IL-12 expression type gene recombinant herpes simplex virus

Abstract

The technical problem is to develop a novel armed oncolytic herpes simplex virus (HSV) with G47 delta as a skeleton. Provided is a pharmaceutical composition containing an IL-12 expression-type genetically recombinant herpes simplex virus (HSV) that has a gene encoding a fusion polypeptide in which a 35 kDa light chain (p35) and a 40 kDa heavy chain (p40) of interleukin 12 (IL-12) are linked by two or more elastin motifs, and that has the following characteristics (a) to (c). (a) the ICP6 gene is deleted or inactivated; (b) deletion or inactivation of the gamma34.5 gene; and (c) the alpha 47 gene is deleted or inactivated.

Description

Technical Field

[0001] This invention relates to herpesviruses for cancer treatment. More specifically, it relates to functionally augmented herpesviruses for cancer treatment that enhance the therapeutic effect of viral therapies. Background Technology

[0002] Oncolytic herpes simplex virus type 1 (HSV-1) is an effective tool for cancer treatment (Non-Patent Literature 1, 2). The first oncolytic HSV-1 with a deletion in the thymidine kinase gene was reported in 1991 (Non-Patent Literature 3). Subsequently, various oncolytic HSV-1s were validated in clinical trials. T-VEC (Talimogene laherparepvec) is an oncolytic HSV-1 with mutations in the γ34.5 and α47 genes, and was approved in the United States and Europe in 2015 as a drug for inoperable melanoma (Non-Patent Literature 4). G207, one of the first oncolytic HSV-1 viruses used in clinical trials, has deletions in both copies of the γ34.5 gene and a lacZ insertion that inactivates the ICP6 gene (Non-Patent Literature 5). G47Δ was constructed by further generating a deletion in the US11 promoter, which is a duplication of the α47 gene in the G207 genome (Non-Patent Literature 6, Patent Literature 1). The effectiveness of G47Δ for various cancers has been confirmed (Non-Patent Literature 7–16). The safety of G47Δ has been demonstrated in multiple clinical trials in Japan, including glioblastoma (UMIN000002661) (Non-Patent Literature 17), castration-resistant prostate cancer (UMIN000010463), metastatic prostate cancer (jRCTs033210603), olfactory neuroblastoma (UMIN000011636, jRCTs03180325), and malignant pleural mesothelioma (UMIN000034063, jRCTs03180326). Recently, in Japan, G47Δ was approved as a new drug for malignant glioma through a Phase II trial (UMIN000015995) targeting glioblastoma (Non-Patent Literature 18).

[0003] As a next-generation cancer treatment HSV-1, functionally augmented G47Δ viruses that integrate various exogenous genes into the basic G47Δ framework are being developed, such as VEGF-expressing swelling-inhibiting oncolytic viruses that integrate genes encoding vascular endothelial growth factor (VEGF) antagonists (Patent Document 2).

[0004] On the other hand, interleukin-12 (IL-12) is a promising approach for cancer treatment, and it has been confirmed that IL-12 in several mouse strains is expressed by oncolytic HSV-1 (non-patent literature 19-22). Generally, tumor formation and growth depend primarily on the host's inability to mount a strong anti-tumor immune response and the formation of new blood vessels that supply nutrients to the tumor. IL-12 can target both of these processes. IL-12 is a cytokine that activates various immune cells, including NK cells and T cells. In fact, IFNγ is induced and secreted by these cells.

[0005] Functional IL-12 and p70 are heterodimeric glycoproteins composed of two subunits: a 35 kDa light chain (p35) and a 40 kDa heavy chain (p40) (Non-Patent Literature 23, 24). The p70-IL-12Rβ1 complex binds to the second p70, and the resulting complex pre-forms a binding site with high affinity for IL-12Rβ2. The interaction between p40 and IL-12Rβ1 is essential for the stabilization of the p70-IL-12Rβ1 complex (Non-Patent Literature 25). In the preparation of biologically active IL-12, it is necessary to simultaneously transfect these two subunit genes into mammalian cells (Non-Patent Literature 26, 27).

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent No. 4212897

[0009] Patent Document 2: WO2019 / 189643

[0010] Non-patent literature

[0011] Non-patent literature 1: Kirn, D., Martuza, RL & Zwiebel, J. Replication-selective virotherapy for cancer: Biological principles, risk management and future directions. Nat. Med. 7, 781-787 (2001).

[0012] Non-patent literature 2: Fukuhara, H., Ino, Y. & Todo, T. Oncolytic virus therapy: A newera of cancer treatment at dawn. Cancer Sci. 107, 1373-1379 (2016).

[0013] Non-Patent Document 3: Martuza, R.L., Malick, A., Markert, J.M., Ruffner, K.L. & Coen, D.M. Experimental therapy of human glioma by means of a genetically engineered virus mutant. Science. 252, 854 - 856 (1991).

[0014] Non-Patent Document 4: Coffin R. Interview with Robert Coffin, inventor of T-VEC: the first oncolytic immunotherapy approved for the treatment of cancer. Immunotherapy. 8, 103 - 106 (2016).

[0015] Non-Patent Document 5: Mineta, T., Rabkin, S.D., Yazaki, T., Hunter, W.D. & Martuza, R.L. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nat. Med. 1, 938 - 943 (1995).

[0016] Non-Patent Document 6: Todo, T., Martuza, R.L., Rabkin, S.D. & Johnson, P.A. Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing. Proc. Natl. Acad. Sci. U S A. 98, 6396 - 6401 (2001).

[0017] Non-Patent Document 7: Sugawara, K., et al. Efficacy of a third-generation oncolytic herpes virus G47Δ in advanced stage models of human gastric cancer. Mol. Ther. Oncolytics. 17, 205 - 215 (2020).

[0018] Non - Patent Document 8: Yamada, T., Tateishi, R., Iwai, M., Koike, K. & Todo, T. Neoadjuvant use of oncolytic herpes virus G47Δ enhances the antitumor efficacy of radiofrequency ablation. Mol. Ther. Oncolytics. 21, 612 - 623 (2020).

[0019] Non - Patent Document 9: Fukuhara, H., Martuza, R.L., Rabkin, S.D., Ito, Y. & Todo, T. Oncolytic herpes simplex virus vector G47delta in combination with androgen ablation for the treatment of human prostate adenocarcinoma. Clin. Cancer Res. 11, 7886 - 7890 (2005).

[0020] Non - Patent Document 10: Zeng, W.G., et al. An oncolytic herpes simplex virus vector, G47Δ, synergizes with paclitaxel in the treatment of breast cancer. Oncol. Rep. 29, 2355 - 2361 (2013).

[0021] Non - Patent Document 11: Liu, T.C., Castelo - Branco, P., Rabkin, S.D. & Martuza, R.L. Trichostatin A and oncolytic HSV combination therapy shows enhanced antitumoral and antiangiogenic effects. Mol. Ther. 16, 1041 - 1047 (2008).

[0022] Non-Patent Document 12: Liu, T.C., et al. Oncolytic HSV armed with platelet factor 4, an antiangiogenic agent, shows enhanced efficacy. Mol. Ther. 14, 789 - 797 (2006).

[0023] Non-Patent Document 13: Liu, T.C., et al. Dominant-negative fibroblast growth factor receptor expression enhances antitumoral potency of oncolytic herpes simplex virus in neural tumors. Clin. Cancer Res. 12, 6791 - 6799 (2006).

[0024] Non-Patent Document 14: Uchihashi, T., et al. Oncolytic herpes virus G47Δ injected into tongue cancer swiftly traffics in lymphatics and suppresses metastasis. Mol. Ther. Oncolytics. 22, 388 - 389 (2021).

[0025] Non-Patent Document 15: Sugawara, K., et al. Oncolytic herpes virus G47Δ works synergistically with CTLA-4 inhibition through dynamic intratumoral immune modulation. Mol. Ther. Oncolytics. 22, 129 - 142 (2021).

[0026] Non-Patent Document 16: Yajima, S., et al. Efficacy and safety of a third-generation oncolytic herpes virus G47Δ in models of human esophageal carcinoma. Mol. Ther. Oncolytics. 23, 402 - 411 (2021).

[0027] Non-Patent Document 17: Todo, T., et al. A phase I / II study of triple-mutated oncolytic herpes virus G47Δ in patients with progressive glioblastoma. Nat. Commun. 13:4119 (2022).

[0028] Non-Patent Document 18: Todo, T., et al. Intratumoral oncolytic herpes virus G47Δ for residual or recurrent glioblastoma: a phase 2 trial. Nat. Med. 28:1630-1639 (2022).

[0029] Non-Patent Document 19: Wong, R.J., et al. Cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carcinoma. Hum. Gene Ther. 12, 253-265 (2001).

[0030] Non-Patent Document 20: Ino, Y., Saeki, Y., Fukuhara, H. & Todo, T. Triple combination of oncolytic herpes simplex virus-1 vectors armed with interleukin-12, interleukin-18, or soluble B7-1 results in enhanced antitumor efficacy. Clin. Cancer Res. 12, 643-652 (2006).

[0031] Non-Patent Document 21: Cheema, T.A., et al. Multifaceted oncolytic virus therapy for glioblastoma in an immunocompetent cancer stem cell model. Natl. Acad. Sci. USA. 110, 12006-12011 (2013).

[0032] Non-Patent Document 22: Parker, J.N., et al. Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc. Natl. Acad. Sci. U S A. 97, 2208-2213 (2000).

[0033] Non-Patent Document 23:: Gubler, U., et al. Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor. Proc. Natl. Acad. Sci. U S A. 88, 4143-4147 (1991).

[0034] Non-Patent Document 24: Wolf, S.F., et al. Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells. J. Immunol. 146, 3074-3081 (1991).

[0035] Non-Patent Document 25: Yoon, C., et al. Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12. EMBO J. 19, 3530-3541 (2000).

[0036] Non-Patent Document 26: Kobayashi, M., et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170, 827-845 (1989).

[0037] Non-patent document 27: Stern, AS, et al. Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells. Proc. Natl. Acad. Sci. US A. 87, 6808-6812 (1990).

[0038] Non-patent literature 28: D'Andrea, A., et al. Production of natural killer cellstimulatory factor (interleukin 12) by peripheral blood mononuclear cells. J. Exp. Med. 176, 1387-1398 (1992).

[0039] Non-patent literature 29: Mattner, F., et al. The IL-12 subunit p40 specifically inhibits effects of the IL-12 heterodimer. Eur. J. Immunol. 23, 2202-2208 (1993).

[0040] Non-patent literature 30: York, IA, et al. A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+T lymphocytes. Cell. 77, 525-535 (1994). Summary of the Invention

[0041] The problem the invention aims to solve

[0042] Develop a novel armed oncolytic herpes simplex virus (HSV) with the G47Δ backbone. Since HSV induces specific anti-tumor immunity, it is expected that IL-12 can function as a payload to enhance its effects.

[0043] Solution for solving the problem

[0044] In this study, two mouse IL-12 expression cassettes using the ultra-early cytomegalovirus (CMV) promoter were prepared:

[0045] 1) Fusion peptide expression type: p35 and p40 subunit genes are linked together by the same reading frame to form a single gene, expressing a fusion peptide composed of p40 and p35.

[0046] 2) Co-expression: The p35 and p40 subunit genes are separated by an internal ribosome entry site (IRES) sequence, allowing the co-expression of these two distinct proteins. In this case, although the p40 subunit transcript is present in many cell types, the p35 subunit will not be secreted if the p40 subunit is absent (Non-Patent Document 28). Therefore, the p40 gene is positioned close to the promoter.

[0047] In fusion proteins with two subunits, maintaining the functional tertiary structure of the expressed molecule is a crucial issue. Therefore, a design was developed that inserts two bovine elastin motifs between the p35 and p40 genes, causing the two subunits to fold and interact. On the other hand, IRES ensures that the two separately expressed subunits form molecules that directly function as IL-12 while maintaining their tertiary structure. However, IRES may lead to uneven expression of the two subunit genes, resulting in overexpression of the p40 gene near the promoter. Excess free p40 subunits may inhibit IL-12 activity in mouse models (Non-Patent Literature 29).

[0048] The α47 gene product binds to antigen-presenting transporters (TAPs) and downregulates the expression of major histocompatibility complex (MHC) class I molecules, thus avoiding detection of infected cells by host immune surveillance (Non-Patent Literature 30). G47Δ is an α47-deficient HSV-1 that prevents the downregulation of MHC class I molecules, demonstrating enhanced immunostimulatory capacity (Non-Patent Literature 6). Therefore, it is suitable as a backbone for expressing immunostimulatory genes in humans. In this study, two different mouse IL-12 expression cassettes were inserted into the deficient ICP6 locus of the G47Δ backbone to create two IL-12 expression morphologies: oncolytic HSV-1, T-mfIL12, and T-mIL12-IRES. The differences in their effects should reflect the methods used to induce IL-12 expression in mice. Evaluations were conducted in vitro and in vivo using mouse prostate cancer and neuroblastoma cells. The results showed that T-mfIL12, a fusion-type IL-12, had a higher expression level of functional IL-12 compared to T-mIL12-IRES, demonstrating high antitumor activity.

[0049] This invention provides the following content.

[0050] [1] A pharmaceutical composition comprising an IL-12-expressing recombinant herpes simplex virus (HSV) having a gene encoding a fusion polypeptide consisting of a 35 kDa light chain (p35) and a 40 kDa heavy chain (p40) of interleukin-12 (IL-12) linked by two or more elastin motifs, and having the characteristics of (a) to (c) below.

[0051] (a) ICP6 gene deletion or inactivation;

[0052] (b) γ34.5 gene deletion or inactivation;

[0053] (c) α47 gene deletion or inactivation.

[0054] [2] The pharmaceutical composition according to 1, wherein the fusion polypeptide is sequentially linked from the N-terminal side to p40, two or more elastin motifs, and p35.

[0055] [3] The pharmaceutical composition according to 1 or 2, wherein the gene encoding the fusion polypeptide is inserted into the ICP6 gene locus.

[0056] [4] The pharmaceutical composition according to any one of 1 to 3, wherein the gene encoding the fusion peptide is any of the following polynucleotides,

[0057] (i) A polynucleotide consisting of the sequence of sequence number 1;

[0058] (ii) A polynucleotide consisting of a sequence having more than 90% sequence identity with the sequence of sequence number 1 and encoding a polypeptide that can form active IL-12.

[0059] (iii) A polynucleotide encoding a polypeptide consisting of the sequence of sequence number 2;

[0060] (iv) A polynucleotide encoding a polypeptide that is composed of a sequence having more than 90% sequence identity with the sequence of sequence number 2 and can form an active IL-12.

[0061] (v) A polynucleotide consisting of the sequence number 3;

[0062] (vi) A polynucleotide consisting of a sequence having more than 90% sequence identity with the sequence of sequence number 3 and encoding a polypeptide that can form active IL-12;

[0063] (vii) Encoding a polynucleotide of a polypeptide consisting of the sequence of sequence number 4;

[0064] (viii) A polynucleotide that encodes a polypeptide consisting of a sequence that has more than 90% sequence identity with the sequence of sequence number 4 and can form an active IL-12.

[0065] [5] The pharmaceutical composition according to any one of 1 to 4, wherein the recombinant HSV is derived from G47Δ.

[0066] [6] A pharmaceutical composition according to any one of 1 to 5, used to enhance the effect of IL-12 in the treatment of tumors. A method for enhancing the effect of IL-12 in the treatment of tumors using a recombinant HSV expressing an IL-12 gene or a pharmaceutical composition containing the IL-12 gene as defined in any one of 1 to 5. Use of a recombinant HSV expressing an IL-12 gene as defined in any one of 1 to 5 in the preparation of a pharmaceutical composition for enhancing the effect of IL-12 in the treatment of tumors. A method for enhancing the effect of IL-12 in the treatment of tumors, comprising the step of administering a recombinant HSV expressing an IL-12 gene or a pharmaceutical composition containing the IL-12 gene as defined in any one of 1 to 5 to a subject. Use of a recombinant HSV expressing an IL-12 gene or a pharmaceutical composition containing the IL-12 gene as defined in any one of 1 to 5 for enhancing the effect of IL-12 in the treatment of tumors.

[0067] [7] The pharmaceutical composition according to any one of 1 to 6 is used for local injection. The composition, preparation, use, method, and application according to 6, wherein the pharmaceutical composition or the IL-12-expressing recombinant HSV is used for local injection.

[0068] [8] A method for enhancing the effect of IL-12 in the treatment of tumors, comprising the step of administering to a subject in need a pharmaceutical composition comprising a recombinant HSV expressing the IL-12 gene as described in any one of 1 to 7.

[0069] [9] A pharmaceutical composition comprising a recombinant HSV expressing an IL-12 gene having a gene encoding IL-12 and having the characteristics described in (a) to (c) above, wherein the pharmaceutical composition induces the production of IFNγ through the expression of IL-12, and the replication capacity of the recombinant HSV expressing the IL-12 gene is not reduced. A pharmaceutical composition comprising a recombinant HSV expressing an IL-12 gene having a gene encoding IL-12 and having the characteristics described in (a) to (c) above, for use in a method of tumor treatment, wherein the production of IFNγ is induced through the expression of IL-12, and the replication capacity of the recombinant HSV expressing the IL-12 gene is not reduced. Use of a recombinant HSV expressing an IL-12 gene having a gene encoding IL-12 and having the characteristics described in (a) to (c) above in the preparation of a pharmaceutical composition for tumor treatment inducing the production of IFNγ through the expression of IL-12, wherein the replication capacity of the recombinant HSV expressing the IL-12 gene is not reduced. A method for treating a tumor in which IL-12 expression induces the production of IFNγ without diminishing the replication capacity of the IL-12-expressing recombinant HSV, comprising administering to a subject a IL-12-expressing recombinant HSV having a gene encoding IL-12 and possessing the characteristics described in (a) to (c) above. A pharmaceutical composition for use in a method for treating a tumor in which IL-12 expression induces the production of IFNγ without diminishing the replication capacity of the IL-12-expressing recombinant HSV, comprising an IL-12-expressing recombinant HSV having a gene encoding IL-12 and possessing the characteristics described in (a) to (c) above.

[0070]

[10] A pharmaceutical composition for intravenous administration comprising a recombinant HSV having a gene encoding IL-12 and having the characteristics described in (a) to (c) above. A recombinant HSV having a gene encoding IL-12 and having the characteristics described in (a) to (c) above, or a pharmaceutical composition comprising thereof, for use in a method of treating a tumor by intravenous administration. Use of a recombinant HSV having a gene encoding IL-12 and having the characteristics described in (a) to (c) above in the preparation of a pharmaceutical composition for intravenous administration for treating a tumor. A method of treating a tumor comprising administering a recombinant HSV having a gene encoding IL-12 and having the characteristics described in (a) to (c) above, or a pharmaceutical composition comprising thereof, into a vein of a subject. Use of a pharmaceutical composition comprising a recombinant HSV having a gene encoding IL-12 and having the characteristics described in (a) to (c) above for intravenous administration for treating a tumor.

[0071]

[11] A pharmaceutical composition for intratumoral administration comprising a recombinant HSV having a gene encoding IL-12 and having the characteristics described in (a) to (c) above, the pharmaceutical composition inducing systemic antitumor immunity, thereby achieving at least one of inhibiting metastasis and inhibiting the proliferation of untreated tumors. A recombinant HSV having a gene encoding IL-12 and having the characteristics described in (a) to (c) above, or a pharmaceutical composition comprising thereof, for use in a method of tumor treatment, the method of tumor treatment inducing systemic antitumor immunity through intratumoral administration, thereby achieving at least one of inhibiting metastasis and inhibiting the proliferation of untreated tumors. Use of a recombinant HSV having a gene encoding IL-12 and having the characteristics described in (a) to (c) above in the preparation of a pharmaceutical composition for intratumoral administration, the pharmaceutical composition for intratumoral administration inducing systemic antitumor immunity, thereby achieving at least one of inhibiting metastasis and inhibiting the proliferation of untreated tumors. A method for treating a tumor that induces systemic antitumor immunity, thereby achieving at least one of inhibiting metastasis and inhibiting the proliferation of untreated tumors, comprising administering a recombinant HSV with an IL-12-encoding gene and having the characteristics described in (a) to (c) above, or a pharmaceutical composition comprising thereto, into the tumor of the target. Use of a recombinant HSV with an IL-12-encoding gene and having the characteristics described in (a) to (c) above, or a pharmaceutical composition comprising thereto, for treating a tumor, wherein the pharmaceutical composition, as a pharmaceutical composition for intratumoral administration, induces systemic antitumor immunity, thereby achieving at least one of inhibiting metastasis and inhibiting the proliferation of untreated tumors.

