-2.

[0069] 3) Functionality of the anti-mouse PD-1 antibody To confirm whether the anti-mouse PD-1 antibody loaded on T-J43 had the expected inhibitory activity against PD-1 / PD-L1 binding, measurements were performed using the AlphaLISA Mouse PD-1 and PD-L1 binding Kit (PerkinElmer). First, the J43 / SV-01 plasmid was transiently expressed in HEK293T cells, and the culture supernatant was concentrated to prepare a sample. In addition, commercially available RMP1-14 and J43 antibodies served as positive controls, and a sample similarly treated with the SV-01 plasmid served as a negative control. The results are shown in Figure 1-3-1.

[0070] 3-2) Functional Verification of the Anti-Mouse PD-1 Antibody To confirm whether the anti-mouse PD-1 antibody loaded on T-J43 had the expected inhibitory activity against PD-1 / PD-L1 binding, measurements were performed using the AlphaLISA Mouse PD-1 and PD-L1 Binding Kit (PerkinElmer). First, the J43 / SV-01 plasmid was transiently expressed in HEK293T cells, and the resulting culture supernatant was concentrated. Another sample was prepared by infecting HEK293T cells with T-J43 (MOI = 1), harvesting the culture supernatant 48 hours later, and concentrating it via ultrafiltration (Amicon-Ultra, 30K). A commercially available J43 antibody was also used as a positive control. The results are shown in Figure 1-3-2.

[0071] Because the antibody expression level of T-J43 was not very high, it was not possible to calculate the IC50, but it is thought to be almost the same as that of J43 / SV-01.

[0072] 4) Cytotoxity Assay Neuro2a (3x10 5 The cells (1000 cells / well) were seeded onto a 6-well plate and infected with T-01 or T-J43 at an MOI of 0.1 or 1, respectively, and cultured at 34.5°C for 1 to 4 days. The cell killing effect was calculated as the survival rate relative to the mock cell count on each day. The results are shown in Figures 1-4.

[0073] 5) Replication Assay Vero, Neuro2a (3x10 5 cells / well) and CloneM3 (4x10 5 Cells (cells / well) were seeded onto 6-well plates and infected with T-01 or T-J43 at an MOI of 0.1 for Vero and CloneM3, and at an MOI of 0.3 for Neuro2a, and then cultured at 37°C. After 24 and 48 hours, the cells and medium were harvested, freeze-thawed three times, sonicated for 1 minute, and then titrated. The results are shown in Figures 1-5.

[0074] 6) Infection efficiency (X-gal staining) Vero, Neuro2a (3x10 5 cells / well) and CloneM3 (5x10 5The cells (2.5 × 10 cells / well) were seeded onto a 6-well plate and infected with T-01 or T-J43 at an MOI of 1 or 3, respectively, and cultured at 37°C for 6 hours. X-gal staining was then performed. The results are shown in Figures 1-6.

[0075] 7) Confirmation of DNA structure The constructed anti-mouse PD-1 antibody expressing virus (T-J43) was digested with the restriction enzyme HindIII, and Southern blotting was performed using the LacZ sequence and insert sequence as probes. The results are shown in Figure 1-7-1.

[0076] 7-2) Confirmation of DNA structure The prepared anti-mouse PD-1 antibody expressing virus (T-J43HA), anti-mouse CTLA4 antibody expressing virus (T-4F10HA), and human B7-1Fc expressing virus (T-hB7.1) were digested with the restriction enzyme HindIII, and Southern blotting was performed using the LacZ sequence and insert sequence as probes. The results are shown in Figure 1-7-2.

[0077] 8) Comparison of antitumor effects in a unilateral subcutaneous tumor model in Neuro2a / A / J mice Neuro2a (3x10 6 A / J mice were subcutaneously injected with 4x10 cells / 70μl of T-01, T-J43 virus, or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 4 The tumor diameter was measured after intratumoral administration of 20 μl of pfu (n=9-10 per group). The results are shown in Figure 1-8. There was a significant difference between T-J43 and T-01 (*; P<0.05).

[0078] 9) Comparison of antitumor effects in a bilateral subcutaneous tumor model in Neuro2a / A / J mice Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 T-01, T-J43 virus, or mock cells twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 The tumor diameter was measured. The results are shown in Figure 1-9. Only T-J43 showed a significant difference from the mock group (*; P<0.05) (on the untreated side on day 10).

[0079] 10) Comparison of antitumor effects in a unilateral subcutaneous tumor model of clone M3 / DBA / 2. The antitumor effects in a unilateral subcutaneous tumor model were compared using the following method. 6 T-01, T-J43 6x10 cells / 50μl) was subcutaneously injected into one side of DBA / 2 mice. ↓8 days. The virus was administered twice into the left tumor, on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). (n=5-7 per group) 4 pfu / 20μl or Mock ↓ Tumor diameter measurement

[0080] The results are shown in Figure 1-10. T-J43 tended to be more effective than T-01, although there was no significant difference.

[0081] 11) Comparison of antitumor effects in a bilateral subcutaneous tumor model of clone M3 / DBA / 2. The antitumor effects in a bilateral subcutaneous tumor model were compared using the following method. 6 T-01, T-J43 2x10 cells / 50μl) was subcutaneously injected into both sides of DBA / 2 mice. ↓8 days. The virus was administered twice into the left tumor, on the day when the tumor reached a maximum diameter of approximately 5mm (Day 0) and three days later (Day 3). (n=5-8 per group) 5 pfu / 20μl or Mock ↓ Tumor diameter measurement

[0082] The results are shown in Figure 1-11. On day 13, T-J43 on the treated side showed a significant difference from T-01. On the untreated side, only T-J43 showed a significant difference from the mock.

[0083] 12) Comparison of antitumor effects in Renca / Balb / c mouse unilateral subcutaneous tumor model (anti-PD-1 antibody, anti-CTLA-4 antibody, and their cocktail) Renca (1x10 5 Balb / c mice were subcutaneously injected with 2x10 cells / 50μl. The day the maximum diameter reached approximately 5mm (Day 0) and three days later (Day 3), T-01, T-J43, T-4F10HA virus, and mock were injected twice. 5The mixture was T-J43 and T-4F10HA at 2x10 pfu / 20μl (n=9-10 per group) and the tumor diameter was measured. 5 An equal amount of the mixture prepared at pfu / 20 μl was mixed and administered in an amount of 20 μl. The results are shown in Figures 1-12-1 and 1-12-2.

[0084] 13) Comparison of the antitumor effects of T-01 and J43 antibodies combined with T-J43 alone in a bilateral subcutaneous tumor model in Neuro2a / A / J mice. Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 T-01, T-J43, or mock cells twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 The tumors were intratumorally administered at a dose of pfu / 20 μl (n=10 per group), and simultaneously, 10 μg of J43 antibody or an isotype control (Armenian Hamster IgG, 2A3) was intraperitoneally administered, and the tumor diameter was measured.

[0085] 14) Comparison of serum and intratumoral antibody concentrations following intraperitoneal administration of J43 and intratumoral administration of T-J43HA* in a Neuro2a / A / J unilateral subcutaneous tumor model Neuro2a (3x10 6 T-J43 or mock cells were injected at 2x10 5 pfu (M), 1x10 6 pfu (H), 5x10 6 Mice were intratumorally administered pfu (XH) / 20 μl (n=3 per group), and anti-PD-1 antibody, J43, or an isotype control (Armenian hamster IgG, 2A3) was intraperitoneally administered at 10, 50, or 200 μg. Blood samples were collected and tumors were harvested 1, 4, and 7 days later. Serum and tumor protein samples were prepared, and serum and intratumor antibody concentrations were measured by ELISA. For ELISA, mouse PD-1 was immobilized and detected using an HRP-conjugated anti-Armenian hamster IgG antibody as the secondary antibody. J43 was used as the standard.