[0072]

[12] A pharmaceutical composition for treating tumors requiring increased immune cell infiltration, comprising a recombinant HSV having an IL-12-encoding gene and the characteristics described in (a) to (c) above. A recombinant HSV having an IL-12-encoding gene and the characteristics described in (a) to (c) above, or a pharmaceutical composition comprising thereof, for use in a method of treating tumors requiring increased immune cell infiltration. Use of a recombinant HSV having an IL-12-encoding gene and the characteristics described in (a) to (c) above in the preparation of a pharmaceutical composition for treating tumors requiring increased immune cell infiltration. A method of treating tumors requiring increased immune cell infiltration, comprising the step of administering a recombinant HSV having an IL-12-encoding gene and the characteristics described in (a) to (c) above, or a pharmaceutical composition comprising thereof, to a subject. Use of a recombinant HSV of an IL-12 expression gene having a gene encoding IL-12 and having the characteristics of (a) to (c) above, or a pharmaceutical composition comprising the HSV, for the treatment of tumors requiring increased infiltration of immune cells.

[0073]

[13] The pharmaceutical composition, preparation, use, method or use according to any one of 9 to 12, wherein the IL-12 expression-type recombinant HSV is the IL-12 expression-type recombinant HSV as defined in any one of 1 to 5.

[0074] The effects of the invention

[0075] If the recombinant HSV with the IL-12 expression gene provided in this application is administered to tumor cells, it can express active IL-12 (maintaining the functional tertiary structure) along with viral replication.

[0076] In addition, the following effects can be achieved.

[0077] • By administering only one virus, it demonstrated the combined effects of systemic administration of IL-12 and viral therapy.

[0078] • Compared to viruses that do not express anything, it showed enhanced therapeutic effects on both drug-treated tumors and distant, untreated tumors.

[0079] • The expressed IL-12 has the same function as IL-12 administered as a protein.

[0080] • Expressed IL-12 can exert a high level of efficacy at lower levels than IL-12 administered as a protein.

[0081] • The expressed IL-12 remains locally in the tumor and is difficult to flow into the bloodstream, thus it is less likely to produce systemic side effects.

[0082] • By combining it with HSV-1 for the treatment of cancer with soluble B7-1 expression, further enhancement of therapeutic effects can be expected.

[0083] • When administered as virally expressed IL-12, it can effectively exert local and systemic anti-tumor immunity through mixed administration of protein IL-12 and non-expressing virus. Attached Figure Description

[0084] [ Figure 1 The structures of T-mfIL12 and T-mIL12-IRES. The cassette (upper segment) represents the DNA structure of HSV-1. Inverted repeat sequences form long chains (U...) each containing an intrinsic sequence. L ) and short chains (U S The structure of oncolytic HSV-1 is as follows. Oncolytic HSV-1 uses the G47Δ backbone, including approximately 1.0 kb deletions in the double copies of the γ34.5 gene, approximately 0.3 kb deletions in the α47 gene, and approximately 0.9 kb deletions in the ICP6 gene. The lacZ gene and the interleukin-12 (IL-12) gene, controlled by the cytomegalovirus (CMV) promoter, are inserted into the deleted ICP6 locus in opposite directions. In T-mfIL12, a cassette containing the mouse IL-12 p35 and p40 genes is linked by two bovine elastin motifs. Mouse IL-12 is expressed as a single fusion peptide and folded for activation. T-mIL12-IRES contains cassettes containing the mouse IL-12 p40 and p35 genes separated by an internal ribosome entry site (IRES) sequence derived from encephalomyocarditis virus (EMCV). Effective co-expression of the p40 and p35 subunits results in active mouse IL-12. T-01 contains an empty cassette without the introduced gene.

[0085] [ Figure 2In vitro replication capacity of T-mfIL12 and T-mIL12-IRES and expression of mouse IL-12. a. In vitro replication analysis. Vero cells were infected with G47Δ, T-01, T-mfIL12 candidate (4 clones), or T-mIL12-IRES candidate (4 clones) at a multiplicity of infection (MOI) of 0.01. Progeny viruses were recovered 48 hours post-infection, and the quantity was determined by plaque assay. No significant difference was observed in replication capacity between T-mfIL12 and T-mfIL12-IRES (p = 0.736, t-test). b. In vitro expression of mouse IL-12. Vero cells were infected with G47Δ, T-01, T-mfIL12 candidate (4 clones), or T-mIL12-IRES candidate (4 clones) at an MOI of 1. The amount of secreted mouse interleukin-12 (IL-12) (p70) was determined by enzyme-linked immunosorbent assay (ELISA). T-mfIL12 expressed significantly higher levels of p70IL-12 than T-mIL12-IRES (p<0.001, t-test). c. Changes in viral yield over time. Vero cells were repeatedly infected with G47△, T-01, T-mfIL12, or T-mIL12-IRES at an MOI of 0.01. Progeny viruses were recovered at 0h, 6h, 24h, and 48h post-infection and titrated by plaque assay. The change in viral yield of T-mfIL12 over time was the same as that of T-mIL12-IRES. d. In vitro expression of mouse IL-12 in mouse tumor cell lines. Neuro2a, PR14-2, or TRAMP-C2 cells were infected with T-mfIL12 or T-mIL12-IRES at an MOI of 1. The amount of secreted mouse IL-12 (p70) was determined by ELISA. T-mfIL12 expressed higher levels of p70IL-12 than T-mIL12-IRES in all three cell lines. All analyses were repeated twice. Bar, SD. **, p<0.001; NS, no significant difference.

[0086] [ Figure 3 In vitro cytotoxic effects of T-mfIL12 and T-mIL12-IRES. In all three mouse cancer cell lines, T-mfIL12 and T-mIL12-IRES showed cytotoxic effects comparable to the control virus T-01 at both MOI = 0.1 (dashed line) and MOI = 1 (solid line). Viable cell counts were performed daily and expressed as a percentage relative to a control with simulated infection. Results are averages from 3-well pairs. bar, SD.

[0087] [ Figure 4The efficacy of T-mfIL12 and T-mIL12-IRES in vivo. a, b, and c are evaluations based on subcutaneous tumor growth inhibition. a. Male C57BL / 6 mice with unilateral subcutaneous TRAMP-C2 tumors (prostate cancer) were treated with T-01, T-mfIL12, or T-mIL12-IRES (5 x 10⁻⁶) on days 0 and 3. 6 Intratumoral inoculation with pfu or mock (n=6). T-mfIL12 showed the highest efficacy. All three viruses were significantly more effective than mock (all p<0.001) (significance not shown in the figure). b. In female A / J mice with bilateral subcutaneous Neuro2a tumors (neuroblastoma), T-01, T-mfIL12, and T-mIL12-IRES (5x10) were administered only to the left tumor on days 0 and 3. 4 Intratumoral inoculation with pfu or mock (n=9-12). T-mfIL12 showed the highest efficacy on both the treated and untreated sides. All three viruses were significantly more effective than mock on the treated side (p=0.001 in T-01, p<0.001 in both T-mfIL12 and T-mIL12-IRES), and T-mfIL12 and T-mIL12-IRES were more effective than mock on the untreated side (p<0.001 for both viruses) (significant differences not shown in the figure). c. In NOG mice with bilateral Neuro2a subcutaneous tumors, T-01, T-mfIL12, or T-mIL12-IRES (1x10⁻¹) were administered only in the left tumor on days 0 and 3. 6 Intratumoral inoculation with T-01, T-mfIL12, or T-mIL12-IRES (n=10) or mock tumors. There was no difference in efficacy between the treated and untreated sides for T-01, T-mfIL12, and T-mIL12-IRES. The effect of IL-12 expression was completely lost on both the treated and untreated sides for T-mfIL12 and T-mIL12-IRES. Tumor volume = length x width x height x 0.52. Results are expressed as mean. bar represents the standard error of the mean (SEM). *, p<0.05; **, p<0.01; ***, p<0.001; binary ANOVA, performed using Bonfrony multiple comparison test. d and e. Evaluation based on survival. Female A / J mice with bilateral subcutaneous Neuro2a tumors were inoculated with T-01, T-mfIL12, or T-mIL12-IRES, 5x10⁻¹⁰, on days 0 and 3 within the left tumor. 5 pfu(d) or 5x10 4Treatment involved intratumoral inoculation with PFU(e) or mock. Mice were euthanized at the time point when the maximum diameter of one tumor reached 22 mm. Both T-mfIL12 and T-mIL12-IRES significantly prolonged the survival of tumor-bearing mice compared to T-01 at both doses (Log-Rank test). One or two mice administered T-mfIL12 showed long-term survival at both doses, but there was no significant difference between T-mfIL12 and T-mIL12-IRES.

[0088] [ Figure 5 Immune response based on T-mfIL12 and T-mIL12-IRES. a. Immunohistochemistry. Bilateral Neuro2a subcutaneous tumors were created in A / J mice, and T-mfIL12, T-mIL12-IRES, and T-01 (2x10⁻¹²) were injected into the left tumor only on days 0 and 3. 5 PFU or mock tumors were harvested on day 6 (n=3 per group). Within the tumors, CD4 was observed in both the treated and untreated sides for all three viruses. + and CD8 + The increase in lymphocyte infiltration was most pronounced in T-mfIL12. Figure 5 a). HSV-1 positive cells were observed in the tumor on the treated side in all viral loads, but not on the untreated side. HE, hematoxylin andeosin. Scale bar, 100 μm. b). In vivo levels of interleukin-12 (IL-12) and interferon-γ (IFNγ). In a unilateral Neuro2a subcutaneous tumor model, T-01, T-mfIL12, and T-mIL12-IRES (2 x 10⁻⁶) were inoculated into the established tumor. 6Mice were tested using either PFU or mock IL-12. Serum and tumor samples were collected on days 1, 3, and 6 (n=3 for each group). IL-12 and IFNγ levels were measured by ELISA. Intratumoral IL-12 levels in T-mfIL12 were significantly higher than those in T-mIL12-IRES at all time points (p=0.018, p=0.016, p=0.046 for days 1, 3, and 6, respectively). Serum IL-12 levels were significantly lower than intratumoral levels. Correlation with intratumoral IL-12 showed that serum IL-12 levels in T-mfIL12 were higher on day 1 than those in T-mIL12-IRES (p=0.047). Furthermore, the IFNγ level of T-mfIL12 was significantly higher than that of T-mIL12-IRES in both tumor and serum on day 1 (tumor and serum, p = 0.014 and p = 0.027, respectively). c. Neuro2a cell-specific immune response. In a unilateral Neuro2a subcutaneous tumor model, T-01, T-mfIL12, and T-mIL12-IRES (5 x 10⁻⁶ cells / mL) were inoculated into the established tumor on days 0 and 3. 4 (PFU) or mock, spleen was removed on day 6. ELISpot analysis showed that spleen cells from mice treated with T-mfIL12 had significantly more IFNγ-secreting cells stimulated by Neuro2a cells compared to those from mice treated with T-01 and T-mIL12-IRES (p = 0.005 and p = 0.004, respectively, relative to T-01 and T-mIL12-IRES). No significant difference was observed among the three viral treatment groups in the number of IL-4-secreting spleen cells. The number of Neuro2a cell-specific IFNγ or IL-4-secreting cells was calculated by subtracting the number of SaI / N response sites from the number of Neuro2a response sites. For b and c, the graph shows the mean. Points represent individual data. bar, SD. *, p < 0.05; **, p < 0.01; NS, no significant difference; one-way ANOVA based on Tukey's multiple comparisons.

[0089] [ Figure 6 A comparison of the in vivo efficacy of direct intratumoral injection of T-mfIL12 and recombinant interleukin-12 (rIL-12). In a bilateral Neuro2a subcutaneous tumor model, rIL-12 and T-01 (5x10⁻¹²) were injected into the left tumor only on days 0 and 4. 4 The presence / absence of PFU and rIL-12, T-mfIL12 (5x10) 4PFU or mock (n=10 per group). Three different doses of rIL-12 were used: 500 ng (a), 50 ng (b), and 1 ng (c). A dose of 50 ng is equivalent to 2 x 10... 6 PFU treatment of T-mfIL12 levels within tumors, 1 ng is equivalent to 5 x 10 4 Intratumoral IL-12 levels after PFU treatment with T-mfIL12. a. With rIL-12 at a dose of 500 ng, on the treatment side, rIL-12, T-01, T-01+rIL-12, and T-mfIL12 were significantly more effective than mock (all p < 0.001 relative to mock). On the non-treatment side, rIL-12 alone did not show a significant antitumor effect compared to mock, but T-01+rIL-12 and T-mfIL12 showed significantly higher efficacy compared to mock (p = 0.039 and p < 0.001, respectively, relative to mock). Furthermore, on the non-treatment side, T-mfIL12 showed a significantly higher efficacy than rIL-12 alone (p = 0.003), while there was no significant difference between T-01+rIL-12 and rIL-12 alone. b. With rIL-12 at a dose of 50 ng, the same results as a were obtained. c. At a dose of 1 ng of rIL-12, no significant effect was observed in either the treated or untreated sides when rIL-12 was used alone. In the treated side, T-mfIL12 showed a significantly higher effect than rIL-12 alone, but there was no significant difference between T-01+rIL-12 and rIL-12 alone. Tumor volume = length x width x height x 0.52. Results are expressed as mean. Standard error of mean (SEM). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significant difference; binary ANOVA, performed using Bonfrani multiple comparison test.

[0090] [ Figure 7Representative gel electrophoresis was performed to observe the structure of the T-BAC / Vec9 plasmid obtained after Cre recombination. DNA from T-BAC (lane 3), T-BAC / Vec9-empty (lane 4), T-BAC / Vec9-fused-mIL12 (lanes 5-10), or T-BAC / Vec9-IRES-mIL12 (lanes 11-16) was digested with HindIII and separated by electrophoresis on a 0.6% (w / v) agarose gel in 1×TB-EDTA buffer at 2.5 V / cm for 18 hours. All bands were as expected: 15kb for T-BAC (lane 3), 11kb, 8kb, and 6.2kb for T-BAC / Vec9-empty (lane 4), bands 1-6 for T-BAC / Vec9 fusion-mIL12 (lanes 5-10), and bands 1-6 for T-BAC / Vec9-IRES-mIL12 (lanes 11-16), with 8kb and 6.2kb respectively. Lanes 1, 2, 17, and 18 were marked with kb.

[0091] [ Figure 8 Interferon-γ secretion from spleen cells stimulated by virally expressed IL-12. The supernatant of Vero cells infected with T-mfIL12 or T-mIL12-IRES at a multiplicity of infection (MOI) = 1 was recovered 48 hours post-infection. Interferon-γ (IFNγ) levels were measured in spleen cells 48 hours after administration of the supernatant, recombinant mouse IL-12, or DMEM (culture medium) (n = 2). IL-12 expressed by both T-mfIL12 and T-mIL12-IRES stimulated spleen cells. ND, not detected.

[0092] [ Figure 9 ] Figure 4 a and Figure 4 Tumor growth curves for each animal (b). In a unilateral TRAMP-C2 subcutaneous tumor model, T-01, T-mfIL12, and T-mIL12-IRES (5×10⁻⁶) were used. 6 PFU or mock were injected into the tumor on days 0 and 3. In bilateral subcutaneous Neuro2a models, T-01, T-mfIL12, and T-mIL12-IRES (5×10⁻⁶) were injected into the tumor. 4 PFU or Mock were injected only into the left tumor on days 0 and 3.

[0093] [ Figure 10 ELISpot analysis of interferon-γ (IFNγ). In established Neuro2a subcutaneous tumors, T-01, T-mfIL12, and T-mIL12-IRES (5 × 10⁻⁶) were inoculated on days 0 and 3. 4Spleens were harvested on day 6 (n=3) using either pfu or mock. Spleen cells were stimulated with Neuro2a cells, SaI / N cells, ConA (positive control), or no stimulant (negative control). a. Graph showing the mean. Dots represent data from individual mice. b. Photographs of wells showing the results of ELISpot analysis. bbar, SD.

[0094] [ Figure 11 ELISpot analysis of interleukin-4 (IL-4). In established Neuro2a subcutaneous tumors, T-01, T-mfIL12, and T-mIL12-IRES (5 × 10⁻⁶) were inoculated on days 0 and 3. 4 Spleen was removed on day 6 using either pfu or mock stimulation. Splenic cells were stimulated with Neuro2a cells, SaI / N cells, ConA (positive control), or no stimulation (negative control). a. Dots indicate data from individual mice. b. Photographs of wells showing ELISpot analysis results. Bar, SD.

[0095] [ Figure 12 [Base sequence data of the inserted nucleotides of T-mfIL12. The underlined portions of the nucleotides in T-mfIL12 correspond to bovine elastin motifs. In T-mIL12-IRES, the underlined portions correspond to IRES arrays.]

[0096] [ Figure 13 [Base sequence data of the inserted nucleotides of T-hIL12. The underlined portion corresponds to the bovine elastin motif.]

[0097] [ Figure 14 The insertion site of the fusion polypeptide and the base sequences before and after it.

[0098] [ Figure 15 ]γ34.5 gene deletion site and the base sequences before and after it (TR) L within the area).

[0099] [ Figure 16 The α47 gene deletion site and the base sequences before and after it.

[0100] [ Figure 17 [Study on viral replication capacity in unilateral subcutaneous tumors of Neuro2a in A / J mice. 5 × 10⁵ cells dispersed in 50 μL DMEM were subcutaneously injected into the left abdomen of A / J mice.] 6 Neuro2a cells were used to create a unilateral subcutaneous tumor. Five days later, 1.0 × 10⁶ cells were injected. 6A single dose of 20 μL of T-01 (n=3 on each test day) or T-mfIL12 (n=3 on each test day) was administered intratumorally to mice. Mice were euthanized on the day of administration and on days 1, 4, 7, and 11 after administration. Tumor portions were excised, weighed, and homogenized with PBS. A portion of the supernatant was titrated to determine viral titer. I bar: standard error

[0101] [ Figure 18 [Comparative experiment on the in vitro cell-killing effects of T-01 virus and T-mfIL12 virus in a subcutaneous tumor model. A. Seed 2.0 x 10⁻⁶ cells / well in a 6-well plate.] 5 RenCa cells were infected with T-01 virus or T-mfIL12 virus at MOIs of 1.0 and 0.1, respectively. Cells were cultured at 34.5°C, and the number of surviving cells was measured every 24 hours until day 4, evaluated as a percentage of surviving cells relative to the mock infection group. In in vitro experimental systems, T-01 virus and T-mfIL12 virus showed comparable cytotoxic effects. 1.0 x 10^6 cells were subcutaneously injected into the left abdomen of BC and BALB / c mice. 5 Subcutaneous tumors were created using RenCa, and when the largest tumor diameter reached 5 mm (day 0), 2 x 10 T-01 virus or T-mfIL12 virus were injected on days 0 and 3. 5 PFU intratumoral administration (n=8 / group). In the T-mfIL12 virus administration group, tumor growth was significantly inhibited compared with the mock virus administration group and the T-01 virus administration group (p=0.0072 and p=0.0202, respectively). * is marked at 0.001≤P<0.05. Additionally, based on titration results, relative to the originally planned intratumoral administration dose of T-01 virus or T-mfIL12 virus 2x10 5 The actual dosage of PFU was 0.6 × 10⁻⁶. 5 and 0.5×10 5 pfu. C. Typical examples of mice on day 9 of the RenCa subcutaneous tumor model experiment. a. mock administration group b. T-01 virus administration group c. T-mfIL12 virus administration group In the T-mfIL12 virus administration group, the tumors disappeared in 3 out of 8 mice.

[0102] [ Figure 19-1 Intravenous administration of T-01 virus and T-mfIL12 virus in RenCa lung metastasis model (5x10) 5 RenCa 2.0 x 10^6 pfu was administered intravenously via the tail vein of BALB / c mice. 5 On day 0, administer 5 x 10 doses of T-01 virus or T-mfIL12 virus intravenously on days 1, 3, and 5. 5PFU. AB, The number of lung metastases was evaluated at 14th time (n = 11 / group). The average number of metastases in the mock group, T-01 virus group, and T-mfIL12 virus group was 205, 42, and 1.9, respectively. The number of metastases was significantly reduced in the T-01 virus and T-mfIL12 virus groups compared to the mock group (p = 0.0210 and p = 0.0017, respectively). In the T-mfIL12 virus group, the number of metastases was significantly reduced compared to the T-01 virus group (p = 0.0159). * is marked at 0.001 ≤ P < 0.05. Additionally, based on the titration results, the actual T-01 virus or T-mfIL12 virus dosage was 2.1 × 10⁻⁶. 5 and 5.9×10 5 PFU. C. Survival time was observed according to the same experimental protocol (n = 12 / group). T-01 virus and T-mfIL12 virus significantly prolonged the survival time of mice compared to the mock virus (p = 0.0016 and p < 0.0001), and T-mfIL12 virus significantly prolonged the survival time of mice compared to T-01 virus (p = 0.0154). Furthermore, based on the titration results, the actual dosage of T-01 virus or T-mfIL12 virus was 5.6 × 10⁻⁶. 5 and 5.0×10 5 pfu.

[0103] [19-2] Intravenous administration of T-01 virus and T-mfIL12 virus in RenCa lung metastasis model (5x10) 6 RenCa 2.0 x 10^6 pfu was administered intravenously via the tail vein of BALB / c mice. 5 (Day 0). AB, on days 1, 3, and 5, administer 5 x 10 doses of T-01 virus or T-mfIL12 virus intravenously. 6 PFU was used to observe survival time (n=12 / group). T-01 virus and T-mfIL12 virus significantly prolonged mouse survival compared to the mock virus (p<0.0001 and p<0.0001, respectively), and T-mfIL12 virus significantly prolonged mouse survival compared to T-01 virus (p=0.0003). Furthermore, based on titration results, the actual dosage of T-01 virus or T-mfIL12 virus administered was 3.3 × 10⁻⁶. 6 and 3.1×10 6PFU. B. Lungs removed at death. a. Mock treatment group b. T-01 virus treatment group c. T-mfIL12 virus treatment group. C. When the start of treatment was delayed to day 9, and days 9, 11, and 13 were set, only T-mfIL12 virus significantly prolonged the survival of mice compared to the mock treatment (p = 0.0054) (n = 10 / group). Additionally, based on titration results, the actual dose of T-01 virus or T-mfIL12 virus was 4.6 × 10⁻⁶. 6 and 5.2×10 6 pfu.

[0104] [ Figure 20 Cytokine secretion activity assay of T-01 and T-mfIL12 viruses administered intravenously in a RenCa lung metastasis model. 2.0 x 10⁻⁶ RenCa was administered intravenously via the tail vein of BALB / c mice. 5 On day 0, administer 5 x 10 doses of T-01 virus or T-mfIL12 virus intravenously on days 1, 3, and 5. 6 PFU was used to remove spleens on day 14. The removed spleens were prepared into single-cell suspensions and seeded in 2x10⁶ wells of each 96-well plate. 5 The secretory activities of mIFNγ and mIL-4 were measured using an ELISpot assay. Three assays were performed independently for each of the three groups. Culture times for both mIFNγ and mIL-4 were 24 h and 48 h. Antigen stimulation was performed using RenCa cells. Enhanced IFNγ secretion was observed in the T-01 and T-mfIL12 virus-treated groups compared to the mock-treated groups under RenCa antigen stimulation (p = 0.0726 and p = 0.0112, respectively), with a significant difference observed in the T-mfIL12 virus group. A significant enhancement in secretion was also observed in the T-mfIL12 virus-treated group compared to the T-01 virus-treated group (p = 0.0251). No significant differences were observed in IL-4 secretory activity between any groups. * is marked at 0.001 ≤ P < 0.05.

[0105] [ Figure 21 A: Survival analysis. MB49 cells (5.0 x 10⁻⁶) were used. 5 The tumor was injected intrabladder into female C57BL / 6 mice at three different doses (2.0 × 10⁻⁶) on days 4 and 7 post-tumor inoculation. 5 1.0×10 6 5.0×10 6 T-mfIL12 was administered intravesically (pfu). Survival data were analyzed using a log-rank test (n=10 for each group). The results showed that the medium and high doses of T-mfIL12 (1x10) were significantly better than those of pfu. 6and 5x10 6 The pfu (pfu) showed statistically significant differences compared to the control (P<0.05 and P<0.01). However, the low dose had no effect (P=0.625). B: Survival analysis. MB49 cells (5.0x10⁻¹) were used in female C57BL / 6 mice. 5 Cells were seeded intrabladder, and PBS or BCG (1.35 mg) was administered intrabladder on day 1. Molck or T-mfIL12 (1.0 x 10⁻⁶ mg) was administered intrabladder on days 4 and 7. 6 PFU). Survival data were analyzed using the log-rank test (n=10 for each group). All treatment groups (BCG+mock, PBS+T-mfIL12, BCG+T-mfIL12) showed significantly strong antitumor effects (P<0.05 log-rank test). Moreover, the combination therapy showed significantly greater antitumor effects than other groups. In the combination therapy group, approximately 30% of mice survived for 50 days (P<0.01 log-rank test). C: Survival analysis. Female C57BL / 6 mice were injected via the tail vein with 1.0×10⁻⁶ PFU. 5 MB49 cells were treated with molck or T-01 and T-mfIL12 (5.0 × 10⁻⁶) on days 1, 3, and 5. 6 According to the Kaplan-Meyer survival analysis, there were significant differences between the two treatment groups and the control group (log-rank test P = 0.04 and P < 0.01, respectively), and there was also a significant difference between T-01 and T-mfIL12 treatments (log-rank test P < 0.05). This demonstrates an improvement in survival rate based on T-mfIL12 treatment.

[0106] [ Figure 22 A: Inhibition of tumor proliferation based on IL-2 / anti-IL-2 mAb complex. On day 5, BALB / c mice with established RenCa subcutaneous tumors were treated with IgG, anti-IL2 mAb (1 mg), IL2 (2 μg), or an IL-2 / anti-IL-2 mAb (2 μg / 1 mg) complex (n = 10 in each group). Tumor proliferation was inhibited by comparison of IL-2 / anti-IL-2 mAb complex with anti-IL2 mAb (P < 0.05, bar ± SD, days 18–23) or control treatment (P < 0.01, bar ± SD, days 12–23). *: P < 0.05 **: P < 0.01. B. Inhibition of tumor proliferation based on combination therapy. On days 0 and 3, IgG + mock, IL-2 / anti-IL-2 mAb complex (2 μg / 1 mg) + mock, IgG + T-mfIL12 (1.0 × 10⁻⁶) were administered. 6PFU or IL-2 / anti-IL-2 mAb (2 μg / 1 mg) complex + T-mfIL12 (1 × 10⁻⁶) 6 BALB / c mice with established RenCa subcutaneous tumors were treated with pfu (n=10 in each group). Subcutaneous RenCa tumors were significantly smaller in the combination therapy group. This was statistically significant at this time point (P<0.0001, bar, SD). *: P<0.05**: P<0.01. C: Histological and immunohistochemical staining of tumors derived from RenCa subcutaneous tumors obtained from the indicated treatment groups. H&E staining of primary tumor tissue derived from RenCa cells of BALB / c mice 2 weeks after treatment with T-mfIL12 and / or IL-2 / anti-IL-2 mAb. Decreased cytology and areas of necrosis were observed in the combination therapy group. Immunohistochemistry of tumors treated with combination therapy also showed increased infiltration of CD4+ (blue dots) and CD8+ (blue dots) T lymphocytes.

[0107] [Figure 23] A: Representative view of actual lung. B: In mouse RCC lung metastases, the antitumor effect of T-mfIL12 dose-dependently inhibited tumor proliferation. BALB / c mice received intravenous injection of RenCa cells on day 0. T-mfIL12 was administered via tail vein on days 1, 3, and 5 (n = 10). Animals died on day 14, and the number of lung metastases was counted. The mean number of lung surface nodules in the control group after the simulated treatment was 2.0 × 10⁻⁶. 5 and 1.0×10 6 The number of animals treated with pfu T-mfIL12 was significantly higher (P<0.05 and P<0.01, respectively; t-test; bar±SD). The dosage was 1.0 × 10⁻⁶. 6 Some animals treated with pfu T-mfIL12 did not show surface nodules. *: P<0.05 **: P<0.01. C: Representative image of actual lung. D: Antitumor immunity was enhanced by simultaneous administration of T-mfIL12 and the IL-2 / anti-IL2 mAb complex. Mice were intravenously inoculated with RenCa cells on day 0. On day 1, 2.0 × 10 5Mice with tumors were treated with T-mfIL12 and a 2 μg / 1 mg IL-2 / anti-IL-2 mAb complex as shown. T-mfIL12 was repeated on days 3 and 5. Lung metastasis was evaluated on day 14. Representative images of the lungs 14 days after RenCa cell inoculation are shown (top). Count of RenCa cell nodules in the lungs on day 14 (bottom). The combination of T-mfIL12 and the IL-2 / anti-IL-2 mAb complex was effective in significantly reducing the number of nodules in the lungs of RenCa cells-carrying Balb / c mice, but the number of nodules was slightly reduced when T-mfIL12 or the IL-2 / anti-IL-2 mAb complex was used alone. *: P < 0.05 **: P < 0.01 (t-test, bar ± SD). E: Investigating the effect of combination therapy on survival using a RenCa cell lung metastasis model. 2 × 10 5 PFU-T-mfIL12 or mock was administered to tumor-bearing mice on days 1, 3, and 5. On day 1, 2 μg / 1 mg of the IL-2 / anti-IL-2 mAb complex was injected intraperitoneally. Survival analysis was performed using the Kaplan-Meyer method, and statistical differences were determined using the log-rank test. The median survival of control animals treated with mock and IgG was 18 days, with 100% mortality on day 24. On the other hand, the median survival for combination therapy, T-mfIL12 alone, and the IL-2 / anti-IL-2 mAb complex alone were 49 days, 38 days, and 25 days, respectively (log-rank test, P < 0.001). All treatment groups (complex + mock, IgG + T-mfIL12, complex + T-mfIL12) showed significantly strong antitumor effects (P < 0.05, log-rank test).

[0108] [ Figure 24 A: Effect of intravenous administration of T-mfIL12 on subcutaneous Neuro2a. Intravenous administration of oncolytic HSV-1 showed a significant antitumor effect, which was further enhanced by IL-12 (days 7–14: mock vs T-01, T-01 vs TmfIL12, p<0.001, SNK; bar, SEM). B: Body weight of mice with subcutaneous tumors treated with the virus. No weight loss was observed in any of the three groups due to intravenous administration, but a slight weight loss was observed on day 2 in the T-mfIL12 group (p<0.05, SNK; bar, SD). C: Viral distribution in the Neuro2a subcutaneous tumor model. The amount of viral DNA in the subcutaneous tumor samples was higher than that in the normal samples according to real-time PCR (bar, SEM).

[0109] [ Figure 25A: Intravenous viral therapy for Neuro2a tumors in the brain. The viral vector was administered intravenously to mice on days 5, 7, and 9 after tumor cell inoculation. The results showed significantly prolonged survival in mice treated with T-mfIL12 and mice treated with mock (p<0.05, Breslow-Gehan-Wilcoxon). B: Intravenous viral therapy for Neuro2a metastatic tumors in A / J mice (treatment regimen 1). The viral vector was administered intravenously to mice on days 1, 4, and 7 after intravenous inoculation of tumor cells. Mice treated with T-mfIL12 or T-01 showed significantly prolonged survival compared to mice treated with mock (T-mfIL12 vs T-01: p<0.05, T-mfIL12 vs mock: p<0.001, T-01 vs mock: p<0.05, respectively, Breslow-Gehan-Wilcoxon). C: Intravenous viral therapy for Neuro2a metastatic tumors in A / J mice (treatment regimen 2). The virus was administered intravenously to mice on days 1, 3, and 5 after tumor cell inoculation. In mice treated with T-mfIL12, survival was significantly prolonged compared to mice treated with mock cells (p<0.01, Breslow-Gehan-Wilcoxon), with surprising survivals exceeding 180 days observed in 2 / 10 of T-01-treated mice and 6 / 10 of T-mfIL12-treated mice. D: Intravenous viral therapy for Neuro2a metastatic tumors in nude mice (treatment regimen 2). The virus was administered intravenously to nude mice lacking T cells on days 1, 3, and 5 after tumor cell inoculation. Mice treated with T-01 and T-mfIL12 showed significantly prolonged survival compared to mice treated with mock cells (p<0.05, p<0.005, Breslow-Gehan-Wilcoxon, respectively).

[0110] [ Figure 26A: Serum IL-12 levels. Serum interleukin (IL)-12 was measured by ELISA in an A / J mouse Neuro2a metastatic tumor model after intravenous administration of three doses (n=3): mock, T-01, and T-mfIL12. In mice administered T-mfIL12, IL-12 levels were high only on day 2. Conversely, IL-12 levels were undetectable at all time points (bar, SEM) in mice administered mock and T-01. B: Serum IFNγ levels. Serum IFNγ was measured by ELISA in an A / J mouse Neuro2a metastatic tumor model after intravenous administration of three doses (n=3): mock, T-01, and T-mfIL12. Evaluated IFNγ levels were lower than initial levels from day 2 to day 6. Significant differences were observed in IFNγ expression among the three groups on day 6 (T-mfIL12 vsT-01: p = 0.019, T-01 vs mock: p = 0.030, T-mfIL12 vs mock: p = 0.007, t-test. bar, SD).

[0111] [ Figure 27 Subcutaneous tumor treatment experiment using a bilateral subcutaneous tumor model in DBA / 2 mice. 1.0 × 10⁻⁶ ppm was subcutaneously injected into the left and right sides of the abdomen of DBA / 2 mice. 6 Subcutaneous tumors were created using Clone M3 malignant melanoma cell line from one mouse per cell. Day 0 was defined as when the largest tumor diameter reached approximately 5 mm. On days 0 and 3, 4.0 × 10⁻⁶ T-01 or T-mfIL12 was directly administered. 4 PFU / 20 μL was injected into the tumor in the left flank (mock, n=14; T-01, n=7; T-mfIL12, n=8). The tumor was measured twice weekly, with measurements taken as maximum diameter × minor diameter × thickness (mm). 3 The tumor volume was calculated and evaluated. Error bar, SEM; *, p<0.05; **, p<0.01; ↑, day of viral administration.

[0112] [ Figure 28 Immunohistochemical staining (A, negative control; B, CD4) + Cells; C, CD8 + Cells). Frozen sections were prepared from excised subcutaneous tumors and immunohistochemically stained with rat anti-mouse CD4 antibody (B) and rat anti-mouse CD8 antibody (C) once. A negative control was performed using 2% BSA / PBS (A). Size bar, 1 mm.

[0113] [ Figure 29 This study used a bilateral subcutaneous tumor model in nude mice for treatment. 1.0 × 10⁻⁶ tumor cells were subcutaneously injected into the left and right sides of the nude mice.6 Subcutaneous tumors were created using Clone M3 malignant melanoma cell line from one mouse per cell. Day 0 was defined as when the largest tumor diameter reached approximately 5 mm. On days 0 and 3, 4.0 × 10⁻⁶ T-01 or T-mfIL12 was directly administered. 4 PFU / 20 μL was injected into the tumor in the left flank (mock, n=10; T-01, n=8; T-mfIL12, n=9). The tumor was measured twice weekly, with measurements taken as maximum diameter × minor diameter × thickness (mm). 3 The tumor volume was calculated and evaluated. Error bar, SEM; **, p<0.01; ↑, day of viral administration.

[0114] [ Figure 30 Analysis of splenic lymphocyte production by IFNγ or IL-4 based on the ELISpot method. In the DBA / 2 mouse Clone M3 subcutaneous tumor model, day 0 was defined as when the maximum diameter of the subcutaneous tumor reached 5 mm. On days 0 and 3, T-01 or T-mfIL12 was directly administered at 4 × 10⁻⁶. 4 PFU / 20 μL was injected into the tumor. On day 14, mice were euthanized at n=3 in each group, and lymphocytes from the isolated spleen were used to measure IFNγ and IL-4 producing lymphocytes using the ELISpot assay. Clone M3 cells, a cell line derived from DBA / 2 mice, were used as antigen stimulation. Error bar, SEM; *, p<0.05; **, p<0.01.

[0115] [ Figure 31 Using F9 5.0 × 10⁶ cells as mouse NSGCT cells 5 129 male mice were subcutaneously injected with a 100 μL cell suspension containing 50 μL of DMEM culture medium and 50 μL of BD matrix gel to establish an F9 model. When the tumor diameter reached approximately φ5 mm, T-01 4.0 × 10⁻⁶ cells were administered intratumorally on days 0 and 3. 4 pfu, T-mfIL 124.0×10 4 PFU and mock. Both the T-01 and T-mfIL12 treatment groups showed significant tumor suppression effects compared to the mock group, and the T-mfIL12 treatment group also showed significant tumor suppression compared to the T-01 treatment group (*P<0.01). The antitumor effect of T-01 was further enhanced by IL-12 expression. Detailed Implementation

[0116] This invention relates to recombinant herpes simplex virus (HSV) expressing the IL-12 expression gene.

[0117] (IL-12 expression pattern)

[0118] This invention relates to a recombinant HSV expressing IL-12.

[0119] In one embodiment, the recombinant HSV integrates a gene encoding interleukin-12 (IL-12). The integrated gene can be arbitrarily configured as long as it can express active IL-12 (maintaining the functional tertiary structure). In a preferred embodiment, a gene encoding a fusion polypeptide linking the 35 kDa light chain (p35) and 40 kDa heavy chain (p40) of IL-2 with two or more elastin motifs is integrated.

[0120] In one embodiment, the animal species from which p35 and p40 are derived is not particularly limited, and can be any animal such as human, mouse, or rat. Preferably, when administering the drug to a human, it is preferably derived from a human.

[0121] In one embodiment, the fusion polypeptide is composed of p355, two or more elastin motifs, and p40 sequentially from the N-terminus, or p40, two or more elastin motifs, and p35 sequentially. The number of elastin motifs is not particularly limited, as long as it is sufficient to link the two subunits and express the active form (maintaining the functional tertiary structure) of IL-12; for example, it can be 2, 3, 4, or 5. The number of elastin motifs is preferably 2. The linking portion of the two subunits sometimes contains several other amino acids in addition to the repetition of the elastin motif (typically VPGVG).

[0122] In one embodiment, the gene encoding the fusion polypeptide is any of the following polynucleotides.

[0123] (i) A polynucleotide consisting of the sequence of sequence number 1;

[0124] (ii) A polynucleotide consisting of a sequence having more than 90% sequence identity with the sequence of sequence number 1 and encoding a polypeptide that can form active IL-12.

[0125] (iii) A polynucleotide encoding a polypeptide consisting of the sequence of sequence number 2;

[0126] (iv) A polynucleotide encoding a polypeptide that is composed of a sequence having more than 90% sequence identity with the sequence of sequence number 2 and can form an active IL-12.

[0127] (v) A polynucleotide consisting of the sequence number 3;

[0128] (vi) A polynucleotide consisting of a sequence having a sequence identity of 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more with the sequence of sequence number 3, and encoding a polypeptide capable of forming active IL-12;

[0129] (vii) Encoding a polynucleotide of a polypeptide consisting of the sequence of sequence number 4;

[0130] (viii) A polynucleotide that encodes a polypeptide that is composed of a sequence having a sequence identity of 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more with the sequence of sequence number 4 and is capable of forming an active IL-12.

[0131] Alternatively, in one embodiment, the fusion polypeptide is any of the following polypeptides.

[0132] (ix) A polypeptide encoded by a polynucleotide consisting of the sequence number 1;

[0133] (x) A polypeptide encoding active IL-12 composed of a sequence having more than 90% sequence identity with the sequence of sequence number 1;

[0134] (xi) A polypeptide consisting of the sequence of sequence number 2;

[0135] (xii) A polypeptide consisting of a sequence that has more than 90% sequence identity with the sequence of sequence number 2 and is capable of forming active IL-12;

[0136] (xiii) A polypeptide encoded by a polynucleotide consisting of the sequence number 3;

[0137] (xiv) A polypeptide encoding an active form of IL-12 composed of a sequence having a sequence identity of 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more with the sequence of sequence number 3.

[0138] (xv) A polypeptide consisting of the sequence of sequence number 4;

[0139] (xvi) A polypeptide consisting of a sequence having a sequence identity of 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more with the sequence of sequence number 4 and capable of forming an active IL-12.

[0140] In sequence list and Figure 12 In the examples described below, the base and amino acid sequences of the polypeptide derived from mouse p35 and p40, which is fused with two elastin motifs, are shown.

[0141] In sequence list and Figure 13 In the examples described below, the base and amino acid sequences of the polypeptides derived from human p40 and p353, which are fused with two elastin motifs, are shown.

[0142] A highly sequence-identical amino acid sequence can be represented as an amino acid sequence in which one or more amino acids are missing, substituted, or added to the original sequence. When referring to amino acid sequences with one or more missing, substituted, or added amino acids, the number of missing, substituted, or added amino acids is not particularly limited as long as the polypeptide composed of that amino acid sequence has the target binding ability; for example, it can be set to 1-100, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-5, 1-3, or 1-2. The position of the deletion, substitution, or substitution can be at the end of the peptide or in the middle, and can be one or more locations. In addition to natural amino acids, amino acids can also be their derivatives, artificial amino acids, or non-natural amino acids. In one embodiment, the amino acid is preferably a natural amino acid.