[0086] Blood concentration: After intraperitoneal administration of the antibody, a considerable concentration of antibody was detected in the blood. This decreased over time. Although it was also detected after intratumoral administration of the T-J43HA virus, the concentration was significantly lower, about 1 / 10,000 of that observed after intraperitoneal administration of the antibody, and was close to the detection limit (Figure 1-14, top).

[0087] Intratumor concentration (concentration in 0.1 mg of tumor protein (measured in 100 μl of a 1 mg / ml solution)): When antibodies were administered intraperitoneally, antibodies were detected in the tumor depending on the dose, and this decreased over time. When T-J43HA virus was administered intratumorally, antibodies were detected even at low titers, but the concentration was similar to that detected in the mock group, and H(1x10 6 pfu) and XH(5x10 6 On day 1, antibodies of approximately 13ng / ml and 25ng / ml were detected in the groups administered IgG (pfu) (Figure 1-14, bottom).

[0088] In this experiment, T-J43HA, whose Fc portion was designed as Armenian Hamster IgG, was used for comparison with the commercially available antibody J43.

[0089] B) Construction of anti-human and mouse PD-L1 antibody-expressing viruses (T-ATZ) 1) Amino acid sequences of anti-human and mouse PD-L1 antibodies The amino acid sequences for expressing anti-human and mouse PD-L1 antibodies (T-ATZ) were designed based on the sequence information of atezolizumab (KEGG entry # D10773; TECENTRIQ (Genentech)) (Figure 2-1-1). Based on this amino acid sequence, a cDNA sequence was designed and an artificial synthetic gene was ordered.

[0090] 2) Confirmation of antibody expression

[0091] 2-2) Confirmation of anti-mouse PD-L1 antibody expression The expression of the anti-mouse PD-L1 antibody loaded onto T-ATZ was confirmed in mouse cell lines. The mouse neuroblastoma cell line neuro2a, mouse melanoma cell line cloneM3, mouse kidney cancer cell line Renca, and the African green monkey kidney epithelial cell line Vero (as a control cell) were infected at an MOI of 2. The culture supernatant was collected 18 hours later and concentrated by ultrafiltration, and the expression of the loaded anti-PD-L1 antibody was confirmed by ELISA (Figure 2-2-2).

[0092] 2-3) Confirmation of anti-human PD-L1 antibody expression The expression of the anti-human PD-L1 antibody loaded onto T-ATZ was confirmed in human cell lines. The human glioma cell line U87MG, human melanoma cell line Mewo, human renal cancer cell line OSRC2, and the African green monkey kidney epithelial cell line Vero (as a control cell) were infected at an MOI of 2. The culture supernatant was collected 18 hours later and concentrated by ultrafiltration, and the expression of the loaded anti-PD-L1 antibody was confirmed by ELISA (Figure 2-2-3).

[0093] 3) Functionality of anti-mouse PD-L1 antibodies To confirm whether the anti-human / mouse PD-L1 antibodies loaded onto T-ATZ had the expected inhibitory activity against PD-1 / PD-L1 binding, measurements were performed using the AlphaLISA Mouse PD-1 and PD-L1 binding Kit (PerkinElmer). First, the ATZ / SV-01 plasmid was transiently expressed in HEK293T cells, and the culture supernatant was concentrated to prepare a sample. A sample similarly treated with the SV-01 plasmid was also used as a negative control. The results are shown in Figure 2-3-1.

[0094] 3-2) Functionality of anti-human PD-L1 antibodies To confirm whether the anti-human / mouse PD-L1 antibodies loaded onto T-ATZ had the expected inhibitory activity against PD-1 / PD-L1 binding, measurements were performed using the AlphaLISA Human PD-1 and PD-L1 binding Kit (PerkinElmer). First, the ATZ / SV-01 plasmid was transiently expressed in HEK293T cells, and the culture supernatant was concentrated to serve as a sample. A sample similarly treated with the SV-01 plasmid was also used as a negative control. The results are shown in Figure 2-3-2.

[0095] 3-3) Functional verification of anti-mouse PD-L1 antibody To confirm whether the anti-human / mouse PD-L1 antibody loaded on T-ATZ had the expected inhibitory activity against PD-1 / PD-L1 binding, measurements were performed using the AlphaLISA Mouse PD-1 and PD-L1 binding Kit (PerkinElmer). HEK293T cells were infected with T-ATZ at an MOI of 1, and the culture supernatant concentrated 48 hours later was used as a sample. Commercially available atezolizumab (ATZ) was used as a positive control. Culture supernatant from a similar infection with T-01 was used as a negative control. The results are shown in Figure 2-3-3.

[0096] 3-4) Functional verification of anti-human PD-L1 antibodies To confirm whether the anti-human / mouse PD-L1 antibodies incorporated into T-ATZ had the expected inhibitory activity against PD-1 / PD-L1 binding, measurements were performed using the AlphaLISA Human PD-1 and PD-L1 binding Kit (PerkinElmer). HEK293T cells were infected with T-ATZ at an MOI of 1, and the culture supernatant was concentrated 48 hours later to serve as a sample. Commercially available atezolizumab (ATZ) was used as a positive control. Culture supernatant from a similarly infected cell line, T-01, was used as a negative control. The results are shown in Figure 2-3-4.

[0097] 4) Cytotoxity Assay CloneM3 (2x10 5The cells (cells / well) were seeded onto a 6-well plate, infected with T-01 or T-ATZ at an MOI of 0.1 or 1, respectively, and cultured at 34.5°C for 1 to 4 days. The results are shown in Figures 2-4.

[0098] 5) Replication Assay Vero (3x10 5 cells / well) and CloneM3 (4x10 5 Cells (cells / well) were seeded onto 6-well plates and infected with T-01 or T-ATZ at an MOI of 0.02 for Vero and 0.1 for CloneM3, and then cultured at 37°C. After 24 and 48 hours, the cells and medium were harvested, frozen and thawed three times, sonicated for 1 minute, and then titrated. The results are shown in Figures 2-5.

[0099] 6) Infection efficiency (X-gal staining) Vero, Neuro2a (3x10 5 The cells (2.5 × 10 cells / well) were seeded onto a 6-well plate, infected with T-01 or T-ATZ at an MOI of 1 or 3, respectively, and cultured at 37°C for 6 hours. X-gal staining was then performed. The results are shown in Figures 2-6.

[0100] 7) Confirmation of DNA structure The constructed anti-human and mouse PD-L1 antibody-expressing viruses (T-ATZ) were digested with the restriction enzyme HindIII, and Southern blotting was performed using the LacZ sequence and insert sequence as probes. The results are shown in Figure 2-7.

[0101] 8) Comparison of antitumor effects in a unilateral subcutaneous tumor model in Neuro2a / A / J mice. The antitumor effects in a unilateral subcutaneous tumor model (first round) were compared using the following method. Neuro2a (5x10 6 T-01, T-ATZ 4x10 cells / 50μl) was subcutaneously injected into A / J mice for 4 days. The virus was administered intratumorally twice (n=6 per group) on the day when the tumor diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 4 pfu / 20μl Mock ↓ Measurement of tumor diameter

[0102] Although there was no significant difference with T-01, it tended to be more effective (Figure 2-8).