[0143] As a method for inserting a gene encoding a fusion polypeptide into an HSV, known methods can be used. For example, DNA encoding IL-12 can be inserted into an expression vector using known methods (such as using restriction endonucleases), and the expression vector can be transfected into a gene-recombinant HSV. The expression vector may further include a promoter, an origin of replication, a selection marker gene, etc., to regulate the expression of the target gene. The promoter and origin of replication can be appropriately selected based on the type of gene-recombinant HSV and the vector into which the gene is introduced.

[0144] Genetic recombination in viruses with large genome sizes, such as HSV, can utilize systems based on, for example, the T-BAC system described in Fukuhara, H. et al. (2005). Cancer Research, 65, 10663-10668, WO2005 / 103237. For HSV, using the T-BAC system, an HSV capable of expressing the target IL-12 in tumor cells can be easily produced by a method comprising the following steps: First, inserting a BAC plasmid into the HSV genome, the BAC plasmid having a loxP site and an FRT site, and inserting an expression cassette containing at least one marker gene between the loxP site and the FRT site; Second, preparing a shuttle vector containing at least one marker gene, a loxP site, and an FRT site, respectively, and inserting the shuttle vector into the loxP site of the HSV genome using Cre recombinase; and Third, co-infecting the host with the HSV genome and a vector capable of expressing Flp recombinase, excising the region of the genome sandwiched by the FRT site, and generating a recombinant HSV of the target gene.

[0145] (Recombinant HSV)

[0146] In one implementation, the gene encoding the fusion polypeptide is introduced into a recombinant HSV. Recombinant gene refers to gene recombination for tumor treatment, in addition to integrating a gene encoding IL-12.

[0147] In one implementation, the recombinant HSV is not particularly limited as long as it is a virus capable of being used for tumor treatment, possessing the ability to infect tumor cells, selectively replicate within tumor cells, destroy tumor cells during viral replication, infect surrounding tumor cells, and further replicate. Known viruses can be used. Such viruses are mostly mutants of naturally occurring viruses that have been genetically modified to enhance tumor selectivity. Attenuation and the addition of modifications to enhance anti-tumor activity (such as the integration of suicide genes) can also be performed as needed.

[0148] For example, mutants of HSV-1 or HSV-2 can be listed as recombinant HSVs that can be used in one implementation.

[0149] In one embodiment, the recombinant HSV is an HSV-1 mutant. HSV-1 is classified as an enveloped double-stranded DNA virus and possesses the following characteristics advantageous for cancer treatment: 1) It can infect all types of human cells; 2) The viral life cycle and genome sequence are well-defined; 3) The function of most viral genes is known, enabling gene manipulation; 4) The viral genome is large (approximately 152 kb), thus allowing for the insertion of large genes or multiple genes. Furthermore, HSV-1 possesses the following advantages suitable for clinical application: 5) It can kill all cells with a relatively low multiple of infection (MOI); 6) Antiviral drugs that inhibit proliferation are available.

[0150] 7) Anti-HSV-1 antibodies in the blood do not affect the spread of viral infection from cell to cell, so repeated administration is possible; 8) Because mice and monkeys that show sensitivity to HSV-1 exist, preclinical evaluation of safety and efficacy can be carried out in animals; 9) Viral DNA does not enter the host cell genome but exists outside the chromosome.

[0151] The HSV-1 genome consists of 82% long unique regions (U). L The genome consists of two intrinsic sequence regions: a short unique region (Us) and a short unique region (Us) comprising 12% of the genome, and inverted repeat sequences located at their respective ends: terminal repeats (TRs) and inverted repeats (IRs) (Table 1, Todo, T. (2008). A journal and virtual library 13, 2060-2064). The L and S regions can each be independently acquired in two directions, therefore the HSV-1 genomic DNA consists of four isoforms. In this genome, the TR... L RL1, RL2, U L UL1~UL56, TR S RS1, U S In US1 to US12, a total of 84 genes are encoded unidirectionally, about half of which are genes not needed for viral replication. By partially deleting these non-essential genes, pathogenicity can be weakened and introduced (Carson, J. et al. (2010). Drugs of the future 35, 183-195).

[0152] [Table 1]

[0153]

[0154] * Indicates the earliest genes

[0155] When the recombinant HSV is an HSV-1 mutant, it may have any one or more of the following characteristics.

[0156] - Tumor cell-specific replication ability is acquired through the inactivation of enzymes associated with viral DNA synthesis, such as thymidine kinase (TK), ribonucleotide reductase (RR), and uracil-N-glycosylase (UNG or UDG).

[0157] - By deleting the gene γ34.5, which encodes the protein ICP34.5, which is associated with the pathogenicity of HSV-1, tumor cell-specific replication ability is acquired.

[0158] -An anti-tumor effect is achieved by causing α47 deficiency.

[0159] - Deletion or inactivation of genes used to improve safety by preventing reversion to wild type (e.g., endogenous γ34.5 gene, α47 gene (ICP47 gene), α0 gene (ICP0 gene), U L 41 gene (vhs gene), U L (56 gene deletions or inactivations).

[0160] - By expressing immune-stimulating genes (IL-4, IL-10, GM-CSF, IL-12, soluble B7.1, etc.), anti-tumor immunity is enhanced and survival is prolonged.

[0161] - By expressing genes for angiogenesis inhibitors such as platelet factor 4, platelet-reactive protein, endostatin, dominant-negative FGF, and angiostatin, the angiogenesis inhibition and anti-tumor effects are enhanced.

[0162] - Enhanced anti-tumor effects by expressing tumor-invasive local metalloproteinase inhibitors.

[0163] - It promotes viral infection by overexpressing metalloproteinases.

[0164] - By controlling viral genes with tumor- or tissue-specific promoters (promoters of calcium opsonin, E2F-responsive cell cycle-dependent promoter B-myb, nestin promoter, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), MUC-1, Musashi enhancers / promoters, etc.), the ability of tumor cells to replicate viruses is enhanced.

[0165] - Swelling is inhibited by expressing a vascular endothelial growth factor (VEGF) antagonist (Patent Document 2).

[0166] In one embodiment, the recombinant HSV is preferably an HSV-1 having any one or more of the features described in (a) to (c) below, and more preferably an HSV-1 having all of the features described in (a) to (c) below. HSV-1 mutants having these features can be produced with reference to WO2011 / 101912 and Patent Document 2, etc.

[0167] (a) The ICP6 gene (UL39 gene) is deleted or inactivated, or expressed under the control of a tumor-specific promoter or a tissue-specific promoter;

[0168] (b) γ34.5 gene deletion or inactivation;

[0169] (c) α47 gene deletion or inactivation.

[0170] Regarding the characteristics described in (a) and (b) above, by lacking ICP6, an enzyme important for nucleotide metabolism and viral DNA synthesis in non-dividing cells, and / or γ34.5, which functions as a phosphorylated PKR enzyme produced during antiviral infection, this virus cannot proliferate in normal cells but exhibits high division activity, resulting in increased RR activity, and can only proliferate in tumor cells where PKR phosphorylation is inhibited. Furthermore, regarding the characteristic described in (c) above, the protein encoded by the α47 gene inhibits the expression of MHC class I on the host cell surface by suppressing TAP, thereby enabling it to evade host immune surveillance. Therefore, in α47 gene-deficient HSV-1, by maintaining MHC class I expression in host cells, enhanced stimulation of immune cells can be expected. Furthermore, HSV-1 with the characteristics described in (b) and (c) above is missing α47, and the US11 late promoter, which overlaps with the α47 genome, is also missing. Therefore, the expression of the US11 gene is placed under the control of the ICP47 immediate-early promoter, thus the expression period is advanced, and it has the function of restoring the viral replication ability weakened by the absence of γ34.5 only in tumor cells.

[0171] Gene deletion or inactivation refers to suppressing gene expression by deleting all or part of the gene, or by substituting or modifying some bases, or by inserting unwanted sequences. This can be done using known methods.

[0172] Tumor-specific or tissue-specific promoters are promoters that specifically enable the expression of genes under their control in desired tumor cells or tissues. Known promoters can be used depending on the gene. For example, the telomerase reverse transcriptase promoter (hTERT promoter) or the E2F promoter can be used.

[0173] Examples of HSVs containing the deletion or inactivation of the γ34.5 gene, ICP6 gene, and / or α47 gene described in (a) to (c) above include: G207, which has both copies of the γ34.5 gene deleted and the ICP6 gene inactivated; and G47Δ, which contains both copies of the γ34.5 gene deleted, the ICP6 gene inactivated, and the α47 gene deleted. Therefore, a recombinant HSV according to one embodiment can also be produced by further modifying G207 and G47Δ.

[0174] In particular, G47Δ is an oncolytic HSV-1 that improves safety and significantly enhances selective viral replication in tumor cells and induces antitumor immunity. From a therapeutic perspective, it can also be safely administered at high doses to the human brain. In June 2021, a regenerative medicine product (generic name: Teserpaturev, trade name: Delytact Injection) was approved in Japan for the indication of malignant glioma.

[0175] In one embodiment, the gene encoding the fusion polypeptide is inserted into the ICP6 locus of the HVS. That is, it is inserted into a site where the IPC6 gene is deleted or inactivated.

[0176] Inserting a gene encoding a fusion polypeptide at a site where the IPC6 gene is deleted or inactivated refers to, for example, a virus having a sequence with high identity to sequence number 7 in the sequence listing.

[0177] Serial number 7 and Figure 14 The diagram shows the insertion site of the region encoding the fusion polypeptide in the virus produced in the experiments described in the Examples section, along with the base sequences before and after it. Figure 14 In the middle, the dashed line (1..226) indicates that the report contains U L U of the 39 gene promoter L The sequence upstream of the coding region of gene 39, with underscores (227..1545) indicating U. LThe amino acid coding regions of gene 39 are shown in the diagram. Wavy lines (1546..1606) represent sequences generated during gene recombination (Note 1: including the loxP sequence (1564..1566)). Dashed lines (1607..4659) represent the amino acid coding regions of the lacZ gene. The following underlined lines (4660..4719) represent sequences derived from plasmid vectors (Note 2: sequences from the 3' side of lacZ in pcDNA6-E / Uni-lacZ). Dashed lines (4726..4845) also represent sequences derived from plasmid vectors (Note 2: SV40polyA and BGH from pVP22 / myc-His2). The surrounding sequence of polyA), TGA (4799..4801) represents the stop codon of the ICP6-lacZ fusion protein, the double wavy line (4846..5010) represents the SV40 polyA sequence derived from pVP22 / myc-His2, the double dashed line (5011..5235) represents the complementary sequence of BGH polyA derived from pVP22 / myc-His2, and the dashed line (5236..5330) represents the sequence derived from the plasmid vector (SV40 polyA and BGH derived from pVP22 / myc-His2). The surrounding sequence of polyA), the following single-dotted underline (5342..5431) represents the complementary sequence on the 3' side of hIL12 cDNA, the following underline (5432..7042) represents the amino acid coding region of the hIL12 gene (derived from pORF-hIL12G2, the double-lined TTA (5432..5434) represents the complementary sequence of the stop codon, the complementary sequence of p35, the complementary sequence of the hinge region (thick dashed line, 6029..6058), the complementary sequence of p40, the double-lined CAT (7040..7042) represents the complementary sequence of the start codon), and the double-lined (7190..7697) represents the complementary sequence of the CMV promoter. The wavy lines before and after the complement sequence (7057..7189, 7698..7747) indicate sequences derived from the plasmid used (MCS); the double-dotted lines (7774..7799) indicate sequences adjacent to the CMV promoter sequence of pVP22 / myc-His2; the dashed lines (7800..7851) indicate sequences generated during gene recombination (including synthetic oligomer sequences of MCS contained in the pLLZF2→pLLZMF2 production process); the wavy lines (7852..7892) indicate the complementary sequences of the linker sequence and the FRT sequence (7857..7859) used in gene recombination; and the dashed lines indicate the sequence downstream of the 892bp deletion on the 3' side of ICP6. *E. coli* β-galactosidase is expressed as a fusion protein with the N-terminus of ICP6.

[0178] The virus possesses the characteristics described in (b) above, for example, in the sequence sequence of sequence number 8 in the sequence listing, a sequence that causes the deletion of all or part of the region of the γ34.5 gene, or the substitution, modification, or insertion of unwanted sequences, thereby inhibiting the expression of the γ34.5 gene. Additionally, it refers to having an IR corresponding to the region of the γ34.5 gene in sequence number 8. L The expression of the γ34.5 gene is suppressed by deleting all or part of its base sequence, or by substituting or modifying some bases, or by inserting unwanted sequences. Preferably, the expression of both genes is suppressed by deleting all or part of their base sequences, or by substituting or modifying some bases, or by inserting unwanted sequences within the regions of the γ34.5 genes.

[0179] Serial number 8 and Figure 15 The γ34.5 gene and its preceding and following base sequences (TR) of the strain derived from G47Δ are shown. L (within the area). Figure 15 In the diagram, the shaded area from the start codon to the stop codon represents the coding region of the γ34.5 gene. Underlined sections indicate deletion sites in the γ34.5 gene. From the deletion site, the non-coding region on the 3' side of the γ34.5 gene up to the stop codon (double underline) 130 bp downstream is translated into a meaningless amino acid sequence. This sequence in TRL (accession number 487-1233 of GU734771.1) is completely identical to the complementary sequence of a portion of IRL (accession number 124965-125711 of GU734771.1).

[0180] The virus having the aforementioned characteristic (c) means, for example, having a sequence in sequence number 9 of the sequence listing that causes the deletion of all or part of the region of the α47 gene, or the substitution, modification, or insertion of unwanted sequences of some bases, thereby inhibiting the expression of the α47 gene.

[0181] Serial number 9 and Figure 16 The image shows the α47 gene and its preceding and following base sequences from the strain from which G47Δ originates. The α47 gene encodes on the complementary strand. The sequence in sequence number 9 is located at positions 218..484. Figure 16 The shaded area, from the start codon to the stop codon, represents the coding region of the γ34.5 gene. Figure 16 The underlined part indicates a missing site in the basic G47Δ framework. Deletions originating from the translation start site do not generate new ORFs.

[0182] Recombinant HSVs can be produced, for example, by referring to patent documents 1 and 2, non-patent document 1, and international publication WO2005 / 103237.

[0183] (Pharmaceutical Composition)

[0184] In one embodiment, a recombinant HSV having a gene encoding the fusion polypeptide of one embodiment described above and having undergone gene recombination for tumor treatment is an active ingredient in a pharmaceutical composition for treating tumors. When the pharmaceutical composition is administered to a subject, the recombinant HSV specifically proliferates in tumor cells and expresses IL-12 locally in the tumor whenever the virus replicates within the tumor.

[0185] Administration of the pharmaceutical composition according to one embodiment is expected to significantly improve the therapeutic efficacy of viral therapy through the immunomodulatory effects of IL-12, more specifically, through promoting lymphocyte infiltration into the tumor, through inducing IFNγ production, or through both inhibiting tumor proliferation. Furthermore, significant improvements in therapeutic efficacy are also expected for tumors requiring increased immune cell infiltration, or for tumors with low immunogenicity, reduced immunogenicity, or those difficult for the host immune system to recognize (immunologically cold tumors). Moreover, through synergistic effects with immune checkpoint inhibition therapy, significant improvements in therapeutic efficacy are expected for both primary and metastatic tumors.

[0186] The following effects can be further expected from administration of the pharmaceutical composition according to one embodiment.

[0187] Although the production of IFNγ is induced by the expression of IL-12, the activity of the virus as an active ingredient is not inhibited, and its replication ability is not reduced.

[0188] • Through IL-12 expression, it more effectively induces systemic anti-tumor immunity, achieving results even in tumors without direct viral administration. Furthermore, it significantly reduces metastasis, thus showing effectiveness in metastatic lesions. It also enhances cellular immune activity against antigen stimulation.

[0189] In addition, it suggests that T cells are involved in the antitumor immune response induced by the pharmaceutical composition of one embodiment.

[0190] Tumors for which a pharmaceutical composition is expected to be applicable in one embodiment can be listed below, but are not limited to these.

[0191] Brain tumors, gliomas, eye tumors, neuroblastomas, retinoblastomas, primary malignant lymphomas of the central nervous system, meningiomas, pituitary adenomas, schwannomas, craniopharyngiomas, intraocular tumors, retinoblastomas, choroidal malignant melanomas, intraocular malignant lymphomas, tumors of the ocular adnexa, eyelid tumors, lacrimal gland carcinomas, lymphomas of the ocular adnexa, orbital sarcomas, conjunctival tumors, optic nerve tumors, gliomas, oral cancer, tongue cancer, pharyngeal cancer, thyroid cancer, auditory organ cancer, adenoid cystic carcinoma, olfactory neuroblastoma, sarcomas of the head and neck, lung cancer, breast cancer, thymoma, thymic carcinoma, malignant pleural mesothelioma, sarcomas of the trunk, cardiac sarcomas, pulmonary neuroendocrine tumors, SMARCA4 of the chest. Defective tumors, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, colorectal cancer (colon cancer, rectal cancer), small bowel 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 (cholangiocarcinoma (including intrahepatic bile duct cancer), gallbladder cancer, duodenal papillary carcinoma), pancreatic cancer, pancreatic and gastrointestinal neuroendocrine tumors, liver tumors, pancreatic neuroendocrine tumors, renal cell carcinoma, renal pelvis and ureter cancer, bladder cancer, urothelial carcinoma, kidney tumors, prostate cancer, breast cancer, cervical cancer, uterine corpus cancer, (endometrial cancer), uterine sarcoma, ovarian cancer and fallopian tube cancer, vaginal cancer, vulvar cancer, malignant melanoma ( Skin), basal cell carcinoma, acanthoma, cutaneous lymphoma, soft tissue sarcoma (adults), desmoidoma, osteosarcoma (children), Ewing sarcoma (children), soft tissue sarcoma (children), rhabdomyosarcoma (children), classification of sarcoma, undifferentiated / unclassified sarcoma, osteosarcoma, chondrosarcoma, chordoma, Ewing sarcoma, liposarcoma, fibrosarcoma, myxofibrosarcoma, undifferentiated pleomorphic sarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, epithelioid sarcoma, alveolar soft tissue sarcoma, clear cell sarcoma (lightly stained cell sarcoma), extraosseous Ewing sarcoma, acute myeloid leukemia, acute lymphoblastic leukemia / lymphoblastic lymphoma, chronic osteosarcoma. Myeloid leukemia, myelodysplastic syndrome, malignant lymphoma, B-cell lymphoma, T / NK-cell lymphoma, Hodgkin's lymphoma, cutaneous lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, adult T-cell leukemia lymphoma, multiple myeloma, acute promyelocytic leukemia, follicular lymphoma, MALT lymphoma, lymphoid morphological lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, peripheral T-cell lymphoma, Burkitt lymphoma, extranodal NK / T-cell lymphoma, cancer of unknown primary origin, hereditary tumors / familial tumors, neuroendocrine carcinoma, germ cell tumor, paraganglioma, germ cell tumor, testicular cell tumor, ovarian germ cell tumor, extragonadal embryonal cell tumor, neuroendocrine tumor.Examples of tumors for which particularly promising effects can be found include neuroblastoma, prostate cancer, kidney cancer, metastatic kidney cancer, bladder cancer, metastatic bladder cancer, malignant melanoma, and non-seminomatous germ cell tumors. Animal experiments have shown that administration of recombinant HSV expressing the IL-12 gene can exert significant anti-tumor effects on these tumors.

[0192] From another perspective, diseases for which the pharmaceutical composition is effective as an implementation method can be listed as cancers for which IL-12 is recognized or under investigation, or cancers that are the target of immunotherapy. Specific examples of such cancers are listed below, but are not limited to these.

[0193] 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, esophageal cancer, small cell lung cancer, hepatocellular carcinoma, glioblastoma, urothelial carcinoma, ovarian cancer, bladder cancer, prostate cancer, cervical cancer, endometrial cancer, soft tissue sarcoma, primary central nervous system lymphoma, primary testicular lymphoma, pancreatic cancer, biliary tract cancer.