[0103] 9) Comparison of antitumor effects in a bilateral subcutaneous tumor model in Neuro2a / A / J mice. The antitumor effects in a bilateral subcutaneous tumor model were compared using the following method. Neuro2a (5x10 6 T-01, T-ATZ 2x10 cells / 50μl) were subcutaneously injected into both sides of A / J mice. ↓4 days. The virus was administered twice into the left flank tumor, on the day when the tumor reached a maximum diameter of approximately 5mm (Day 0) and 3 days later (Day 3). (n=10 per group) 5 pfu / 20μl Mock ↓ Measurement of tumor diameter

[0104] Only T-ATZ showed a significant difference from mock on both the treated and untreated sides (Figure 2-9).

[0105] 10) Comparison of antitumor effects in a unilateral subcutaneous tumor model in cloneM3 / DBA / 2 mice. The antitumor effects in a unilateral subcutaneous tumor model (first round) were compared using the following method. 6 T-01, T-ATZ 2x10 cells / 50μl) was subcutaneously injected into one side of DBA / 2 mice for 10 days. The virus was administered twice into the left flank tumor, on the day when the tumor reached a maximum diameter of approximately 5mm (Day 0) and three days later (Day 3). (n=10-12 per group) 5 pfu / 20μl Mock ↓ Measurement of tumor diameter

[0106] There was a significant difference from T-01 (Figure 2-10).

[0107] 11) Comparison of antitumor effects in DBA / 2 mouse / clone M3 bilateral subcutaneous tumor model. Antitumor effects in bilateral subcutaneous tumor models were compared using the following method. 6 T-01, T-ATZ 2x10 cells / 50μl) was subcutaneously injected into both sides of DBA / 2 mice. ↓8 days. The virus was administered twice into the left flank tumor, on the day when the tumor reached a maximum diameter of approximately 5mm (Day 0) and three days later (Day 3). (n=8-10 per group) 5 pfu / 20μl Mock ↓ Measurement of tumor diameter

[0108] In the untreated side, only T-ATZ showed a significant difference from mock on day 11 (Figure 2-11).

[0109] 12) Comparison of antitumor effects in Renca / Balb / c mouse unilateral subcutaneous tumor model (anti-PD-L1 antibody) Renca (1x10 5 Balb / c mice were subcutaneously injected with 2 × 10 cells / 50 μl. The cells were then injected twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3), with T-01, T-ATZ virus, or mock. 5 The tumor was administered intratumorally at a dose of pfu / 20 μl (n=9-10 per group), and the tumor diameter was measured (Figure 2-12).

[0110] 13) Comparison of the antitumor effects of T-01 and ATZ antibody combined with T-ATZ alone in a bilateral subcutaneous tumor model in Neuro2a / A / J mice. Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 T-01, T-ATZ, or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20 μl was administered intratumorally (n=10 per group), and simultaneously, 10 μg of ATZ antibody or isotype control (human IgG1) was administered intraperitoneally, and tumor diameter was measured (Fig. 2-13-1, B13-2, B13-3, B13-4).

[0111] 14) Comparison of serum and intratumoral antibody concentrations following intraperitoneal administration of ATZ and intratumoral administration of T-ATZ in a Neuro2a / A / J unilateral subcutaneous tumor model Neuro2a (3x10 6 cells / 70 μl) were subcutaneously injected unilaterally into A / J mice, and on the day when the maximum diameter reached approximately 5 mm (Day 0), 4 × 10 T-ATZ or mock was administered. 4 pfu (L), 2x10 5 pfu (M), 1x10 6Mice were intratumorally administered pfu(H) / 20μl (n=3 per group), or intraperitoneally administered 10, 50, or 200μg of anti-PD-L1 antibody, ATZ, or isotype control (human IgG1). Blood and tumor samples were collected 1, 4, and 7 days later. Serum and tumor protein samples were prepared, and serum and intratumor antibody concentrations were measured by ELISA. For ELISA, mouse PD-L1 was immobilized and an HRP-conjugated anti-human Fc antibody was used as the secondary antibody. ATZ was used as the standard.

[0112] Blood concentration: After intraperitoneal administration of the antibody, a considerable concentration of antibody was detected in the blood. This decreased over time. In the ATZ 200 μg group, even a 1 / 1000 dilution exceeded the measurement range, so it was classified as ND. On the other hand, no antibody was detected after intratumoral administration of the T-ATZ virus (Figure 2-14, top).

[0113] Intraperitoneal administration: When antibodies were administered intraperitoneally, antibodies were detected in the tumor depending on the dose, and this decreased over time. When the T-ATZ virus was administered intratumorally, antibodies were detected in the tumor, although in small amounts (Figure 2-14, bottom).

[0114] C) Construction of anti-mouse CTLA4 antibody expressing viruses (T-9H10 and 4F10) 1) Anti-mouse CTLA4 antibody expressing virus (T-9H10) For the anti-mouse CTLA4 antibody, the antibody coding region was cloned from hybridoma 9H10.11G3, and after sequencing, an amino acid sequence for expression of anti-mouse CTLA4 antibody (4F10) was designed (Figure 3-1-1). Based on this amino acid sequence, a cDNA sequence was designed and an artificially synthesized gene was ordered.

[0115] 1-2) Anti-mouse CTLA4 antibody expressing virus (T-4F10) The coding region for the anti-mouse CTLA4 antibody was cloned from the hybridoma UC10-4F10-11, and after sequencing, an amino acid sequence for expression of the anti-mouse CTLA4 antibody (4F10) was designed (Figure 3-1-2). Based on this amino acid sequence, a cDNA sequence was designed and an artificially synthesized gene was ordered.

[0116] 1-3) Anti-mouse CTLA4 antibody expressing virus (T-4F10HA) The coding region for the anti-mouse CTLA4 antibody was cloned from the hybridoma UC10-4F10-11, and after sequencing, an amino acid sequence for expression of the anti-mouse CTLA4 antibody (4F10) was designed (Figure 3-1-3). Based on this amino acid sequence, a cDNA sequence was designed and an artificially synthesized gene was ordered. In this case, the constant regions of the H and L chains were derived from Armenian Ham IgG, and the virus was named 4F10HA.

[0117] 2) Confirmation of antibody expression HEK293T cells were infected with T-9H10, 4F10, and T-01 at an MOI of 0.1, and the supernatant was collected 48 hours later and concentrated by ultrafiltration (Amicon Ultra-30K). ELISA for human Fc was then performed (human Fc antibody was immobilized and detected with HRP-human Fc antibody) (Figure 3-2-1, A).

[0118] HEK293T cells were infected with T-4F10HA at an MOI of 1, and the culture supernatant was concentrated 48 hours later. Mouse CTLA-4 was immobilized and detected with an HRP-conjugated anti-Armenian hamster IgG antibody as the secondary antibody. The commercially available anti-CTLA-4 antibody, 4F10, was used as a standard (Figure 3-2-1, B).

[0119] 2-2) Confirmation of anti-mouse CTLA4 antibody expression The expression of the anti-mouse CTLA4 antibody loaded onto T-4F10HA was confirmed in mouse cell lines. The mouse neuroblastoma cell line neuro2a, mouse melanoma cell line cloneM3, mouse kidney cancer cell line Renca, and the African green monkey kidney epithelial cell line Vero (as a control cell) were infected at an MOI of 2, and the culture supernatant was collected 18 hours later and concentrated by ultrafiltration, after which the expression of the loaded anti-CTLA-4 antibody was confirmed by ELISA (Figure 3-2-2).

[0120] 3) Functional verification of anti-mouse PD-1 antibody To confirm whether the anti-mouse CTLA4 antibody loaded on T-4F10HA has the expected inhibitory activity against CTLA4 / CD80 binding, we established an AlphaLISA (Mouse CTLA4 / mouse CD80) binding assay system using biotinylated mouse CTLA-4 and His-tagged mouse CD80. HEK293T cells were infected with T-4F10HA (MOI = 1), and the culture supernatant was collected 48 hours later and concentrated by ultrafiltration (Amicon-Ultra, 30K). The commercially available 4F10 antibody was used as a positive control (Figure 3-3).

[0121] 4) Cytotoxicity Assay: Neuro2a (2x10 5 cells / well) and CloneM3 (1.6x10 5 cells / well) were seeded onto 6-well plates and infected with T-01, T-4F10, or T-9H10 at an MOI of 0.1 or 1, respectively, and cultured at 34.5°C for 1 to 4 days (Figures 3-4).

[0122] 5) Replication Assay Nuero2a and Vero (3x10 5 cells / well) and CloneM3 (4x10 5 Cells (cells / well) were seeded onto 6-well plates and infected with T-01, T-4F10, or T-9H10 at an MOI of 0.3 for Neuro2a, 0.02 for Vero, and 0.1 for CloneM3, and then cultured at 37°C. After 24 and 48 hours, the cells and medium were harvested, frozen and thawed three times, sonicated for 1 minute, and titrated (Figures 3-5).

[0123] 6) Infection efficiency (X-gal staining) Vero and Neuro2a (3x10 5 cells / well), CloneM3 (5x10 5 cells / well) were seeded onto a 6-well plate and infected with T-01, T-4F10, or T-9H10 at an MOI of 1 or 3, respectively, and cultured at 37°C for 6 hours. X-gal staining was then performed (Figure 3-6-1).

[0124] 6-2) Infection efficiency (X-gal staining) Vero and Neuro2a (3x10 5 The cells (cells / well) were seeded onto a 6-well plate, infected with T-01 or T-4F10HA at an MOI of 1 or 3, respectively, and cultured at 37°C for 6 hours. X-gal staining was then performed (Figure 3-6-2).

[0125] 7) Confirmation of DNA structure The constructed anti-mouse CTLA-4 antibody expressing viruses (T-9H10, T-4F10) were digested with the restriction enzyme HindIII, and Southern blotting was performed using the insert sequence as a probe. The DNA structure of T-4F10HA is shown in Figure 1-7-2.

[0126] 8) Comparison of antitumor effects in a unilateral subcutaneous tumor model in Neuro2a / A / J mice. The antitumor effects in a unilateral subcutaneous tumor model were compared using the following method. Neuro2a (5x10 6 T-01, T-9H10 4x10 cells / 50μl) were subcutaneously injected into A / J mice for 5 days. The virus was administered intratumorally twice (on Day 0, when the tumor reached a maximum diameter of approximately 5mm, and three days later (Day 3) (n=7-9 per group). 4 pfu / 20μl Mock ↓ Measurement of tumor diameter

[0127] Only T-9H10 showed a significant difference from the mock (Figure 3-8).

[0128] 9) Comparison of antitumor effects in a bilateral subcutaneous tumor model in Neuro2a / A / J mice. The antitumor effects in a bilateral subcutaneous tumor model were compared using the following method. Neuro2a (5x10 6 T-01, T-9H10 2x10 cells / 50μl) was subcutaneously injected into both sides of the tumor in A / J mice. ↓4 days. The virus was administered twice into the left flank tumor, on the day when the tumor reached a maximum diameter of approximately 5mm (Day 0) and three days later (Day 3). (n=8 per group) 5 pfu / 20μl Mock ↓ Measurement of tumor diameter

[0129] Only T-9H10 showed a significant difference from the mock (Figure 3-9).

[0130] 10) Comparison of antitumor effects in a unilateral subcutaneous tumor model in cloneM3 / DBA / 2 mice. The antitumor effects in a unilateral subcutaneous tumor model were compared using the following method. 6 DBA / 2 mice were injected subcutaneously with 2x10 cells / 50μl of T-01, T-9H10 for 8 days. The virus was then injected into the left flank tumor twice: on Day 0 when the tumor reached a maximum diameter of approximately 5mm and three days later (Day 3). (n=7-8 per group) 5 pfu / 20μl Mock ↓ Measurement of tumor diameter

[0131] Only T-9H10 showed a significant difference from the mock (Figure 3-10).

[0132] 11) Comparison of antitumor effects in a bilateral subcutaneous tumor model in cloneM3 / DBA / 2 mice. The antitumor effects in a bilateral subcutaneous tumor model were compared using the following method. 6 DBA / 2 mice were injected subcutaneously with 2x10 cells / 50μl of T-01, T-9H10 for 8 days. The virus was then injected into the left flank tumor twice: on Day 0 when the tumor reached a maximum diameter of approximately 5mm and three days later (Day 3). (n=7-9 per group) 5 pfu / 20μl Mock ↓ Measurement of tumor diameter

[0133] There was a significant difference from T-01 (Figure 3-11).

[0134] 12) Comparison of antitumor effects in a Renca / Balb / c mouse unilateral subcutaneous tumor model (anti-CTLA-4 antibody) as shown in Figure 1-12.

[0135] 13) Comparison of the antitumor effects of T-01 and 4F10 antibody combination therapy and T-4F10HA alone in a Neuro2a / A / J mouse bilateral subcutaneous tumor model. Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 T-01, T-4F10HA, or mock cells twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5pfu / 20 μl was administered intratumorally (n=10 per group), and simultaneously, 1 μg of 4F10 antibody or isotype control (Armenian Hamster IgG, 2A3) was administered intraperitoneally, and tumor diameter was measured (Fig. 3-12-1, C12-2, C12-3, C12-4).

[0136] 14) Comparison of serum and intratumoral antibody concentrations following intraperitoneal administration of 4F10 and intratumoral administration of T-4F10HA in a Neuro2a / A / J unilateral subcutaneous tumor model Neuro2a (3x10 6 cells / 70 μl) were subcutaneously injected unilaterally into A / J mice, and on the day when the maximum diameter reached approximately 5 mm (Day 0), 4 × 10 T-4F10HA or mock cells were injected. 4 pfu (L), 2x10 5 pfu (M), 1x10 6 pfu (H), 5x10 6 Mice were given intratumoral injections of pfu (XH) / 20 μl (n=3 per group), or intraperitoneal injections of 1, 5, or 25 μg of anti-CTLA4 antibody, 4F10, or an isotype control (Armenian hamster IgG, 2A3). Blood samples were collected and tumors were harvested 1, 4, and 7 days later. Serum and tumor protein samples were prepared, and serum and intratumoral antibody concentrations were measured by ELISA. For ELISA, mouse CTLA4 was immobilized and an HRP-conjugated anti-Armenian hamster IgG antibody was used as the secondary antibody. 4F10 was used as the standard.

[0137] Blood concentration: After intraperitoneal administration of the antibody, a considerable concentration of antibody was detected in the blood. This decreased over time. Since no antibody was detected after intratumoral administration of the T-4F10 virus, an increased amount of virus was used, and a fairly low concentration was detected. However, this value was significantly lower than that of the 1 μg intraperitoneal administration (Figure 3-13, top).