[0194] Unresectable malignant melanoma; unresectable advanced or recurrent non-small cell lung cancer; maintenance therapy after radical chemoradiotherapy for unresectable locally advanced non-small cell lung cancer; unresectable or metastatic renal cell carcinoma; unresectable or metastatic renal cell carcinoma; recurrent or refractory classical Hodgkin lymphoma; head and neck cancer with recurrence or distant metastasis; unresectable advanced or recurrent gastric cancer that has worsened after chemotherapy; unresectable advanced or recurrent malignant pleural mesothelioma that has worsened after chemotherapy; unresectable advanced or recurrent cancer that has worsened after chemotherapy. High-frequency microsatellite instability (MSI-High) colorectal cancer, unresectable advanced or recurrent esophageal cancer that has progressed after chemotherapy, unresectable urothelial carcinoma that has progressed after chemotherapy, advanced or recurrent high-frequency microsatellite instability (MSI-High) solid tumors that have progressed after chemotherapy (limited to cases where standard treatment is difficult), unresectable or metastatic renal cell carcinoma, advanced small cell lung cancer, PD-L1 positive hormone receptor negative and HER2 negative unresectable or recurrent breast cancer, and unresectable Merkel cell carcinoma.

[0195] From another perspective, tumors for which the drug composition is effective as an implementation method can be listed as those for which viral therapy is considered particularly effective, such as: nervous system tumors (e.g., astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma, schwannoma, neurofibrosarcoma, neuroblastoma), pituitary tumors (e.g., pituitary adenoma), medulloblastoma, melanoma, brain tumors, prostate cancer, head and neck cancer, esophageal cancer, renal cell carcinoma, pancreatic cancer, breast cancer, lung cancer, colon cancer, colorectal cancer, gastric cancer, skin cancer, ovarian cancer, bladder cancer, sarcomas (e.g., osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma), squamous cell carcinoma, neuroectodermal tumors, thyroid tumors, lymphoma, hepatocellular carcinoma, mesothelioma, epithelioid carcinoma, benign tumors (e.g., benign thyroid tumors or benign prostatic hyperplasia), etc.

[0196] Regarding the present invention and one embodiment, when referred to as treatment in connection with a disease or condition, the term includes, except as specifically described, prevention, reduction of the risk of disease, treatment, inhibition of progression, and prevention of recurrence.

[0197] The method of administration of the pharmaceutical composition according to one embodiment is not particularly limited. For example, it can be administered intravenously, intra-arterially, intravenously, intraperitoneally, intrathoracically, intraspinally, subcutaneously, intradermally, intramuscularly, or onto mucosal surfaces (e.g., the eyes, nasal cavity, lungs, oral cavity, intestines, rectum, vagina, and urinary tract surfaces). Preferably, it is administered directly to the tumor tissue via injection, endoscopy, or surgery. Local administration prevents viral positivity in tissues outside the localized administration site, and since IL-12 is expressed locally in the tumor, the risk of side effects is reduced compared to systemic administration of IL-12.

[0198] In another preferred embodiment, the pharmaceutical composition is administered intravenously. Even with intravenous administration, the virus can be delivered to the tumor and exert its antitumor effect, with less pronounced progressive toxicity and neurotoxicity expected from intravenous administration. Furthermore, intravenous administration can be expected to provide an antitumor effect relative to systemic metastatic tumors.

[0199] One embodiment of the pharmaceutical composition can be formulated using known formulation methods for administering viruses into a mammal, including a human. For example, it may contain an adjuvant or any carrier, or it may be diluted using only a physiologically permissible solution such as sterile saline or sterile buffered saline without adding an adjuvant or carrier. Frozen formulations, dried formulations, lyophilized formulations, etc., suitable for long-term storage can also be prepared. Additionally, additives permitted as pharmaceuticals may be included.

[0200] One embodiment of the pharmaceutical composition comprises a therapeutically effective amount of one embodiment of the recombinant HSV. A therapeutically effective amount refers to the amount of the substance that can alleviate one or more symptoms of the treated tumor to a certain extent; more specifically, it refers to an amount that exhibits at least one of reducing tumor size, inhibiting (delaying or stopping) tumor metastasis, inhibiting (delaying or stopping) tumor proliferation, and alleviating one or more tumor-related symptoms. Those skilled in the art can appropriately determine the specific dosage based on the severity of symptoms, the age, sex, weight, sensitivity differences of the recipient, method of administration, timing of administration, dosing interval, nature of the formulation, promoter strength, etc.

[0201] One embodiment of the pharmaceutical composition can be administered, for example, by injection, approximately 10... 1 ~ Approximately 10 12 Plaque forming units (pfu), preferably about 10 7 ~ Approximately 10 10 pfu, further optimized to approximately 10 8 PFU ~ Approximately 5 x 10 9 The drug composition may be administered in one or more doses. The frequency of administration of the drug composition in one embodiment may be appropriately set according to the target patient and the target disease. For example, the initial two doses may be administered within two weeks, followed by doses at intervals of three to five weeks.

[0202] The pharmaceutical composition of one embodiment may further contain other active ingredients, or may be used in combination with pharmaceutical compositions containing other active ingredients. Combination use refers to administering multiple active ingredients or pharmaceutical compositions simultaneously, sequentially, or at intervals. Examples of substances that can be combined include BCG (attenuated bovine tuberculosis bacillus) and complexes of IL-2 antibodies and anti-IL-2 antibodies. Such combination therapy can achieve a synergistic anti-tumor effect compared to administering them individually. Furthermore, combination therapy can lead to increased infiltration of CD4+ T cells and CD8+ T cells in the peritumoral region.

[0203] Furthermore, the pharmaceutical composition of one embodiment can be used concurrently with other tumor treatments, such as surgery (tumor resection, etc.), chemotherapy, radiotherapy, immunotherapy, hormone therapy, and combinations thereof. Viral therapy based on the pharmaceutical composition of one embodiment, because it is based on a different mechanism than existing tumor treatments, can enhance tumor treatment efficacy, for example, by combining it with other treatments such as radiotherapy and chemotherapy.

[0204] One embodiment of the pharmaceutical composition can be administered in combination as multiple HSVs expressing different immune checkpoint inhibitory antibodies. Alternatively, it can be administered in combination with a soluble B7-1-expressing HSV-1 for cancer treatment. When administered in combination, it can be mixed or administered sequentially.

[0205] One embodiment also relates to a treatment method for tumors using a virus with added IL-12 expression function. Another embodiment also relates to the use of functionally augmented recombinant HSV in the preparation of a medicament for treating tumors. The method and use can be practiced with reference to the descriptions related to the above-described pharmaceutical compositions.

[0206] In a preferred embodiment, the pharmaceutical composition is used to enhance the effect of IL-12 in the treatment of tumors. Administration of IL-12 as a virally expressed IL-12 can effectively stimulate both local and systemic antitumor immunity by delivering IL-12 as a protein.

[0207] In addition, this application provides a method for enhancing the effect of IL-12 in the treatment of tumors, which includes the step of administering a pharmaceutical composition containing a substance that introduces the IL-12 gene into an oncolytic HSV to a subject in need of it.

[0208] Example

[0209] [Example 1]

[0210] (method)

[0211] cell

[0212] TRAMP-C2 cells were obtained from the American Type Culture Collection (ATCC; Rockville, USA). These cells were cultured in DMEM supplemented with 5% (v / v) fetal bovine serum (FCS), 5% (v / v) Nu-serum IV, and 10 nM dihydrotestosterone. Pr14-2 cells were provided by Dr. Jeffery Green (NCI, Bethesda, USA) and cultured in DMEM supplemented with 10% (v / v) FCS. Vero (African green monkey kidney) cells and Neuro2a cells were obtained from ATCC and maintained in DMEM supplemented with 10% (v / v) FCS and 2 mM glutamine.

[0213] Virus construction

[0214] Previously, recombinant HSV-1 could be constructed using homologous recombination technology. However, due to the large genome size of HSV-1, the selection and observation of the structure of the target recombinant, which has a very low probability, is a time-consuming and labor-intensive process. To minimize such accidental factors, we first constructed the G47Δ-BAC system using a bacterial artificial chromosome (BAC) and two recombinases. Using G47Δ as a backbone, we were able to develop multiple armed oncolytic HSV-1 viruses in parallel, rapidly, and precisely (Non-Patent Literature 31). However, we later found that the replication capacity of the G47Δ-BAC product was lower than that of G47Δ. We believed that the reason might be due to an unknown mutation in the G47Δ-BAC plasmid. Therefore, based on the same concept, we reconstructed the entire system and added improvements such as several additional useful cloning sites. Using this T-BAC system, armed oncolytic HSV-1 derived from G47Δ with replication capacity comparable to G47Δ and containing the desired insertion gene can be produced.

[0215] First, parental T-BAC virus was prepared by homologous recombination with pBAC-ICP6EF (Non-Patent Literature 31), a plasmid containing the G47Δ DNA and the ICP6 coding region. Vero cells were transfected with Lipofectamine (11668027, Thermo Fisher Scientific, USA) according to the manufacturer's instructions, using 0.9 μg of a 1:1:1 mixture of G47Δ DNA purified by the Na / I method, pBAC-ICP6EF (undigested), and pBAC-ICP6EF linearized by Asc I digestion. Recombinant viruses that formed GFP-positive plaques at 30–50% cell killing effect were selected and further passaged in Vero cells. After repeating GFP-positive and lacZ-negative selection three times, several candidates with sufficient replication were screened, and the parental T-BAC virus was selected. This step is important because there are unexpected mutations in the modified virus that reduce viral yield. Circular viral DNA was isolated from infected Vero cells using the Hirt method and electroporated into *E. coli* DH10B (18290015, ThermoFisher Scientific, USA). Antibiotic-resistant colonies were isolated, T-BAC plasmid DNA was purified, and its structure was observed by digestion with endonucleases.

[0216] Second, two plasmids were prepared for filling the mouse IL-12 expression cassette into the shuttle vector (pVec9). The fusion mouse IL-12 gene of T-mfIL12 (Sequence No. 1) was derived from the pORF-mIL-12 plasmid (porf-mil12, InvivoGen, San Diego, USA), which consists of p35 and p40 subunits linked by a bovine elastin motif. The fusion mouse IL-12 (Sequence No. 2) was expressed as a single peptide with a signal sequence in the p35 subunit. The pVec9-fused-mIL12 plasmid was developed using a plasmid containing the mouse IL-12 p35 and p40 genes in a single gene form within the same reading frame. A 1.7kb PCR fragment of pORF-mIL-12 (primers: 5'-GCTAGCCTGAGATCACCGCG-3' (SEQ ID NO: 5), 5'-GCTAGCATCCGTTGCATCCTA-3' (SEQ ID NO: 6)) was digested with Nhe I and inserted into the Avr II site of pVec9 to create pVec9-fused-mIL12. Similarly, using the IRES sequence derived from the 5' untranslated region of encephalomyocarditis virus (EMCV), a single isolated expression cassette was used to construct the pVec9-IRES-mIL12 plasmid. The IRES-type mouse IL-12 gene of T-mIL12-IRES was derived from the plasmid pHCIL12-tk (Non-Patent Literature 32). The constructs were observed by sequencing. A 2.3kb SpeI-StuI fragment containing the pIL-12-p40-IRES-p35 plasmid was inserted into the Avr II-Stu I site of pVec9 to generate pVec9-IRES-mIL12.

[0217] Third, a mixture of T-BAC plasmid (1.5 μg) and pVec9-fused-mIL12 or pVec9-IRES-mIL12 (150 ng each) was incubated in 10 μl of Cre recombinase (MO298, NEB, USA) at 37°C for 30 minutes, followed by electroporation into *E. coli* DH10B. To select for samples containing the mutant BAC plasmid, bacteria were streaked onto LB plates containing Cm (15 μg / ml) and Kan (10 μg / ml) and incubated overnight at 37°C. The DNA structure of the recombinant T-BAC / Vec9 plasmid was observed by gel analysis after endonuclease digestion. Transfection was performed on Vero cells using 2 μg of T-BAC / Vec9 DNA and 0.5 μg of the Flp-expressing plasmid pOG44 (V600520, Thermo Fisher Scientific, USA) with 15 μl of Lipofectamine, following the manufacturer's instructions. Transfected cells were cultured overnight at 37°C in DMEM containing 10% (v / v) FCS. On the second day, the medium was replaced with DMEM containing 1% heat-inactivated fetal bovine serum (IFCS). Culture was continued for several days until plaques appeared. Progeny viruses that were GFP-negative and lacZ-positive after X-gal staining were screened under an inverted fluorescence microscope. Three limiting dilutions were performed to select one clone. The recombinant virus was collected, and the viral DNA structure was observed by digestion with endonucleases. The base sequences of the transgenic cassettes of the recombinant viruses were determined (primers: 5'-CGCAAATGGGCGTAGCGTG-3' (Sequence No. 10), 5'-TAGAGGCACAGTCGAGG-3' (Sequence No. 11), 5'-ACCCGCCCAAGAACTTGCAG-3' (Sequence No. 12), 5'-GGATCGACCCTGCAGGGAAC-3' (Sequence No. 13)). Primers were designed to align the nucleotides between the CMV promoter and polyA. These viruses were titrated on Vero cells using plaque assays.

[0218] Replication analysis and cytotoxicity assays

[0219] To perform replication analysis, Vero cells were packed at 3x10⁻⁶ cells per cell. 5Cells were seeded at a density of 6-well plates. Two wells were infected with each of four clones of G47Δ, T-01, T-mfIL12, or T-mIL12-IRES at an MOI of 0.01. Forty-eight hours after infection, cells were lysed by three cycles of freezing and thawing. Progeny viruses were measured on Vero cells using a plaque assay as described previously (Non-Patent Literature 6). In another experiment, two wells were infected in Vero cells with each of G47Δ, T-01, T-mfIL12, or T-mIL12-IRES at an MOI of 0.01. Progeny viruses were recovered at 0, 6, 24, and 48 hours post-infection and titrated using a plaque assay. Results are expressed as the arithmetic mean of two replicates. Cell killing efficacy assays were performed as described above (Non-Patent Literature 6). Cells were seeded in 6-well plates and cultured overnight at 37°C. Then, the cells were inoculated with virus one hour later. After removing the inoculum, the cells were cultured in DMEM. The number of viable cells was counted daily using a Coulter counter (Z1single, Beckman Coulter, USA) and expressed as a percentage relative to the mock-infected control.

[0220] In vitro IL-12 expression assay

[0221] Vero, Neuro2a, PR14-2, or TRAMP-C2 cells were seeded into 24-well plates (1x10⁻⁶ cells per well). 5 Cells / well were cultured at 37°C for 24 hours. Two wells of cells were then infected with each virus at MOI=1 and cultured at 39.5°C for 48 hours. All viruses used in this study were derived from HSV-1 strain F and were therefore temperature-sensitive, not replicating at 39.5°C (Non-Patent Literature 5). The supernatant was recovered, and the concentration of IL-12 was determined using a mouse IL12p70 immunoassay (M1270, R&D Systems Inc., IL) at a detection limit of 7.8 pg / ml. Results are expressed as the arithmetic mean of two replicates. To detect the p35 and p40 subunits separately, mouse IL12A (IL12p35) ELISA kits (SEA059Mu, Cloud-Clone, TX) and mouse IL12B (IL12p40) ELISA kits (SEA058Mu, Cloud-Clone, TX) were used. The expression ratio of p35 subunit to p40 subunit was adjusted according to the control group (recombinant mouse IL-12, 095-05331, Wako, Japan).

[0222] Interferon-γ release analysis

[0223] The supernatant from Vero cells infected with T-mfIL12 or T-mIL12-IRES at MOI=1 was recovered 48 hours post-infection. Diluted using VP-SFM medium (11681020, ThermoFisher Scientific). Spleens from A / J mice were collected, and cell suspensions were prepared. The supernatant or recombinant mouse IL-12 (rIL12, 095-05331, Wako, Japan) was applied to spleen cells for 48 hours. Interferon-γ (IFNγ) levels were measured twice using a mouse IFNγ Uncoated ELISA kit (88-7314, ThermoFisher Scientific).

[0224] Animal experiments.

[0225] Six-week-old male C57BL / 6 mice, female A / J mice, and female NOG (NOD / shi-scid.IL-2RγKO) mice were purchased from CLEA Japan (Tokyo, Japan). All animals were placed in cages of no more than five. Subcutaneous tumor therapy was performed as described previously (Non-Patent Literature 31). Subcutaneous tumors were generated by transplanting 5 x 10T TRAMP-C2 prostate cancer cells into the left ventral region of male C57BL / 6 mice. 6 Neuro2a neuroblastoma cells (5 x 10⁻⁶) were transplanted into the two flanks of female A / J mice or NOG mice. 6 The tumor was prepared by administering 20 μl of mock, viral suspension, or recombinant mouse IL-12p70 (#577004, BioLegend; rIL-12) to the left tumor 5–7 days after transplantation, when the tumor reached a diameter of approximately 5 mm. The administration was repeated after 3 or 4 days. Tumor growth was determined by measuring tumor volume (length x width x height x 0.52) twice per week. Mice were euthanized when the tumor reached a maximum diameter of 22 mm.

[0226] Immunohistochemistry.

[0227] Bilateral Neuro2a subcutaneous tumors were induced in A / J mice. On days 0 and 3, T-mfIL12, T-mIL12-IRES, and T-01 (2x10⁻¹²) were injected into only the left tumor. 5Tumors were harvested on day 6 using either PFU or mock tumors (n=3 in each group). Tumors were fixed overnight in 20% formaldehyde, embedded in paraffin, and sectioned at 4 μm. Sections were immunostained with anti-CD8 antibody (98941, CST, Massachusetts, USA), anti-CD4 antibody (25229, CST, Massachusetts, USA), or anti-HSV-1 antibody (ab9533, abcam, Cambridge, UK), and developed with diaminobenzidine.

[0228] IL-12 and IFNγ levels in the body

[0229] In the unilateral subcutaneous Neuro2a model, T-01, T-mfIL12, and T-mIL12-IRES (2x10) were used. 6 PFU or mock ELISA was administered into tumors established on day 0. Serum and tumor samples were collected from 3 mice in each group on days 1, 3, and 6. Mouse IL12 and mouse IFNγ levels were measured in duplicate using a mouse IL12p70 immunoassay (M1270, R&D Systems Inc., IL) and a mouse IFNγ uncoated ELISA (88-7314, ThermoFisher Scientific). Additionally, for the T-mfIL12 and T-mIL12-IRES groups, tumor samples were collected on day 0 after viral injection (n=4). Matrix IL-12 concentrations in mock and viral suspensions were measured by ELISA: IL-12 was not detected in mock and T-01, and 20 μl (2 x 10⁻⁶) of T-mfIL12 and T-mIL12-IRES were... 6 The amounts of IL-12 in the pfu were calculated to be 157.3 pg and 5.2 pg, respectively.

[0230] Enzyme-linked immunospot (ELISpot) analysis

[0231] In a unilateral subcutaneous Neuro2a model, T-01, T-mfIL12, and T-mIL12-IRES (5x10) were injected intratumorally on days 0 and 3. 4 (PFU) or mock. On day 6, spleens were aseptically collected from 3 mice in each group to prepare cell suspensions. Spleen cells were co-cultured with Neuro2a cells or Sal / N cells derived from A / J mice (negative control) for use with mouse interferon γ-ELISpot. PLUS (3321-4HPW-2, Mabtech AB, Sweden) and mouse interleukin-4 (mIL-4) ELISpot PLUS ELISpot analysis of (3311-4HPW-2, Mabtech AB, Sweden). The analysis was repeated 3 times.

[0232] Statistics and repeatability.

[0233] Data comparisons between treatment groups were performed using Student's t-test, Tukey's univariate ANOVA with multiple comparisons, or Bonfroni's bivariate ANOVA with multiple comparisons. Survival assays were performed using Kaplan-Meier analysis. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using JMP Proversion 11.0.0 (SAS Institute, USA). Experiments were repeated twice for replication analysis, cytotoxicity studies, and in vitro expression analysis. Tumor samples were collected from three mice in each group to determine in vivo IL-12 and IFNγ levels. Nine to twelve mice were used per group in in vivo animal experiments. For IFNγ and IL-4 ELISpot analysis, three mice were treated per group, and the analysis was repeated three times.

[0234] (result)

[0235] Construction of T-mfIL12 and T-mIL12-IRES

[0236] We previously constructed an oncolytic HSV-1 with a G47Δ backbone, armed with mouse interleukin-18 and soluble mouse B7-1 using a bacterial artificial chromosome (BAC) and two recombinase systems (Cre / loxP and Flp / FRT) (Non-Patent Literature 29). This system was then modified and reconstructed (T-BAC system), improving replication capacity and enabling the production of armed oncolytic HSV-1 derived from G47Δ with the desired genes. Using the T-BAC system and two mouse IL-12 expression cassettes, two IL-12-expressing oncolytic HSV-1 variants, T-mfIL12 and T-mIL12-IRES, were produced. Each virus, in addition to an 894bp deletion of the ICP6 gene containing the expression cassette, also exhibited a double-copy deletion of the γ34.5 gene and a deletion of the α47 gene. Figure 1The lacZ gene was configured after the ICP6 promoter, and the expression cassette controlled by the CMV promoter was configured in reverse. The length from the ATG to the stop codon of the mouse IL-12 gene in T-mfIL12 is 1620 bp. The upstream p35 gene is linked to the downstream p40 gene, thus expressing mouse IL-12 as a fusion protein. The mouse IL-12 gene in T-mIL12-IRES contains a 1008 bp p40 subunit immediately preceding the IRES sequence, and a 648 bp p35 subunit inside; both subunits are expressed simultaneously but separately. The T-01 virus, used as a control, was constructed using a shuttle vector with an empty cassette. Parallel construction of these three viruses revealed that over 99% of the viral plaques formed after recombination were positive for lacZ expression. The four clones of each recombinant HSV-1 were isolated by three limiting dilutions, and the constructs were observed by restriction endonuclease digestion. Figure 7 It was confirmed that in the transgenic cassettes of T-mfIL12 and T-mIL12-IRES, the sequences of the p35 and p40 subunits of T-mfIL12 are identical to those of T-mIL12-IRES. Figure 12 ).