[0138] Intratumoral concentration (concentration in 0.1 mg of tumor protein (measured in 100 μl of a 1 mg / ml solution)): When antibodies were administered intraperitoneally, antibodies were detected in the tumor depending on the dose, and this decreased over time. When T-4F10HA virus was administered intratumorally, the concentration was below the detection limit, and was detectable in the XH (5x10^6 pfu) administration group, where the maximum amount of tumor protein was introduced (calculated as concentration per tumor) (Figure 3-13, bottom).

[0139] I) Combination effect of ICI-expressing anti-cancer HSV-1 I-1) Combination effect of anti-CTLA-4 antibody (T-9H10) and anti-PD-1 antibody (T-J43) expressing HSV-1 in a bilateral subcutaneous tumor model in Neuro2a / A / J mice. Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 T-9H10, T-J43, or mock cells twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 The mixture was administered intratumorally at a concentration of 2x10 pfu / 20μl (n=13 per group), and the tumor diameter was measured. 5 Equal amounts of the mixture prepared at pfu / 20 μl were mixed and administered in an amount of 20 μl (Figs. 4-1-1 and 4-1-2).

[0140] I-2) Effect of the combination of anti-CTLA-4 antibody (T-9H10) and anti-PD-L1 antibody (T-ATZ) expressing HSV-1 in a Neuro2a / A / J mouse bilateral subcutaneous tumor model. Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 T-9H10, T-ATZ, or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20 μl (n=10 per group) and the tumor diameter was measured. 5 Equal amounts of the mixture prepared at pfu / 20 μl were mixed and administered in an amount of 20 μl (Figs. 4-1 and 4-1-2).

[0141] I-3) Effect of the combination of anti-CTLA-4 antibody (T-9H10) and anti-PD-1 antibody (T-J43) expressing HSV-1 in a bilateral subcutaneous tumor model in clone M3 / DBA / 2 mice. 6 DBA / 2 mice were subcutaneously injected with 2 × 10 T-9H10, T-J43, or mock cells twice: on Day 0 when the maximum diameter reached approximately 5 mm and 3 days later (Day 3). 5 pfu / 20 μl (n=8 per group) and the tumor diameter was measured. 5 Equal amounts of the mixture prepared at pfu / 20 μl were mixed and administered in an amount of 20 μl (Figure 4-3).

[0142] I-4) Effect of the combination of anti-CTLA-4 antibody (T-9H10) and anti-PD-L1 antibody (T-ATZ) expressing HSV-1 in a bilateral subcutaneous tumor model in clone M3 / DBA / 2 mice. clone M3 (1x10 6 DBA / 2 mice were subcutaneously injected with 2 × 10 T-9H10, T-ATZ, or mock twice: on Day 0 when the maximum diameter reached approximately 5 mm and 3 days later (Day 3). 5 pfu / 20 μl (n=8 per group) and tumor diameter was measured. 5 An equal volume mixture of the mixtures prepared at pfu / 20 μl was administered in an amount of 20 μl (Figure 4-4).

[0143] II) Comparison of the effects of combined and sequential administration of ICI-expressing anti-cancer HSV-1 II-1) Therapeutic effects of a cocktail (mix) and sequential administration of anti-CTLA-4 antibody-expressing (T-4F10HA) and anti-PD-1 antibody-expressing (T-J43) HSV-1 in a bilateral subcutaneous tumor model in Neuro2a / A / J mice Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2x10 T-4F10HA, T-J43, or mock cells twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5pfu / 20μl (n=10 per group) and tumor diameter was measured. 5 The mixture was administered in equal amounts at pfu / 20 μl. Sequential administration consisted of administering T-4F10HA on day 0 and T-J43 on day 3, or vice versa (Figures 5-1-1 and 5-1-2).

[0144] II-2) Therapeutic effects of a cocktail and sequential administration of anti-CTLA-4 antibody-expressing (T-4F10HA) and anti-PD-L1 antibody-expressing (T-ATZ) HSV-1 in a bilateral subcutaneous tumor model in Neuro2a / A / J mice. 6 A / J mice were injected subcutaneously on both sides with 2x10 T-4F10HA, T-ATZ, or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20 μl (n=10 per group) and tumor diameter was measured. 5 The mixture was administered in equal amounts at pfu / 20 μl. Sequential administration consisted of administering T-4F10HA on day 0 and T-ATZ on day 3, or vice versa (Figures 5-2 and 5-2-2).

[0145] II-3) Therapeutic effects of a cocktail and sequential administration of anti-CTLA-4 antibody-expressing (T-4F10HA) and anti-PD-1 antibody-expressing (T-J43) HSV-1 in a bilateral subcutaneous tumor model in clone M3 / DBA / 2 mice. 6 A / J mice were injected subcutaneously on both sides with 2x10 T-4F10HA, T-J43, or mock cells twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20 μl (n=7 per group) and tumor diameter was measured. 5The mixture was administered in equal amounts at pfu / 20 μl. Sequential administration consisted of administering T-4F10HA on day 0 and T-J43 on day 3, or vice versa (Figures 5-3-1 and 5-3-2).

[0146] II-4) Therapeutic effects of a cocktail and sequential administration of anti-CTLA-4 antibody-expressing (T-4F10HA) and anti-PD-L1 antibody-expressing (T-ATZ) HSV-1 in a bilateral subcutaneous tumor model in clone M3 / DBA / 2 mice. 6 A / J mice were injected subcutaneously on both sides with 2x10 T-4F10HA, T-ATZ, or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20 μl (n=6-9 per group) and tumor diameter was measured. 5 The mixture was administered in equal amounts at pfu / 20 μl. Sequential administration consisted of administering T-4F10HA on day 0 and T-ATZ on day 3, or vice versa (Figures 5-4-1 and 5-4-2).

[0147] D) Construction of anti-human PD-1 antibody-expressing virus (T-Niv) 1) Amino acid sequence of anti-human PD-1 antibody The amino acid sequence for expression of anti-human PD-1 antibody (T-Niv) was designed from the sequence information of the anti-human PD-1 antibody, Nivolumab (KEGG entry# D10316; OPDIVO (Ono)) (Figure 6-1-1). Based on this amino acid sequence, a cDNA sequence was designed and an artificially synthesized gene was ordered.

[0148] 2) Confirmation of anti-human PD-1 antibody expression HEK293T cells were infected with T-Niv and T-01 at an MOI of 0.1. After 48 hours, the supernatant was collected and concentrated by ultrafiltration (Amicon Ultra-30K). ELISA for human Fc (human Fc antibody was immobilized and detected with HRP-human Fc antibody) (Figure 6-2-1, A) and PD-1 binding ELISA (human PD-1 was immobilized and detected with HRP-human Fc antibody) (Figure 6-2-1, B) were performed.

[0149] 2-2) Confirmation of anti-human PD-1 antibody expression The expression of the anti-human PD-1 antibody loaded onto T-Niv was confirmed in human cell lines. The human glioma cell line U87MG, human melanoma cell line Mewo, human renal cancer cell line OSRC2, and the African green monkey kidney epithelial cell line Vero (as a control cell) were infected at an MOI of 2. The culture supernatant was collected 18 hours later and concentrated by ultrafiltration, and the expression of the loaded anti-PD-L1 antibody was confirmed by ELISA (Figure 6-2-2).

[0150] 3) Functionality of the anti-human PD-1 antibody To confirm whether the anti-human PD-1 antibody loaded onto T-Niv had the expected inhibitory activity against PD-1 / PD-L1 binding, measurements were performed using the AlphaLISA Human PD-1 and PD-L1 binding Kit (PerkinElmer). First, the Niv / SV-01 plasmid was transiently expressed in HEK293T cells, and the culture supernatant was concentrated to serve as a sample. A sample similarly treated with the SV-01 plasmid was also used as a negative control.