[0237] In vitro characteristics of T-mfIL12 and T-mIL12-IRES

[0238] To evaluate the replication capacity of two viruses expressing IL-12, the replication capacity of Vero cells (3x10⁻¹²) was measured 48 hours after infection with an MOI of 0.01. 5 Number of progeny viruses recovered in cells / wells Figure 2 a) The arithmetic mean of viral yields (±SD) obtained from the four clones of T-mfIL12 and T-mIL12-IRES was 6.8 x 10⁻⁶. 6 (±0.59x10 6 ) and 7.0x10 6 (±0.94x10 6 )pfu (p=0.736, t-test). These yields were compared with those of T-01 (7.6 x 10⁻⁶) used as a control virus. 6 The pfu was the same, but G47Δ, as the parent virus, showed a higher yield (2.0 x 10^6 pfu). 7 The expression level of mouse IL-12 was evaluated by measuring the concentration in the supernatant using ELISA 48 hours after Vero cells were infected with MOI 1. Figure 2b). This ELISA specifically detects the functional morphology (p70) of mouse IL-12. The arithmetic mean (±SD) of IL-12 concentrations in the four clones of T-mfIL12 and the four clones of T-mIL12-IRES were 10.0±0.18 and 1.5±0.39 ng / mL, respectively (p<0.001, t-test), with T-mfIL12 expressing the p70 morphology of IL-12 approximately 7-fold higher than T-mIL12-IRES. The initial clones identified by each recombinant G47Δ were used for further analysis. The corrected expression ratios of the p35 and p40 subunits were 1.8:1 and 10:1 in T-mfIL12 and T-mIL12-IRES, respectively. The viral yield of T-mfIL12 in Vero cells changed over time (0 h, 6 h, 24 h, and 48 h) similarly to that of T-mIL12-IRES over time. Figure 2 c). Both T-mfIL12 and T-mIL12-IRES expressed IL-12 stimulated spleen cells to secrete IFNγ, and biological activity was observed. Figure 8 ).

[0239] The in vitro cytotoxic effects of T-mfIL12 and T-mIL12-IRES were evaluated using the mouse neuroblastoma cell line Neuro2a and the mouse prostate cancer cell lines Pr14-2 and TRAMP-C2. In all three mouse cancer cell lines, both T-mfIL12 and T-mIL12-IRES showed cytotoxic effects comparable to the control virus T-01 at both MOI 0.1 and MOI 1. Figure 9 The expression of mouse IL-12 based on T-mfIL12 and T-mIL12-IRES was further evaluated in three cell lines. With the MOI set to 1, the concentration in the supernatant was determined by ELISA 48 hours after infection. Figure 2 d). In all three cell lines, T-mfIL12 showed higher expression of mouse IL-12 than T-mIL12-IRES.

[0240] The efficacy of T-mfIL12 and T-mIL12-IRES in vivo

[0241] The efficacy of two IL-12-expressing viruses in vivo was evaluated using two immunodeficient mouse tumor models, TRAMP-C2 tumor in syngeneic C57BL / 6 mice, and Neuro2a tumor in syngeneic A / J mice. Figure 4 a and 4b Figure 9 When the established subcutaneous TRAMP-C2 tumors reached approximately 5 mm in diameter, T-01, T-mfIL12, and T-mIL12-IRES (5 x 10) were injected intratumorally twice on days 0 and 3.6 PFU or mock. C57BL / 6 mice and TRAMP-C2 cells derived from C57BL / 6 showed comparative resistance to HSV-1 infection (Non-Patent Literature 33). Dosage (5 x 10) 6 The pfu was determined based on our prior research (non-patent literature 9). In this TRAMP-C2 model, T-mfIL12 treatment showed the highest efficacy, with tumors significantly smaller on day 25 compared to T-01 and T-mIL12-IRES treatments (p = 0.003 and p = 0.029, respectively, relative to T-01 and T-mIL12-IRES, t-test). Figure 4 a). Between groups, all three viruses were significantly more effective than the mock virus (p < 0.001 for all three viruses, ANOVA), but only T-mfIL12 was significantly more effective than T-01 (p = 0.027, ANOVA). Figure 4 a) There was no significant difference between T-mIL12-IRES and T-01.

[0242] Neuro2a cells with low immunogenicity were transplanted into the bilateral flanks of A / J mice. At the time point when the established Neuro2a subcutaneous tumors reached approximately 5 mm in diameter, T-01, T-mfIL12, and T-mIL12-IRES (5 x 10⁻⁶ cells / mL) were injected twice into the left tumor on days 0 and 3. 4 PFU or mock. A / J mice and Neuro2a cells derived from A / J mice are sensitive to HSV-1 infection (Non-Patent Literature 33). Dosage (5 x 10⁻⁶) 4 The pfu was determined based on experience in the model (Non-Patent Literature 20). T-mfIL12 and T-mIL12-IRES were both on the treatment side on day 11 (T-mfIL12 and T-mIL12-IRES were p<0.001 and p=0.016, respectively, t test) and on the non-treatment side on day 11 (T-mfIL12 and T-mIL12-IRES were p<0.001 and p=0.018, respectively, t test); Figure 4 (b) Significantly inhibited tumor proliferation compared to T-01. Between groups, all three viruses were significantly more effective than mock on the treatment side (p = 0.001 in T-01, p < 0.001 in both T-mfIL12 and T-mIL12-IRES, ANOVA). On the non-treatment side, T-mfIL12 and T-mIL12-IRES were more effective than mock (p < 0.001 in both viruses, ANOVA). Figure 4B). Furthermore, both T-mfIL12 and T-mIL12-IRES showed significantly higher efficacy than T-01 on the treatment side (T-mfIL12 and T-mIL12-IRES, p<0.001 and p=0.006, respectively, ANOVA), and similarly on the non-treatment side (T-mfIL12 and T-mIL12-IRES, p<0.001 and p=0.011, respectively, ANOVA). Moreover, on day 13, T-mfIL12 caused significant tumor proliferation inhibition compared to T-mIL12-IRES on both the treatment and non-treatment sides (p=0.043 on the treatment side and p=0.040 on the non-treatment side, t-test, ... Figure 4 b). Between groups, T-mfIL12 was more effective than T-mIL12-IRES on the non-treatment side (p = 0.018, ANOVA); Figure 4 b). Data related to tumor growth in each animal are documented in Figure 9 .

[0243] To observe the enhanced efficacy of T-mfIL12 and T-mIL12-IRES compared to T-01, based on the immune effect of virally expressed IL-12, the efficacy was further evaluated in NOG mice that excluded not only NK activity but also the effects of adaptive immunity. Figure 4 c). At the time point when the bilateral Neuro2a subcutaneous tumors reached approximately 5 mm in diameter, T-01, T-mfIL12, and T-mIL12-IRES (1x10) were inoculated into the left tumor only on days 0 and 3. 6 PFU or mock. On the treatment side, all three viruses showed significant efficacy compared to mock (p<0.001 vs mock for all viruses, binary ANOVA). However, regarding the effect on IL-12 expression, T-mfIL12 and T-mIL12-IRES were completely absent in NOG mice, and there was no difference in efficacy among the three viruses T-01, T-mfIL12, and T-mIL12-IRES. None of the three viruses were effective in the contralateral untreated tumor (PFU). Figure 4 c).

[0244] The efficacy was then evaluated using the survival rate of A / J mice carrying bilateral Neuro2a subcutaneous tumors. When the established Neuro2a subcutaneous tumors reached approximately 5 mm in diameter, T-01, T-mfIL12, T-mIL12-IRES, or mock were injected into the left tumor on days 0 and 3. Mice were euthanized when one of the tumors reached a diameter of 22 mm. Two doses (5 x 10⁻⁶) were used in different experiments. 5 PFU and 5x10 4PFU. Using 5x10 5 In the case of PFU, T-mfIL12 and T-mIL12-IRES significantly prolonged the survival of tumor-bearing mice compared with T-01 (p = 0.023 and p = 0.002 for T-mfIL12 and T-mIL12-IRES, respectively, Log-Rank test). Figure 4 d). However, only the T-mfIL12 group showed survival at day 60 (2 / 11). Using 5x10 4 In the case of PFU, both T-mfIL12 and T-mIL12-IRES significantly prolonged the survival of cancer-bearing mice compared with T-01 (for T-mfIL12 and T-mIL12-IRES, p = 0.015 and p = 0.046, respectively, Log-Rank test). Figure 4 e. Supplementary Data 4). One to two mice treated with T-mfIL12 showed long-term survival at both doses, but there was no significant difference between T-mfIL12 and T-mIL12-IRES (5 x 10⁻⁶). 5 pfu[ Figure 4 In d], p = 0.974, 5 x 10 4 pfu[ Figure 4 [e] p = 0.265 (Log-Rank test). In summary, the experimental results show that T-mfIL12 exhibits higher in vivo efficacy than T-mIL12-IRES.

[0245] Furthermore, immunohistochemical evaluation was performed to determine whether IL-12-expressing viruses promote lymphocyte infiltration into tumors. Bilateral Neuro2a subcutaneous tumors were created in A / J mice, and on days 0 and 3, T-mfIL12, T-mIL12-IRES, and T-01 (2x10⁻¹²) were injected into only the left tumor. 5 PFU (Plasma Activated Fumes) or mock tumor samples were collected on day 6. Within the tumor, CD4+ was observed on both the treated and untreated sides. + and CD8 + Increased lymphocyte infiltration was more pronounced in T-mfIL12 and T-mIL12-IRES compared to T-01. Figure 5 a). On the treated side, HSV-1 positive cells were observed within the tumor with all viruses, but on the untreated side, none of the viruses induced HSV-1 positivity. Figure 5 a).

[0246] In vivo levels of IL-12 and IFNγ induced by T-mfIL12 and T-mIL12-IRES

[0247] To evaluate the in vivo gene expression and function of T-mfIL12 and T-mIL12-IRES, in vivo IL-12 and IFNγ levels were measured in a unilateral Neuro2a subcutaneous tumor model. T-01, T-mfIL12, and T-mIL12-IRES (2 x 10⁻⁶ cells / mL) were seeded into the established tumor cells. 6 PFU or mock tests were performed, and serum and tumor samples were collected on days 1, 3, and 6 (n=3 for each group). Within the tumor, IL-12 levels peaked on day 1, with T-mfIL12 and T-mIL12-IRES measured at 2590±1450 pg / ml and 228±115 pg / ml (mean ± SD), respectively. Both IL-12 levels gradually decreased before day 3 and day 6. Figure 5 (b) On day 0 after viral injection, the intratumoral IL-12 level in T-mfIL12 was 7.7 ± 9.9 pg / ml, while it was undetectable in T-mIL12-IRES. This indicates that the IL-12 detected after day 1 was due to virus-induced IL-12 expression. The intratumoral IL-12 level in T-mfIL12 was significantly higher than that in T-mIL12-IRES at all time points (p = 0.018, p = 0.016, and p = 0.046 on days 1, 3, and 6, respectively, univariate ANOVA). The serum IL-12 level was significantly lower than the intratumoral level (approximately 2 log lower). Correlation with intratumoral IL-12, the serum IL-12 level in T-mfIL12 was higher than that in T-mIL12-IRES on day 1 (p = 0.047, univariate ANOVA). IFNγ was detected in the tumor from day 1 for all three viruses and continued to increase on day 3. Figure 5 b). IFNγ was detected in serum only in viruses expressing IL-12, peaking on day 1. The IFNγ level of T-mfIL12 was significantly higher in both tumors and serum on day 1 than that of T-mIL12-IRES (p = 0.014 in tumors and p = 0.027 in serum, univariate ANOVA).

[0248] Specific antitumor immune responses based on T-mfIL12 and T-mIL12-IRES.

[0249] To evaluate the specific antitumor immune response induced by T-mfIL12 and T-mIL12-IRES, an ELISpot analysis was performed in a unilateral Neuro2a subcutaneous tumor model. Within the established tumor, T-01, T-mfIL12, and T-mIL12-IRES (5 x 10⁻⁶ cells / mL) were inoculated on days 0 and 3. 4PFU or mock, spleen was removed on day 6. ELISpot analysis showed that spleen cells from T-mfIL12-treated mice secreted significantly more IFNγ stimulated by Neuro2a cells compared to spleen cells from T-01- and T-mIL12-IRES-treated mice (p = 0.005 and p = 0.004, respectively vs. T-01 and T-mIL12-IRES, univariate ANOVA). Figure 5 c). No such response was observed in SaI / N cells derived from the A / J mouse system as a control cell. Figure 10 No significant differences were observed in the number of IL-4-secreting spleen cells among the three viral treatment groups. Figure 5 c. Figure 11 ).

[0250] Comparison of in vivo efficacy of T-mfIL12 and intratumoral recombinant IL-12 administration

[0251] To evaluate the advantages of expressing IL-12 in the oncolytic HSV-1 form, the efficacy of T-mfIL12 was compared with direct intratumoral injection of recombinant mouse IL-12 (rIL-12) in A / J mice with bilateral Neuro2a subcutaneous tumors. Figure 6 A report describes how injecting 500 ng of rIL-12 into the tumor of immunodeficient mice with subcutaneous tumors of low immunogenicity caused tumor growth inhibition (Non-Patent Literature 34). According to... Figure 5 Based on the above experiment, it was calculated that injecting 2x10 into the tumor... 6 The highest intratumoral IL-12 level obtained by pfu T-mfIL12 was 45.7 ng / tumor (30.1 ± 9.6, mean ± SEM). Furthermore, 5x10 4 When T-mfIL12 of pfu was injected intratumorally into Neuro2a subcutaneous tumors, the highest intratumoral IL-12 level was measured at 0.668 ng / tumor (0.238 ± 0.147, n = 4). Therefore, three different doses of rIL-12, 500 ng, 50 ng, and 1 ng, were tested. When the established tumor diameter reached approximately 5 mm, rIL-12 and T-01 (5 x 10) were injected intratumorally into only the left tumor on days 0 and 4. 4 pfu), T-01 (5x10) 4 pfu)+rIL-12, T-mfIL12(5x10) 4PFU or mock (n=10 per group). At a dose of 500 ng of rIL-12, on the treatment side, rIL-12, T-01, T-01+rIL-12 and T-mfIL12 were all significantly more potent than mock (all p<0.001 vs mock, binary ANOVA); Figure 6 a). Furthermore, on the non-treatment side, while rIL-12 alone did not show a significant antitumor effect compared to mock, T-01+rIL-12 and T-mfIL12 showed significantly higher effects compared to mock (p = 0.039 and p < 0.001 vs mock, respectively, BIA). Moreover, on the non-treatment side, T-mfIL12 showed a significantly higher effect than rIL-12 alone (p = 0.003, BIA), while T-01+rIL-12 showed no significant difference compared to rIL-12 alone. The same results were obtained when rIL-12 was used at a dose of 50 ng. Figure 6 (b) When the dose of 1 ng that showed IL-12 levels in tumors treated with T-mfIL12 was used in this experiment to administer rIL-12, rIL-12 alone did not show significant effects in either the treated or untreated sides. T-mfIL12 was significantly more effective in the treated side compared to rIL-12 alone (p<0.001, binary dispersion analysis). Figure 6 c) There was no significant difference between T-01+rIL-12 and rIL-12 alone. These results suggest that virally expressed IL-12 is more effective than recombinant IL-12 in acting on both local and systemic anti-tumor immunity.

[0252] (Inspection)

[0253] In this study, we compared the efficacy of two armed oncolytic HSV-1 molecules expressed using different methods, while controlling the same mouse IL-12 molecule with the same ultra-early CMV promoter. T-mfIL12 expressed mouse IL-12 as a single active fusion peptide linking the p35 and p40 subunits with a bovine elastin motif. T-mIL12-IRES co-expressed both the p35 and p40 subunits, with the IRES sequence inserted between the two subunit genes. We found that fusion expression significantly and efficiently produced IL-12 in various cell lines compared to co-expression of both subunits, while the expression method had no effect on the replication capacity or cytotoxicity of armed oncolytic HSV-1. Compared to the control virus T-01, both T-mfIL12 and T-mIL12-IRES showed significantly higher efficacy. However, T-mfIL12 reflected a higher level of active IL-12 expression than T-mIL12-IRES, demonstrating significantly higher efficacy in two different syngeneic mouse tumor models in vivo. In NOG mice, the enhanced efficacy disappeared, indicating that the virally expressed IL-12 functioned in an immune-mediated manner. In the low-immunogenic Neuro2a tumor model of HSV-1-sensitive A / J mice, IFNγ assay, ELISpot analysis, and immunohistochemistry showed that the amount of IL-12 in tumors treated with T-mfIL12 was significantly higher than that in tumors treated with T-mIL12-IRES, leading to an enhanced anti-tumor immune response. This tumor model is considered one of the most suitable models for evaluating the function of the immunomodulatory payload of oncolytic HSV-1, and therefore it was used in this study.

[0254] In addition to direct cell killing, oncolytic HSV-1 also induces tumor-specific immune responses (Non-Patent Literature 35-37). One method to improve the effectiveness of oncolytic HSV-1 is to arm the virus with immunostimulatory molecules. In some studies, different immunostimulatory delivery genes have been loaded onto the same oncolytic HSV-1 backbone for evaluation. In mice with subcutaneous squamous cell carcinoma, oncolytic HSV-1 expressing mouse IL-12 (NV1042) showed a higher efficacy than oncolytic HSV-1 expressing mouse GM-CSF (NV1034) (Non-Patent Literature 19). Mice administered NV1042 had a higher proportion of rejection of re-attacked tumor cells compared to mice administered NV1034 (Non-Patent Literature 19). In two mouse prostate cancer models, NV1042 showed a higher efficacy than control HSV-1 (Non-Patent Literature 38), but NV1034 did not show a significant enhancement. We used the same HSV-1 backbone lacking the γ34.5 and ICP6 genes to create three oncolytic HSV-1 strains armed with mouse soluble B7-1, mouse IL-12, or mouse IL-18 (referred to as vHsv-B7-1-Ig, vHsv-IL-12, and vHsv-IL-18, respectively) (Non-Patent Literature 20). In A / J mice with Neuro2a subcutaneous tumors, the combination of vHsv-B7-1-Ig, vHsv-IL-12, and vHsv-IL-18 showed higher efficacy than either the virus alone or in combination of two viruses, but vHsv-IL-12 alone was the most effective of the three strains. These studies suggest that IL-12 is currently a good choice as a transgene when arming oncolytic HSV-1 with a single immunostimulatory insert (transgene). Systemic administration of IL-12 has been shown to cause severe toxicity, including death, in clinical trials (Non-Patent Literature 39, 40). Therefore, from a safety perspective, local expression of IL-12 using oncolytic HSV-1 as a distribution tool is also a suitable method (Non-Patent Literature 41). Recently, several clinical trials of IL-12 gene therapy using adenovirus vectors have been conducted (Non-Patent Literature 42, 43).

[0255] In addition to the above, IL-12, as a transgene for arming other oncolytic HSV-1s, has been tested not only in preclinical but also for clinical purposes. G47Δ-mIL12, like T-mfIL12, contains the mouse IL-12 gene in the G47Δ backbone and was fabricated using our previous G47Δ-BAC system (Non-Patent Literature 21, 31). In an immunodeficient mouse glioblastoma stem cell model, G47Δ-mIL12 attacked mouse glioblastoma cells, increased IFNγ secretion, inhibited angiogenesis, and reduced the number of regulatory T cells within the tumor (Non-Patent Literature 21). The antitumor effect based on the combination of G47Δ-mIL12 with anti-PD-1 and anti-CTLA-4 antibodies is CD4-dependent. + and CD8 + T cells and macrophages (Non-Patent Literature 44, 45) (Non-Patent Literature 46). Although temozolomide therapy antagonizes G47Δ-mIL12, the combination of G47Δ-mIL12 with a systemic VEGFR tyrosine kinase inhibitor (Non-Patent Literature 47) or angiostatin-expressing G47Δ (Non-Patent Literature 48) improved efficacy in a mouse glioblastoma model. Furthermore, the first-generation γ34.5-deficient oncolytic HSV-1, M002, armed with mouse IL-12, showed higher efficacy than the parent virus and G207 in preclinical brain tumor models (Non-Patent Literature 22, 49). Subsequently, for the purpose of conducting clinical trials in patients with high-grade gliomas, M032, expressing human but not mouse IL-12, was constructed using the same framework and strategy as M002 (Non-Patent Literature 50). Intracerebral administration of M032 (maximum 10) was observed. 8 Safety of pfu in trials using non-human primates (Non-Patent Literature 51). M032 is being used in a Phase I clinical trial in patients with recurrent malignant gliomas (NCT02062827). Methods for inserting the human IL-12 gene into the HSV-1 backbone lacking ICP47 and ICP34.5 (Δ47 / Δ34.5 / IL12) and methods for acting mouse IL-12 (R-115) on fully toxic and HER2-redirected oncolytic HSV-1 have also been reported (Non-Patent Literature 53, 54).

[0256] NV1042 and G47Δ-mIL12 use the mouse IL-12 gene expressed as a single fusion protein (Non-Patent Literature 19, 21), while M002, M032, Δ47 / Δ34.5 / IL12, and R-115 use mouse or human IL-12 genes co-expressed as dual proteins via IRES-separated p35 and p40 subunit genes and regulated by the Early Growth Response-1 promoter or CMV promoter. Because IL-12 is species-specific, after obtaining preclinical data using oncolytic HSV-1 armed with mouse IL-12, a new oncolytic HSV-1 armed with human IL-12 needs to be constructed to initiate clinical trials. Since clinical development requires time, manpower, and funding, it is difficult to halt or change course once initiated. Therefore, it is essential to comprehensively evaluate whether the oncolytic virus entering clinical development is optimal in all aspects, including safety, efficacy, preparation, stability, clinical usability, regulatory compliance, and environmental impact. In this study, as one aspect, the optimal method for expressing IL-12 as a payload of oncolytic HSV-1 was evaluated. The results showed that expressing the IL-12 gene as a single fusion peptide was significantly superior to co-expressing both subunits. The results of this study demonstrate the successful production of a G47Δ-derived oncolytic HSV-1 (T-hIL12) expressing human IL-12 in a functional fusion peptide form using the T-BAC system, a modified version of the G47Δ-BAC system (Non-Patent Document 31) capable of rapidly and accurately inserting the desired gene into the G47Δ-deleted ICP6 locus. In the T-hfIL12-expressed fusion human IL-12 (base sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4), the p40 and p35 subunits are linked by an elastin motif. T-hIL12 has completed a comprehensive characterization evaluation, including the function of expressed human IL-12, and is currently being used in a physician-led Phase I / II clinical trial in Japan for patients with advanced malignant melanoma (jRCT2033190086).

[0257] (Other cited references)

[0258] Non-Patent Document 31: Fukuhara, H., Ino, Y., Kuroda, T., Martuza, R. L. & Todo, T. Triplegene-deleted oncolytic herpes simplex virus vector double-armed with interleukin 18 and soluble B7-1 constructed by bacterial artificial chromosome-mediated system. Cancer Res. 65, 10663-10668 (2005).

[0259] Non-Patent Document 32: Toda, M., Martuza, R. L., Kojima, H. & Rabkin, S. D. In situ cancer vaccination: an IL-12 defective vector / replication-competent herpes simplex virus combination induces local and systemic antitumor activity. J. Immunol. 160, 4457-4464 (1998).

[0260] Non-Patent Document 33: Lopez, C. Genetics of natural resistance to herpesvirus infections in mice. Nature 258, 152-153 (1975).

[0261] Non-Patent Document 34: Caminschi, I., et al. Interleukin-12 induces an effective antitumor response in malignant mesothelioma. Am. J. Respir. Cell Mol. Biol. 19, 738-746 (1998).

[0262] Non-Patent Document 35: Toda, M., Rabkin, S.D., Kojima, H. & Martuza, R.L. Herpes simplex virus as an in situ cancer vaccine for the induction of specific anti-tumor immunity. Hum. Gene Ther. 10, 385-393 (1999).

[0263] Non-Patent Document 36: Liu, B.L., et al. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Ther. 10, 292-303 (2003).

[0264] Non-Patent Document 37: Thomas, D.L. & Fraser, N.W. HSV-1 therapy of primary tumors reduces the number of metastases in an immune-competent model of metastatic breast cancer. Mol. Ther. 8, 543-551 (2003).

[0265] Non-Patent Document 38: Varghese, S., et al. Enhanced therapeutic efficacy of IL-12, but not GM-CSF, expressing oncolytic herpes simplex virus for transgenic mouse derived prostate cancers. Cancer Gene Ther. 13, 253-265 (2006).

[0266] Non-Patent Document 39: Atkins, M.B., et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin. Cancer Res. 3, 409-417 (1997).

[0267] Non-Patent Document 40: Cohen, J. IL-12 deaths: explanation and a puzzle. Science. 270, 908 (1995).

[0268] Non-Patent Document 41: Alatrash, G., et al. Clinical and immunologic effects of subcutaneously administered interleukin-12 and interferon alfa-2b: phase I trial of patients with metastatic renal cell carcinoma or malignant melanoma. J. Clin. Oncol. 22, 2891-2900 (2004).

[0269] Non-Patent Document 42: Chiocca, E. A., et al. Regulatable interleukin-12 gene therapy in patients with recurrent high-grade glioma: Results of a phase 1 trial. Sci. Transl. Med. 11(505), eaaw5680 (2019).

[0270] Non-Patent Document 43: Chiocca, E. A., et al. Combined immunotherapy with controlled interleukin-12 gene therapy and immune checkpoint blockade in recurrent glioblastoma: An open-label, multi-institutional phase I trial. Neuro. Oncol. 24, 951-963 (2022).

[0271] Non-patent literature 44: Saha, D., Martuza, RL & Rabkin, SD Macrophage polarization contributes to glioblastoma eradication by combination immunovirotherapy and immune checkpoint blockade. Cancer Cell. 32, 253-267.e5 (2017).

[0272] Non-patent literature 45: Saha, D., Martuza, RL & Rabkin, SDCuring glioblastoma: oncolytic HSV-IL12 and checkpoint blockade. Oncoscience. 4, 67-69 (2017).

[0273] Non-patent literature 46: Saha, D., Martuza, RL & Rabkin, SDTemozolomide antagonizesoncolytic immunovirotherapy in glioblastoma. J. Immunother. Cancer. 8, e000345 (2020).

[0274] Non-patent literature 47: Saha, D., et al. Combinatorial effects of VEGFR kinaseinhibitor axitinib and oncolytic virotherapy in mouse and human glioblastomastem-like cell models. Clin. Cancer Res. 24, 3409-3422 (2018).

[0275] Non-patent literature 48: Zhang, W., et al. Combination of oncolytic herpes simplexviruses armed with angiostatin and IL-12enhances antitumor efficacy in human glioblastoma models. Neoplasia. 15, 591-599 (2013).

[0276] Non-Patent Document 49: Markert, J.M., et al. Preclinical evaluation of a genetically engineered herpes simplex virus expressing interleukin-12. J. Viol. 86, 5304-5313 (2012).

[0277] Non-Patent Document 50: Patel, D.M., et al. Design of a Phase I Clinical Trial to Evaluate M032, a Genetically Engineered HSV-1 Expressing IL-12, in Patients with Recurrent / Progressive Glioblastoma Multiforme, Anaplastic Astrocytoma, or Gliosarcoma. Hum. Gene Ther. Clin. Dev. 27 69-78 (2016).

[0278] [Non-Patent Document 51] Roth, J.C., et al. Evaluation of the safety and biodistribution of M032, an attenuated herpes simplex virus type 1 expressing hIL-12, after intracerebral administration to aotus nonhuman primates. Hum. Gene Ther. Clin. Dev. 25 16-27 (2014).

[0279] Non-Patent Document 52: Haghighi-Najafabadi, N., et al. Oncolytic herpes simplex virus type-1 expressing IL-12 efficiently replicates and kills human colorectal cancer cells. Microb. Pathog. 160, 105164 (2021).

[0280] Non-patent literature 53: Menotti, L., et al. HSV as A Platform for the Generation of Retargeted, Armed, and Reporter-Expressing Oncolytic Viruses. Viruses 10, 352 (2018).

[0281] Non-patent literature 54: Lioni, V., et al.Afully-virulent retargeted oncolytic HSVarmed with IL-12elicits local immunity and vaccine therapy towards distanttumors.PLos Pathog.14,e1007209(2018).

[0282] [Example 2: In vivo viral replication experiment]

[0283] To investigate whether T-mfIL12 with an inserted exogenous gene can replicate in vivo with comparable capacity to T-01, T-01 or T-mfIL12 was administered to unilateral subcutaneous tumors of Neuro2a in A / J mice, and the viral titer in the tumors was measured on the day of administration and 1, 4, 7, and 11 days after administration.

[0284] In vivo, T-01 and T-mfIL12 showed almost equivalent replication capacity within subcutaneous tumors of Neuro2a. Figure 17 Regardless of the virus, with day 0 (T-01: 2.94 × 10⁻⁶) 4 PFU / tumor SEM = 5.80 × 10⁻⁶ 3 T-mfIL12: 1.14×10 5 PFU / tumor SEM = 2.17 × 10⁻⁶ 4 Compared to (p = 0.0194), on day 1 (T-01: 1.18 × 10⁻⁶), the difference was 1.18 × 10⁻⁶. 6 PFU / tumor SEM = 5.38 × 10⁻⁶ 5 T-mfIL12: 3.22×10 5 PFU / tumor SEM = 1.63 × 10⁻⁶ 5 At p = 0.2020, a slight increase in titer was observed, but it gradually decreased until day 4 (T-01: 1.83 × 10⁻⁶). 4 PFU / tumor SEM = 1.77 × 10⁻⁶ 4 T-mfIL12: 2.30×10 4PFU / tumor SEM = 1.17 × 10⁻⁶ 4 p = 0.8331), Day 7 (T-01: 7.51 × 10 3 PFU / tumor SEM = 5.20 × 10⁻⁶ 3 T-mfIL12: 1.26×10 4 PFU / tumor SEM = 1.14 × 10⁻⁶ 4 (p = 0.7062), Day 11 (T-01: 0 pfu / tumor SEM = 0.00, T-mfIL12: 1.69 × 10⁻⁶) 2 PFU / tumor SEM = 1.69 × 10⁻⁶ 2 (p=0.3739) Although there were concerns that IFNγ mediated by IL-12 expression might inhibit the activity of the virus itself and reduce its replication ability, no such situation was observed.

[0285] [Example 3: Kidney Cancer]

[0286] 1. In vitro cell-killing and antitumor effects of T-mfIL12 virus

[0287] Previous studies have shown that the T-01 virus exhibits anti-tumor effects against renal cell carcinoma even in vivo. Therefore, this study compared the T-mfIL12 virus (mIL-12-expressing type), created to enhance anti-tumor effects by activating T and NK cells in vivo, with the T-01 virus to investigate whether this enhances the anti-tumor effect. First, the cytotoxic effects of both viruses were compared in vitro using RenCa cells. Results showed that in studies infected with any virus at an MOI of 1.0, almost all cells died on day 2, indicating comparable cytotoxic effects in vitro. Figure 18 A). Next, in the RenCa subcutaneous tumor model (mouse renal cancer cells RenCa 1.0×10⁻⁶), 5 50 μl / mouse was subcutaneously administered to both feet of BALB / c mice, with day 0 defined as the day the maximum tumor diameter reached 5 mm or more. Intratumoral administration was 2 x 10-1 5 The antitumor effects of PFU-based T-01 virus or T-mfIL12 virus were compared. The mean tumor volume on day 9 in the mock administration group was 992 mmHg. 3 The average tumor volume in the T-01 virus or T-mfIL12 virus administration group on day 9 was 609 mm. 3 and 342mm 3In the T-mfIL12 virus administration group, a significant difference in tumor volume was observed compared to the mock administration group. Furthermore, in the mock administration group, mice died on day 11 due to tumor-derived hemorrhage and consumption, but no deaths were observed in the T-01 virus or T-mfIL12 virus administration groups until day 15. On day 13, the average tumor volume in both the T-01 virus and T-mfIL12 virus administration groups was 974 mmHg. 3 and 445mm 3 In a subcutaneous tumor model, T-mfIL12 virus significantly inhibited tumor growth compared to T-01 virus. Figure 18 B, C).

[0288] 2. Antitumor effects of T-mfIL12 virus in distant tumors

[0289] In HSV-1-based cancer viral therapy, in addition to the direct cell-killing effect of the virus, the induction of systemic anti-tumor immunity is said to be related to efficacy. Therefore, this study investigated whether the effect was also obtained in cancer lesions where the virus was not directly administered. Furthermore, it was investigated whether T-mfIL12 virus, which is believed to more effectively utilize anti-tumor immunity by expressing IL-12, showed enhanced anti-tumor efficacy in distal tumors where the virus was not directly administered, compared to T-01 virus. In the Neuro2a subcutaneous tumor model, subcutaneous tumors were created bilaterally in mice, with only the left tumor receiving a 5x10 [virus / treatment]. 5 Intratumoral administration of PFU-T-01 virus or T-mfIL12 virus. In this Neuro2a bilateral subcutaneous tumor model, for the tumor on the treatment side where the virus was directly administered, the mean tumor volume on day 15 in the mock, T-01 virus, and T-mfIL12 virus administration groups was 2838 mmHg. 3 526mm 3 30mm 3 The results of the significance test showed that both the T-01 virus and T-mfIL12 virus administration groups significantly inhibited tumor growth compared to the mock group. In the T-mfIL12 virus administration group, tumor growth was also significantly inhibited compared to the T-01 virus administration group. Figure 18 (D-ac). The average tumor volume on day 15 of the contralateral tumor without direct viral administration was 4037 mmHg in the mock, T-01 virus, and T-mfIL12 virus administration groups. 3 2443mm 3 1377mm 3Tumor enlargement was reduced in both the T-01 and T-mfIL12 virus administration groups, particularly in the T-mfIL12 virus administration group, where a significant difference was observed compared to the mock administration group. Figure 18 D-bc).

[0290] 3. Antitumor effect of T-mfIL12 virus in RenCa lung metastasis model

[0291] Next, in the RenCa lung metastasis model (RenCa cells 5.0 x 10⁻⁶), 5 This study compared the antitumor effects of intravenous administration of 5 x 10⁻⁶ cells / 200 μl / mfIL12 virus to BALB / c mice (day 0 was defined as the day of intravenous administration). 5 Or 5x10 6 The number of lung metastases was evaluated using PFU-T-01 or T-mfIL12 virus (n = 10⁻¹² / group). Survival was also evaluated separately using the same experimental protocol. Virus dosage was 5 x 10⁻¹². 5 Under PFU administration, the average number of lung metastases on day 14 was 205, 42, and 1.9 in the mock, T-01 virus, and T-mfIL12 virus administration groups, respectively. Figure 19-1 (AB). In the T-01 virus and T-mfIL12 virus administration groups, the number of metastases was significantly reduced compared to the mock group. The number of metastases was also significantly reduced in the T-mfIL12 virus group compared to the T-01 virus group. Furthermore, in the survival observation experiment, at a virus dosage of 5 x 10⁻⁶, 5 Under PFU conditions, the mean survival days in the mock, T-01 virus, and T-mfIL12 virus administration groups were 31 days, 39 days, and 46 days, respectively. The results of the significance test showed that T-01 virus and T-mfIL12 virus significantly prolonged the survival time of mice compared to the mock group, and T-mfIL12 virus also significantly prolonged the survival time of mice compared to T-01 virus. Figure 19-1 C). Next, the viral load was 5 x 10⁻⁶. 6 Under PFU conditions (the actual dosage of T-01 virus or T-mfIL12 virus is 3.4 x 10⁻⁶), the dosage is 10⁻⁶. 6 and 2.7x10 6The number of lung metastases on day 14 was 326, 7.1, and 0.4 in the mock, T-01, and T-mfIL12 virus administration groups, respectively. In the T-01 and T-mfIL12 virus administration groups, the number of metastases was significantly reduced compared to the mock group (p = 0.0045 and p = 0.0024), and further reduced compared to T-01 virus (p = 0.0023). In the survival observation experiment, the average survival days in the mock, T-01, and T-mfIL12 virus administration groups were 24, 42, and 55 days, respectively. T-01 virus and T-mfIL12 virus significantly prolonged the survival of mice compared to the mock group, and T-mfIL12 virus significantly prolonged the survival of mice compared to T-01 virus (p = 0.0023). Figure 19-2 AB).

[0292] 4. Antitumor effect of T-mfIL12 virus in mice with established lung metastases in RenCa

[0293] Furthermore, to investigate the therapeutic effect of intravenous administration of the virus in mice with established lung metastases, the survival time of mice was observed by delaying the start of treatment. In clinical practice, lung metastases are observed via X-ray examination, but it is considered difficult to create a model with a completely identical progression. Therefore, to determine the treatment start date, preliminary experiments were conducted, starting at least from the point when the tumor is visually observed. RenCa 2.0x10 was administered intravenously on day 0. 5 200 μl / mouse. Lungs of mice were harvested on days 3, 5, 7, and 9 for ink staining to investigate whether metastatic lesions could be visually observed on the lung surface (n = 3 / day). In this preliminary experiment, no lung metastatic lesions were visually observed on the lung surface of mice harvested on days 3, 5, and 7 in any individual. On the other hand, 16, 54, and 20 lung metastatic lesions were observed on the lung surface of mice harvested on day 9, respectively. Therefore, in the study of the treatment effect under established lung metastasis, the treatment start date was set as day 9, with intravenous administration of 5 x 10 μl. 6 The survival of patients with PFU-treated T-01 or T-mfIL12 viruses was evaluated. With treatment initiation delayed to day 9, the mean survival days in the mock, T-01, and T-mfIL12 virus groups were 24 days, 26 days, and 28 days, respectively, with only T-mfIL12 virus significantly prolonging survival. Figure 19-2 C).

[0294] 5. Evaluation of cytokine secretion activity in mouse spleen cells after intravenous administration of T-mfIL12 virus using the ELISpot method

[0295] To evaluate the in vivo immunomodulatory activity following intravenous administration of T-01 or T-mfIL12 virus in the RenCa lung metastasis model, cytokine secretion activity in the spleen was assessed on day 14. In the RenCa lung metastasis model, similar to the standard treatment model, 5 x 10⁵ cells / day were administered intravenously on days 1, 3, and 5. 6 PFU-containing T-01 virus or T-mfIL12 virus was used to euthanize mice on day 14, and spleens were aseptically removed. The secretory activity of interferon-γ (IFNγ) and interleukin-4 (IL-4) in splenocytes was evaluated using enzyme-linked immunospot (ELISpot) assay. Spleens were homogenized in DMEM (DMEM-5) containing 5% heat-inactivated FBS and passed twice through a 70 μm filter. After centrifugation at 300 RCF and 4°C for 5 minutes, the supernatant was removed, and 5 ml / spleen of ACK lysis buffer (Lonza, MD, USA) was added. The cells were incubated at room temperature for 10 minutes, centrifuged at 300 RCF and 4°C for 5 minutes, and the supernatant was removed. The cells were washed twice with HL-1 solution containing 2 mM L-glutamine and 1% penicillin / streptomycin (P0781, Sigma-Aldrich, MO, USA), and the cell count was determined. Cells were prepared in 2x10⁻⁶ cells / spleen. 6 / ml. Add 100μl (2x10⁶ ml) to a 96-well plate. 5 (Number of cells) of spleen cells were cultured at 37°C for 24 hours with 100 μl of active stimulation solution. For each group of 3 mice, mouse IFNγ (mIFNγ) was measured independently three times using the ELISpot kit (BD ELISPOT Mouse IFNγELISPT Kit, BD Biosciences, CA, USA), and mouse IL-4 (mIL-4) was measured using the corresponding antibody (BD ELISPOT Mouse IL-4ELISPOT Set, BD Biosciences, CA, USA). IL-4 was measured after 48 hours of culture. For antigen stimulation, RPMI 1640 with 0.05 mg / ml mitomycin C (Sigma-Aldrich, MO, USA) was added, and the cells were cultured at 37°C for 30 minutes, washed three times with DMEM-5, and diluted with HL-1 to 5 x 10⁻⁶. 6The substance was obtained at / ml. As a positive control, concanavalin A (WakoPure Chemical Industries, Osaka) was used at 2 μg / ml; as a negative control, only culture medium was used. Under RenCa antigen stimulation, the number of spots showing IFNγ secretion was observed to be 187, 211, and 234 per well in the mock, T-01 virus, and T-mfIL12 virus administration groups, respectively. Enhanced IFNγ secretion was observed in the T-01 virus and T-mfIL12 virus administration groups, with a significant enhancement observed in the T-mfIL12 virus group. A significant enhancement in secretion was also observed in the T-mfIL12 virus administration group compared to the T-01 virus administration group. Regarding IL-4 secretion activity, no significant difference was observed in any group. Figure 20 A). In summary, this suggests enhanced tumor-specific cellular immunity against RenCa cells stimulated by antigens in the T-mfIL12 virus administration group.