[0151] The results of measuring the PD-1 / PD-L1 binding inhibitory activity using culture supernatants transfected with Niv / SV-01 and SV-01 (empty vector) are shown in Figure 6-3-1, A. The IC50 value, calculated as the concentration of human Fc of Niv / SV-01 used in this experiment, was 23.64 ng / ml (Figure 6-3-1, B).

[0152] 3-2) Functionality of the anti-human PD-1 antibody To confirm whether the anti-human PD-1 antibody loaded on T-Niv had the expected inhibitory activity against PD-1 / PD-L1 binding, measurements were performed using the AlphaLISA Human PD-1 and PD-L1 binding Kit (PerkinElmer). HEK293T cells were infected with T-Niv (MOI = 1), and the culture supernatant was collected 48 hours later and concentrated by ultrafiltration (Amicon-Ultra, 30K). Commercially available nivolumab was used as a positive control (Figure 6-3-2).

[0153] 4) Cytotoxity Assay Vero (2x10 5 cells / well) were seeded onto 6-well plates and infected with T-01 or T-Niv at an MOI of 0.01 or 0.1, respectively, and cultured at 34.5°C for 1 to 4 days (Figure 6-4).

[0154] 5) Replication Assay Vero (3x10 5 cells / well) were seeded onto a 6-well plate, infected with T-01 or T-Niv at an MOI of 0.01, and cultured at 37°C. After 24 and 48 hours, the cells and medium were harvested, frozen and thawed three times, sonicated for 1 minute, and then titrated (Figure 6-5).

[0155] 6) Infection efficiency (X-gal staining) Vero cells were seeded on a 6-well plate and infected with T-01 or T-Niv at an MOI of 1 or 3, respectively, and cultured at 37°C for 6 hours. X-gal staining was then performed (Figure 6-6).

[0156] 7) Confirmation of DNA structure The constructed anti-human PD-1 antibody-expressing virus (T-Niv) was digested with the restriction enzyme HindIII, and Southern blotting was performed using the LacZ sequence and insert sequence as probes (Figures 6-7).

[0157] E) Construction of anti-human CTLA4 antibody-expressing virus 1) Amino acid sequence of anti-human CTLA4 antibody (T-Ipi) The amino acid sequence for expressing anti-human CTLA4 antibody (Ipi) was designed from the sequence information of the anti-human CTLA4 antibody ipilimumab (KEGG entry #D04603; Bristol-Myers Squibb, YERVOY) (Figure 7-1-1). Based on this amino acid sequence, a cDNA sequence was designed and an artificially synthesized gene was ordered.

[0158] 1-2) Amino acid sequence of anti-human CTLA4 antibody (T-Tic) The amino acid sequence for expression of anti-human CTLA4 antibody (Tic) was designed from the sequence information of ticilimubumab (Tremelimumab) (JP Patent Publication No. 2012-167120) (Figure 7-1-2). Based on this amino acid sequence, a cDNA sequence was designed and an artificially synthesized gene was ordered.

[0159] 2) Confirmation of anti-human CTLA-4 antibody expression Ipi / SV-01 and Tic / SV-01 plasmids were transiently expressed in HEK293T cells, and the culture supernatant was concentrated to prepare a sample (Figure 7-2-1, A). A sample similarly treated with the SV-01 plasmid was used as a negative control. ELISA against human Fc (human Fc antibody was immobilized and detected with HRP-human Fc antibody) was performed.

[0160] HEK293T cells were infected with T-Ipi at an MOI of 1, and the supernatant was collected 48 hours later. After concentration by ultrafiltration (Amicon Ultra-30K), a CTLA-4 binding ELISA was performed (human CTLA-4 was immobilized and detected with HRP-Human Fc antibody, with ipilimumab as the standard) (Figure 7-2-1, B).

[0161] HEK293T cells were infected with T-Tic at an MOI of 1, and the supernatant was collected 48 hours later. After concentration by ultrafiltration (Amicon Ultra-30K), a CTLA-4 binding ELISA was performed (human CTLA-4 was immobilized and detected with HRP-Human Fc antibody, with ticilimumab as the standard) (Figure 7-2-1, C).

[0162] 2-2) Confirmation of anti-human CTLA-4 antibody expression The expression of the anti-human CTLA-4 antibody loaded onto T-Ipi and T-Tic was confirmed in human cell lines. The human glioma cell line U87MG, human melanoma cell line Mewo, human renal cancer cell line OSRC2, and the African green monkey kidney epithelial cell line Vero (as a control cell) were infected at an MOI of 2. The culture supernatant was collected 18 hours later and concentrated by ultrafiltration, and the expression of the loaded anti-CTLA-4 antibody was confirmed by ELISA (Figure 7-2-2).

[0163] 3) Functionality of the anti-human CTLA4 antibody To confirm whether the anti-human CTLA4 antibody loaded on T-Ipi had the expected inhibitory activity against CTLA-4 / CD80 (B7-1) binding, measurements were performed using the AlphaLISA Human CTLA-4 / CD80 binding kit (PerkinElmer). First, the Ipi / SV-01 plasmid was transiently expressed in HEK293T cells, and the culture supernatant was concentrated to prepare a sample. A sample similarly treated with the SV-01 plasmid was also used as a negative control (Figure 7-3-1).

[0164] 3-2) Functionality of the anti-human CTLA4 antibody To confirm whether the anti-human CTLA4 antibody loaded onto T-Tic had the expected inhibitory activity against CTLA-4 / CD80 (B7-1) binding, measurements were performed using the AlphaLISA Human CTLA-4 / CD80 binding kit (PerkinElmer). First, the Tic / SV-01 plasmid was transiently expressed in HEK293T cells, and the culture supernatant was concentrated to prepare a sample. A sample similarly treated with the SV-01 plasmid was also used as a negative control (Figure 7-3-2).

[0165] 3-3) Functionality of anti-human CTLA-4 antibodies To confirm whether the anti-human CTLA-4 antibodies loaded on T-Ipi and T-Tic had the expected CTLA-4 / CD80 binding inhibitory activity, measurements were performed using the AlphaLISA Human CTLA-4 / CD80 binding kit (PerkinElmer). HEK293T cells were infected with T-Ipi and T-Tic (MOI = 1). 48 hours later, the culture supernatant was collected and concentrated by ultrafiltration (amicon-ultra, 30K). Commercially available ipilimumab and ticilimumab were used as positive controls.

[0166] 4) Cytotoxity Assay Vero (2x10 5 cells / well) were seeded onto 6-well plates and infected with T-01, T-Ipi, or T-Tic at an MOI of 0.01 or 0.1, respectively, and cultured at 34.5°C for 1 to 4 days (Figure 7-4).

[0167] 5) Replication Assay Vero (3x10 5 cells / well) were seeded onto a 6-well plate and infected with T-01, T-Ipi, or T-Tic at an MOI of 0.01 and cultured at 37°C. After 24 and 48 hours, the cells and medium were harvested, frozen and thawed three times, sonicated for 1 minute, and then titrated (Figure 7-5).

[0168] 6) Infection efficiency (X-gal staining) Vero cells were seeded on a 6-well plate and infected with T-01, T-Ipi, or T-Tic at an MOI of 1 or 3, respectively, and cultured at 37°C for 6 hours. X-gal staining was then performed (Figure 7-6).

[0169] 7) Confirmation of DNA structure The anti-human CTLA4 antibody-expressing viruses (T-Ipi, T-Tic) prepared were digested with the restriction enzyme HindIII, and Southern blotting was performed using the LacZ sequence and insert sequence as probes (Figure 7-7).

[0170] III) Effect of combined and sequential administration of immune checkpoint inhibitor antibody-expressing virus and immune stimulatory factor-expressing virus. A) Neuro2a / A / J mouse bilateral subcutaneous tumor model. 1) Anti-CTLA4 antibody (T-4F10HA) x soluble B7-1-expressing virus Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 cells / 70 μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20μl (n=11-12 per group) and tumor diameter was measured. The combination (Mix) consisted of 2x10 T-4F10HA and T-mB7.1 (an oncolytic HSV-1 expressing soluble mouse B7-1, Fukuhara, H. et al., Cancer Res. 65, 10663-10668 (2005)). 5 The mixture was administered in equal amounts at pfu / 20 μl. Sequential administration consisted of administering T-4F10HA on day 0 and T-mB7.1 on day 3, or vice versa (Figure 11A-1).

[0171] 2) Anti-CTLA4 antibody (T-4F10HA) x IL12-expressing virus Neuro2a (2.5x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 cells / 70 μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20 μl (n=11-12 per group) and tumor diameter was measured. For the combination (Mix), T-4F10HA and T-mfIL12 (oncolytic HSV-1 expressing mouse IL-12, Non-Patent Document 2: Fukuhara, H. et al., Commun Med (Lond) 3, 40 (2023)) were administered at 2x10 5 Equal amounts of the mixtures prepared at pfu / 20 μl were mixed and administered in a volume of 20 μl. For sequential administration, T-4F10HA was administered on day 0 and T-mfIL12 on day 3, or vice versa (Figure 11-A-2).

[0172] 3) Anti-PD-1 antibody (T-J43) x soluble B7-1 expressing virus Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 cells / 70 μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20μl (n=9-10 per group) and tumor diameter was measured. 5 The mixture was administered in equal amounts at pfu / 20 μl. Sequential administration consisted of administering T-J43 on day 0 and T-mB7.1 on day 3, or vice versa (Figure 11-A-3).

[0173] 4) Anti-PD-1 antibody (T-J43) x IL12-expressing virus Neuro2a (2.5x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 cells / 70 μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5pfu / 20μl (n=11-12 per group) and tumor diameter was measured. 5 Equal amounts of the mixtures prepared at pfu / 20 μl were mixed and administered in a volume of 20 μl. For sequential administration, T-J43 was administered on day 0 and T-mfIL12 on day 3, or vice versa (Figure 11-A-4).

[0174] 5) Anti-PD-L1 antibody (T-ATZ) x soluble B7-1 expressing virus Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 cells / 70 μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20μl (n=9-10 per group) and tumor diameter was measured. 5 The mice were administered 20 μl of a mixture of equal amounts of T-ATZ and T-mB7.1 on day 3, or vice versa (Figure 11A-5).

[0175] 6) Anti-PD-L1 antibody (T-ATZ) x IL12-expressing virus Neuro2a (3x10 6 A / J mice were injected subcutaneously on both sides with 2 × 10 cells / 70 μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20 μl (n=9-11 per group) and tumor diameter was measured. 5 Equal amounts of the mixtures prepared at pfu / 20 μl were mixed and administered in a volume of 20 μl. Sequential administration consisted of administering T-ATZ on day 0 and T-mfIL12 on day 3, or vice versa (Figure 11-A-6).

[0176] B) cloneM3 / DBA / 2 mouse bilateral subcutaneous tumor model 1) Anti-CTLA4 antibody (T-4F10HA) x soluble B7-1 expressing virus cloneM3 (1x10 6A / J mice were subcutaneously injected with 2 × 10 cells / 50 μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5 mm (Day 0) and 3 days later (Day 3). 5 pfu / 20 μl (n=9 per group) and tumor diameter was measured. 5 Equal amounts of the mixtures prepared at pfu / 20 μl were mixed and administered in a volume of 20 μl. For sequential administration, T-4F10HA was administered on day 0 and T-mB7.1 on day 3, or vice versa (Figure 11-B-1).

[0177] 2) Anti-CTLA4 antibody (T-4F10HA) x IL-12 expressing virus clone M3 (1 x 10 6 DBA / 2 mice were subcutaneously injected with 2x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 5 pfu / 20 μl (n=10 per group) and tumor diameter was measured. 5 Equal amounts of the mixtures prepared at pfu / 20 μl were mixed and administered in a volume of 20 μl. Sequential administration consisted of administering T-4F10HA on day 0 and T-mfIL12 on day 3, or vice versa (Figure 11-B-2).

[0178] 3) Anti-PD-1 antibody (T-J43) x soluble B7-1 expressing virus clone M3 (1x10 6 DBA / 2 mice were subcutaneously injected with 2x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 5 pfu / 20μl (n=9 per group) was administered intratumorally, and tumor diameter was measured. 5 The mixture was administered in equal amounts at pfu / 20 μl. Sequential administration consisted of administering T-J43 on day 0 and T-mB7.1 on day 3, or vice versa (Figure 11-B-3).

[0179] 4) Anti-PD-1 antibody (T-J43) x IL-12 expressing virus clone M3 (1x10 6 DBA / 2 mice were subcutaneously injected with 2x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 5 pfu / 20 μl (n=10 per group) and tumor diameter was measured. 5 Equal amounts of the mixtures prepared at pfu / 20 μl were mixed and administered in a volume of 20 μl. Sequential administration consisted of administering T-J43 on day 0 and T-mfIL12 on day 3, or vice versa (Figure 11-B-4).

[0180] 5) Anti-PD-L1 antibody (T-ATZ) x soluble B7-1 expressing virus cloneM3 (1x10 6 DBA / 2 mice were subcutaneously injected with 2x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 5 pfu / 20μl (n=9-10 per group) and tumor diameter was measured. 5 The mice were administered 20 μl of a mixture of equal amounts of T-ATZ and T-mB7.1 on day 3, or vice versa (Figure 11-B-5).

[0181] 6) Anti-PD-L1 antibody (T-ATZ) x IL12 expressing virus clone M3 (1x10 6 DBA / 2 mice were subcutaneously injected with 2x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 5 pfu / 20μl (n=8-10 per group) and tumor diameter was measured. 5The mixture was administered in equal amounts at pfu / 20 μl. Sequential administration consisted of T-ATZ on day 0 and T-mfIL12 on day 3, or vice versa (Figure 11-B-6).

[0182] C) Renca / Balb / c mouse bilateral subcutaneous tumor model 1) Anti-CTLA4 antibody (T-4F10HA) x soluble B7-1 expressing virus Renca (1x10 5 Balb / c mice were subcutaneously injected with 1x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 6 pfu / 20μl (n=9-10 per group) and tumor diameter was measured. 6 The mixture was administered in equal amounts at pfu / 20 μl. Sequential administration consisted of administering T-4F10HA on day 0 and T-mB7.1 on day 3, or vice versa (Figure 11C-1).

[0183] 2) Anti-CTLA4 antibody (T-4F10HA) x IL-12 expressing virus Renca (1x10 5 Balb / c mice were subcutaneously injected with 1x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 6 pfu / 20 μl (n=10 per group) and tumor diameter was measured. 6 Equal amounts of the mixtures prepared at pfu / 20 μl were mixed and administered in a volume of 20 μl. Sequential administration consisted of administering T-4F10HA on day 0 and T-mfIL12 on day 3, or vice versa (Figure 11-C-2).

[0184] 3) Anti-PD-1 antibody (T-J43) x Renca virus expressing soluble B7-1 (1x10 5 Balb / c mice were subcutaneously injected with 1x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 6pfu / 20μl (n=10 per group) and tumor diameter was measured. 6 Equal amounts of the mixtures prepared at pfu / 20 μl were mixed and administered in a volume of 20 μl. For sequential administration, T-J43 was administered on day 0 and T-mB7.1 on day 3, or vice versa (Figure 11-C-3).

[0185] 4) Anti-PD-1 antibody (T-J43) x IL-12 expressing virus Renca (1x10 5 Balb / c mice were subcutaneously injected with 1x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 6 pfu / 20 μl (n=10 per group) and tumor diameter was measured. 6 Equal amounts of the mixtures prepared at pfu / 20 μl were mixed and administered in a volume of 20 μl. For sequential administration, T-J43 was administered on day 0 and T-mfIL12 on day 3, or vice versa (Figure 11-C-4).

[0186] 5) Anti-PD-L1 antibody (T-ATZ) x soluble B7-1 expressing virus Renca (1x10 5 Balb / c mice were subcutaneously injected with 1x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 6 pfu / 20μl (n=9-10 per group) and tumor diameter was measured. 6 The mice were administered 20 μl of a mixture of equal amounts of T-ATZ and T-mB7.1 on day 0 and day 3, respectively (Figure 11C-5).

[0187] 6) Anti-PD-L1 antibody (T-ATZ) x IL12-expressing virus Renca (1x10 5Balb / c mice were subcutaneously injected with 1x10 cells / 50μl of therapeutic virus or mock twice: on the day when the maximum diameter reached approximately 5mm (Day 0) and 3 days later (Day 3). 6 pfu / 20μl (n=9-10 per group) and tumor diameter was measured. 6 The mixture was administered in equal amounts at pfu / 20 μl. Sequential administration consisted of T-ATZ on day 0 and T-mfIL12 on day 3, or vice versa (Figure 11C-6).

[0188] IV) Effect of administration of a triple cocktail of viruses expressing immune checkpoint inhibitor antibodies and immune stimulators. A) Neuro2a (2x10) subcutaneous tumor model in Neuro2a / A / J mice 6 A / J mice were injected subcutaneously on both sides with 8x10 cells / 70μl of the therapeutic virus, its control virus (non-loaded T-01), or mock-injected three times: on the day when the maximum diameter reached approximately 5mm (Day 0), and 3 and 7 days later (Day 7). 4 The three viruses were administered intratumorally at a dose of 8x10 pfu / 20μl (n=9 per group), and tumor diameter was measured. 4 Equal amounts of the mixture prepared at pfu / 20 μl were mixed and administered in an amount of 20 μl (FIG. 12-A).

[0189] B) cloneM3 / DBA / 2 mouse bilateral subcutaneous tumor model cloneM3 (1x10 6 DBA / 2 mice were subcutaneously injected with 2x10 cells / 50μl of the therapeutic virus, its control virus (non-loaded T-01), or mock-injected three times: on the day when the maximum diameter reached approximately 5mm (Day 0), and 4 and 7 days later (Day 7). 5 pfu / 20 μl (n=8-10 per group) and tumor diameter was measured. 5 Equal amounts of the mixture prepared at pfu / 20 μl were mixed and administered in an amount of 20 μl (FIG. 12-B).

[0190] C) Renca / Balb / c mouse bilateral subcutaneous tumor model, Renca (1x10 5 Balb / c mice were subcutaneously injected with 1x10 cells / 50μl of the therapeutic virus, its control virus (non-loaded T-01), or mock-injected three times: on the day when the maximum diameter reached approximately 5mm (Day 0), and 4 and 7 days later (Day 7). 6 The three viruses were administered intratumorally at a dose of 1x10 pfu / 20 μl (n=8-9 per group), and tumor diameter was measured. 6 Equal amounts of the mixture prepared at pfu / 20 μl were mixed and administered in an amount of 20 μl (FIG. 12-C).

[0191] [Sequences listed in the sequence listing] SEQ ID NO:1 IgK leader sequence SEQ ID NO:2 Signal peptidase recognition sequence SEQ ID NO:3 VH, anti-mouse PD-1 antibody SEQ ID NO:4 CH, human IgG4 SEQ ID NO:5 Furin recognition sequence SEQ ID NO:6 FMDV-2A SEQ ID NO:7 VL, anti-mouse PD-1 antibody SEQ ID NO:8 CL, human λ SEQ ID NO:9 CH, Armenian Ham IgG SEQ ID NO:10 CL, Armenian Ham Ig κ SEQ ID NO:11 VH, derived from Atezolizumab SEQ ID NO:12 CH, human IgG1 N298A SEQ ID NO:13 VL, derived from Atezolizumab SEQ ID NO:14 CL, human κ SEQ ID NO:15 VH, anti-mouse CTLA4 antibody, derived from hybridoma 9H10.11G3 SEQ ID NO:16 CH, human IgG1 SEQ ID NO:17 VL, anti-mouse CTLA4 antibody, hybridoma 9H10.SEQ ID NO:18 VH, anti-mouse CTLA4 antibody, from hybridoma UC10-4F10-11 SEQ ID NO:19 VL, anti-mouse CTLA4 antibody, from hybridoma UC10-4F10-11 SEQ ID NO:20 VH, anti-mouse CTLA4 antibody, from hybridoma UC10-4F10-11 SEQ ID NO:21 VL, anti-mouse CTLA4 antibody, from hybridoma UC10-4F10-11 SEQ ID NO:22 VH, from Nivolmab SEQ ID NO:23 CH, human IgG4 S224P SEQ ID NO:24 VL, from Nivolmab SEQ ID NO:25 VH, from Ipilimumab SEQ ID NO:26 VL, from Ipilimumab SEQ ID NO:27 VH, from Ticilimumab SEQ ID NO:28 CH, human IgG4 S224P SEQ ID NO:29 VL, derived from ticilimubmab SEQ ID NO:30 Insertion site of a gene encoding an immune checkpoint inhibitor and the nucleotide sequence surrounding it SEQ ID NO:31 Sequence containing γ34.5 gene deletion site (TR. L region) SEQ ID NO:32 Sequence containing α47 gene deletion site (TR L region)

Claims

1. An oncolytic herpes simplex virus carrying a gene encoding an immune checkpoint inhibitor.

2. The herpes simplex virus according to claim 1, which has the following characteristics (a) to (c): (a) the ICP6 gene is deleted or inactivated, (b) the γ34.5 gene is deleted or inactivated, and (c) the α47 gene is deleted or inactivated.

3. The virus of claim 2, wherein a gene encoding an immune checkpoint inhibitor is inserted into the site where the IPC6 gene is deleted or inactivated.

4. The virus described in claim 1, wherein the immune checkpoint inhibitor is an immune checkpoint inhibitor antibody or a fragment thereof.

5. The virus of claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or a fragment thereof.

6. A pharmaceutical composition comprising a virus according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, which is for local injection.

8. The pharmaceutical composition described in claim 6 or 7, which suppresses blood concentrations of immune checkpoint inhibitors.

9. A combination of the virus according to any one of claims 1 to 5 and at least one oncolytic herpes simplex virus having a gene encoding an immune checkpoint inhibitor different therefrom.

10. A combination of the virus according to any one of claims 1 to 5 with at least one of an oncolytic herpes simplex virus having a gene encoding a soluble B7-1 and an oncolytic herpes simplex virus having a gene encoding IL-12.