[0296] 6. Evaluation of cytokine levels in mouse serum and lung tissue after intravenous administration of T-mfIL12 virus using ELISA

[0297] In the RenCa lung metastasis model, to evaluate cytokine levels in the organism after intravenous administration of T-01 or T-mfIL12 virus, a single intravenous administration of the virus was performed, and cytokine levels in serum and lung tissue were measured over time. In the RenCa lung metastasis model, a single administration of 5 x 10⁵ viruses was administered on day 1. 6Mice were given PFU-treated T-01 virus or T-mfIL12 virus. mIL-12 and mIFNγ levels in serum and lung tissue were measured on days 2, 4, and 6. For serum, collected blood was centrifuged at 2000 RCF for 20 minutes, and the supernatant was collected for evaluation. For lung tissue, the weight of the excised lung tissue was measured, and the tissue was homogenized in EP tubes containing PBS for 3 minutes using a homogenizer, followed by 1 minute of sonication, and then centrifuged at 2000 RCF for 20 minutes. The supernatant was collected for evaluation. For each group of 3 mice, mIL-12 ELISA kits (Quantikine Mouse IL-12p70 Immunoassay R&D, MN, USA) and mIFNγ ELISA kits (Endogen Mouse IFNγ ELISA Kit, Pierce Biotechnology, IL, USA) were independently measured twice. Regarding IL-12, the highest values ​​were observed in both serum and lung tissue on day 2 in the T-mfIL12 virus administration group. On day 2, serum IL-12 levels in the T-mfIL12 virus administration group were approximately 2.0-fold and 1.5-fold higher than those in the mock and T-01 virus administration groups, respectively, showing a significant difference. On day 4, a significant difference was observed only between the T-mfIL12 and mock groups, with a value approximately 1.3-fold higher. Figure 13 Ba). In lung tissue, in the T-mfIL12 virus administration group, IL-12 was detected above the sensitivity level only on days 2 and 4. Figure 20 Bb). Regarding IFNγ, serum IFNγ levels peaked on day 2 in the T-mfIL12 virus administration group and were not measured thereafter. In the other two groups, IFNγ was not detected at any measurement site. Figure 20 Bc). IFNγ levels in lung tissue were highest on day 2 in all groups, with no significant differences observed between groups on days 2 and 4. On day 6, the T-mfIL12 viral load was approximately 1.2 times higher than the mock group, a significant difference was observed. Figure 20 Bd). In lung tissue, IFNγ levels are thought to increase with IL-12 levels, suggesting that these cytokines may also be involved in enhancing the anticancer effects of T-mfIL12 virus in vivo.

[0298] [Example 4-1: Bladder Cancer]

[0299] 1. T-mfIL12 prolongs the survival of mice with isotopic bladder cancer.

[0300] In an isotopic bladder cancer model (mouse bladder cancer cells MB49 5.0x10), 5The ability of T-mfIL12 to control tumor cell proliferation in vivo was investigated in female C57BL / 6 mice (intrabladder inoculation). Kaplan-Mayer survival curves were plotted, and the survival time distribution of treated animals was compared using the log-rank test. Almost all control mice in the simulated treatment died within 24 days after tumor cell inoculation. All deceased mice had grossly visible bladder cancer. In contrast, mice treated with medium and high doses (1.0 x 10⁻⁶) were also included in the control group. 6 and 5.0x10 6 In the group containing T-mfIL12 (pfu), 50% of the mice survived for more than 38 days. Figure 21 A, P = 0.02 and P < 0.01 (log-rank test). However, there was no effect in the low-dose group (P = 0.625 log-rank test). There was no statistically significant difference between the medium-dose and high-dose groups (P = 0.261 log-rank test).

[0301] 2. T-mfIL12 prolonged the survival of mice with isotopic bladder cancer after BCG treatment.

[0302] Furthermore, using the same animal model and treatment regimen, the effects of BCG (1.35 mg) and T-mfIL12 (1.0 x 10⁻⁶) on BCG (1.35 mg) and T-mfIL12 (1.0 x 10⁻⁶) were investigated. 6 The question was whether the combination of PBS (polysaccharide sulfadiazine) and T-mfIL12 was more effective than the single administration of BCG or T-mfIL12. In a bladder cancer model, mice were intravesically inoculated with mock, or administered BCG and T-mfIL12, either single or in combination, on days 1, 4, and 7. All treatment groups (BCG+mock, PBS+T-mfIL12, BCG+T-mfIL12) showed significantly strong antitumor effects (P<0.05 log-rank test). However, according to Bonfroni correction, the BCG+mock group showed no antitumor effect. While no antitumor effect was observed in the BCG+Mock group, an antitumor effect was observed in the BCG+mock group. In the combination administration groups, approximately 30% of the mice survived for 50 days. Figure 21 B. P < 0.01 (log-rank test).

[0303] 3. T-01 or T-mfIL12 prolongs the survival of mice with lung metastases from bladder cancer.

[0304] Inhibiting the proliferation of metastatic tumors is a major challenge in cancer treatment. Therefore, we investigated whether T-01 and T-mfIL12 could inhibit the established lung metastasis model of bladder cancer. Animals treated with T-01 (P = 0.04 log-rank test) or T-mfIL12 (P < 0.01 log-rank test) showed significantly prolonged survival rates compared to the simulated treatment animals. Figure 21 C). The median survival times were 50 days, 39 days, and 26 days, respectively. Furthermore, compared to animals treated with T-01, animals treated with T-mfIL12 had a longer survival time (P < 0.05 log-rank test).

[0305] [Example 4-2: Simultaneous administration of renal cell carcinoma, IL-2 / anti-IL-2 mAb]

[0306] 4. When T-mfIL12 is injected into a tumor and IL-2 / anti-IL-2 mAb complex is administered simultaneously, an effective anti-tumor immune response can be obtained.

[0307] The efficacy of the IL-2 / anti-IL-2 mAb complex was tested in a mouse model of renal cell carcinoma. The IL-2 / anti-IL-2 mAb complex was prepared by incubating 2 μg of mouse IL-2 and 1 mg of anti-IL-2 mAb (S4B6-1) at room temperature for 10 minutes. Tumor proliferation was inhibited by the IL-2 / anti-IL-2 mAb complex compared to anti-IL2 mAb (P<0.05, days 18–23) or control treatment (P<0.01, days 12–23). Figure 22 A). Using the same model, the efficacy of combination therapy with T-mfIL12 and the IL-2 / anti-IL-2mAb complex was investigated. Treatment with T-mfIL12 alone or the IL-2 / anti-IL-2mAb complex alone was superior to mock+IgG, but the combination therapy of T-mfIL12 with the IL-2 / anti-IL-2mAb complex showed a significant improvement in tumor control compared to either T-mfIL12 alone or the IL-2 / anti-IL-2mAb complex alone. Figure 22 B). In subcutaneous tumors that underwent simultaneous intratumoral injection of T-mfIL12 and intraperitoneal injection of IL-2 / anti-IL-2 mAb complex, CD4+ extensively infiltrated. + and CD8 + T lymphocytes. The combination therapy group showed decreased cellularity and areas of necrosis. Immunohistochemistry of the tumors treated with combination therapy also showed increased infiltration of CD4+ (blue dots) and CD8+ (blue dots) T lymphocytes. Figure 22 C).

[0308] 5. Combination therapy with T-mfIL12 and IL-2 / anti-IL-2mAb reduced tumor metastasis.

[0309] In a mouse RenCa lung metastasis model, the study investigated whether T-mfIL12 prevented tumor development. The results showed that the mean number of lung surface nodules in the mock-treated controls was 2.0 × 10⁻⁶. 5 Or 1.0×10 6The average number of animals treated with pfu T-mfIL12 was significantly higher (Figures 23A and 23B, P<0.05 and P<0.01, respectively). Partial 1.0×10 6 No surface nodules were observed in animals treated with pfu T-mfIL12.

[0310] Concomitant administration of T-mfIL12 and the IL-2 / anti-IL-2mAb complex resulted in additive or synergistic effects. Therefore, we investigated whether this combination therapy produced an additive or synergistic effect in halting tumor development in a mouse RenCa lung metastasis model. The results showed that the combination of T-mfIL12 and the IL-2 / anti-IL-2mAb complex was effective, significantly reducing the number of RenCa cells in the lungs of BALB / c mice, while T-mfIL12 or the IL-2 / anti-IL-2mAb complex alone only slightly reduced the number of nodules (Fig. 23C; 23D). These results clearly demonstrate that treatment including concomitant administration of T-mfIL12 and the IL-2 / anti-IL-2mAb complex has a potent protective effect in a lung RenCa cell tumor burden model.

[0311] Furthermore, survival experiments were conducted in mice with the RenCa cell model. Survival rates were significantly improved in the combination-treated animals. The median survival time of control animals treated with mock and IgG was 18 days, with 100% dying on day 24. In contrast, the median survival time for combination therapy, T-mfIL12 alone, or IL-2 / anti-IL-2 mAb complex alone were 49 days, 38 days, and 25 days, respectively (log-rank test, P<0.001) (Figure 23E). Additionally, analysis of liver and kidney tissue specimens showed no histological damage in any of the treatment groups.

[0312] [Example 5: Intravenous administration]

[0313] 1. Intravenous administration of HSV-1 inhibited the proliferation of Neuro2a subcutaneous tumors.

[0314] To evaluate the therapeutic effect of intravenous administration of oncolytic viruses, the antitumor effects of T-01 and T-mfIL12 were tested in A / J mice with Neuro2a subcutaneous tumors. When the subcutaneous tumor diameter reached 5 mm (4 days after tumor cell inoculation), mice were administered a simulated infection extract, T-01, or T-mfIL12 (5 × 10⁻⁶) via tail vein injection on days 0, 2, and 4. 6 pfu). If T-01 (Aver. 4000mm) is administered intravenously on day 14. 3 ), then with mock(6000mm 3Compared to ), tumor proliferation was significantly inhibited (p<0.001, SNK). Furthermore, if T-mfIL12 (Aver. 2100mm) was administered intravenously, tumor proliferation was significantly inhibited (p<0.001, SNK). 3 Compared with T-01, the antitumor effect was enhanced (p<0.001, SNK). Antitumor effects were observed from day 7 onwards, with p-values ​​less than 0.001 for all three groups. These results indicate that intravenous administration of oncolytic HSV-1 has a significant antitumor effect, which is further enhanced by IL-12. Figure 24 A). Mouse body weight was measured three times a week. Except for a slight, transient decrease in body weight in the T-mfIL12 group compared to the mock group on day 2 (p<0.05), intravenous treatment did not cause significant weight loss. Figure 24 B). No significant systemic toxicity or neurotoxicity, or other clinical side effects, believed to be caused by intravenous administration of the virus, were observed.

[0315] 2. HSV-1 is delivered to subcutaneous tumors via intravenous administration.

[0316] To investigate viral distribution after tail vein injection, viral DNA was quantified in subcutaneous tumors and normal tissues. Mice were administered the drug three times on days 0, 2, and 4. Mice died on day 5, and tumors, livers, spleens, lungs, kidneys, and brains (n=5) were collected. Viral load was quantified by real-time PCR. The results are presented below. Figure 24 C. The viral DNA levels detected in the lungs, liver, spleen, kidneys, and brain were 307 pfu / mg, 111 pfu / mg, 297 pfu / mg, 9 pfu / mg, and 7 pfu / mg, respectively. The average viral DNA level detected in subcutaneous tumors was 1140 pfu / mg, significantly higher than in normal tissues. This indicates that oncolytic viruses can be delivered to subcutaneous tumors via intravenous administration.

[0317] 3. T-mfIL12 prolonged the survival of mice with intracranial tumors when administered intravenously.

[0318] To investigate whether intravenous administration of oncolytic virus (ICV) induced a therapeutic effect on brain tumors, A / J mice with Neuro2a tumors in their brains were administered ICV via tail vein injection on days 5, 7, and 9. Figure 25A). As an animal model of intracranial tumors, A / J mice and Neuro2a (neuroblastoma) cells were used. No significant differences were observed in the T-01 administration group (mean 18 days) and the T-mfIL12 administration group (mean 19 days), but in mice administered T-mfIL12 intravenously (mean 14 days, p<0.05, Breslow-Gehan-Wilcoxon), survival was significantly prolonged compared with mice administered the mock (mean 14 days, p<0.05) (p>0.05).

[0319] 4. Intravenous viral therapy has shown antitumor effects against systemic metastatic tumors.

[0320] The study investigated the antitumor effect of intravenous administration on Neuro2a metastatic tumors. Specifically, the virus was administered via tail vein injection on days 1, 4, and 7. Figure 25 B). The median survival of control animals treated with mock was 27.1 days, with 100% dying on day 33. In contrast, mice treated with T-01 had prolonged survival compared to the mock group (p<0.05, Breslow-Gehan-Wilcoxon). Significantly prolonged survival was observed in the T-mfIL12 treatment groups compared to both the T-01 and mock groups (p<0.05 and p<0.001, respectively, in the Breslow-Gehan-Wilcoxon groups).

[0321] Mice were treated with the same dose on days 1, 3, and 5 (treatment regimen 2). Figure 25 As shown in Figure C, the median survival times for mock, T-01, and T-mfIL12 were 35.9 days, 69.8 days, and 123.9 days, respectively. In mice treated with T-mfIL12, a significant increase in mean survival was observed compared to mice treated with mock (p<0.01, Breslow-Gehan-Wilcoxon). No significant difference was observed in mice treated with T-01 compared to the mock or T-mfIL12 groups (p>0.05, Breslow-Gehan-Wilcoxon). Nevertheless, 20% (2 / 10) of the T-01-treated mice and 60% (6 / 10) of the T-mfIL12-treated mice exhibited surprising survival times exceeding 180 days. These long-term survivals, all in a completely disease-free state, demonstrate the effectiveness of intravenous viral therapy in these animals against potential tumor seeding.

[0322] In our study, post-mortem examination of mice that died from Neuro2a metastatic tumors revealed metastases in various organs, including the lungs, liver, kidneys, ovaries, lymph nodes, intestines, and muscles. The number and distribution of metastases varied considerably among animals. Based on these results, we believe that survival time is the optimal benchmark for evaluating treatment efficacy in this experiment.

[0323] 5. T cells play an important role in the anti-tumor immune response induced by T-mfIL12.

[0324] To investigate the involvement of T cells in the T-mfIL12-induced antitumor immune response, athymic mice lacking T lymphocytes were used in a treatment experiment. Athymic mice were treated by intravenous inoculation with Neuro2a cells followed by tail vein injection on days 1, 3, and 5. Figure 25 As shown in Figure D, there was no difference in survival between mice treated with T-01 and those treated with T-mfIL12 (p>0.05, Breslow-Gehan-Wilcoxon). However, a significant difference in survival was observed between mice treated with T-mfIL12 (mean 48.8 days) and mice treated with mock treatment (mean 33.8 days, p<0.005, Breslow-Gehan-Wilcoxon). Furthermore, a prolonged survival was observed in mice treated with T-01 (mean 53.3 days) compared to mice treated with mock treatment (p<0.05, Breslow-Gehan-Wilcoxon). One mouse (1 / 10) treated with T-01 survived for more than 180 days. No enhanced antitumor effect was observed in mice treated with T-mfIL12 compared to mice treated with T-01. These data suggest that T cells are involved in antitumor immune responses, such as those induced by T-mfIL12.

[0325] 6. IL-12 induces significant IFNγ secretion upon intravenous administration.

[0326] Investigating serum IL-12 and IFNγ levels in an A / J mouse Neuro2a metastatic tumor model. Figure 26 A) Following intravenous inoculation of tumor cells, mice were treated three times via intravenous administration on days 1, 3, and 5. Blood samples were collected on days 2, 4, and 6 to measure IL-12 and IFNγ concentrations (n=3, at each time point). On day 2, the day after the initial intravenous administration, the IL-12 level detected in mice treated with T-mfIL12 was 518 pg / ml, which then decreased rapidly. On days 4 and 6, serum IL-12 expression was undetectable (0 pg / ml). In contrast, serum IL-12 was undetectable at all time points in both mock and T-01 administrations.

[0327] like Figure 26As shown in B, in the T-mfIL12-treated group, the IFNγ level was 3989 pg / ml on day 2, and then decreased. On day 4, the IFNγ level was 868 pg / ml, reaching 711 pg / ml on day 5. In T-01-treated mice, the level was 174 pg / ml on day 1, 300 pg / ml on day 4, and 262 pg / ml on day 6. In mock-treated mice, the IFNγ levels at these time points were 115 pg / ml, 58 pg / ml, and 148 pg / ml, respectively. On day 6, there were significant differences in IFNγ expression among the three groups (T-mfIL12 vs T-01: p = 0.019, T-01 vs mock: p = 0.030, T-mfIL12 vs mock: p = 0.007, t-test).

[0328] [Example 6: Malignant melanoma]

[0329] 1. Subcutaneous tumor treatment experiment using a bilateral subcutaneous tumor model in DBA / 2 mice

[0330] Establishment of bilateral subcutaneous tumor models (using mouse malignant melanoma cells CloneM3 1.0×10⁶ cells) 6 50 μL LMEM / mouse was subcutaneously injected into the left and right lateral ventricular regions of syngeneic DBA / 2 mice to induce a response based on mock, T-01, or T-mfIL12 (4 × 10⁻⁶). 4 A treatment trial using pfu / 20 μL was conducted. In this trial, viral therapy was administered to only one (left) subcutaneous tumor in both sides. IL-12 produced by T-mfIL12 enhanced anti-tumor immunity, thus raising the expectation of anti-tumor effects on distant tumors as well. Statistical analysis of the results was performed using Student's t-test.

[0331] On the administration side, T-mfIL12 significantly inhibited the growth of subcutaneous tumors on the administration side compared with mock (day 21, p<0.05; day 25, p<0.05). Figure 27 A). Furthermore, T-mfIL12 significantly inhibited the growth of subcutaneous tumors on the treatment side compared to T-01 (day 17, p<0.01; day 21, p<0.05). Moreover, significant differences were also observed between T-mfIL12 and mock on the non-treatment side (day 17, p<0.05; day 21, p<0.01). Figure 27B). Furthermore, a significant difference was observed between T-mfIL12 and T-01 (day 21, p < 0.05). No significant difference was observed between mock and T-01 in either the administered or unadministered side. These results indicate that T-mfIL12 exhibits a more significant antitumor effect on Clone M3 subcutaneous tumors than mock, not only in the administered side but also in the unadministered side. Moreover, although T-mfIL12 and T-01 have comparable in vitro cytotoxic effects and viral replication capacity, T-mfIL12 shows a significant antitumor effect in vivo in the administered side, suggesting that immune activation induced by IL-12 expression is also involved in enhancing the antitumor effect.

[0332] 2. Observation of tumor-infiltrating lymphocytes based on immunohistochemical staining

[0333] To evaluate the activation of antitumor immunity within tumors, immunohistochemical staining was used to observe the CD4+ tumor-infiltrating lymphocytes in the subcutaneous tumors on the drug-treated side of the Clone M3 subcutaneous tumor model in DBA / 2 mice. + Cells and CD8 + Cells. One slice of subcutaneous tumor tissue excised on day 14 after viral administration, after being trisected, was used for the experiment. Subcutaneous tumor sections were observed at three sites: the lateral (skin side), central, and medial (body side). As a negative control, sections treated with 2% BSA / PBS after one antibody reaction were prepared to confirm the absence of non-specific reactions resulting from a second antibody reaction. Figure 28 A).

[0334] CD4 + Cells and CD8 + Cells were observed in large numbers mainly along the tumor-skin boundary and the outline of blood vessels. Figure 28 (B, C) Compared with the mock group or the T-01 group, CD4 was observed at any site on the skin side, center, and body side of subcutaneous tumors in the T-mfIl12 treatment group. + Increase in cells.

[0335] 3. Treatment experiment using a bilateral subcutaneous tumor model in nude mice

[0336] To investigate whether the enhanced antitumor effect in the T-mfIL12-treated group in the DBA / 2 mouse subcutaneous tumor model, as shown above, was due to the immune enhancement effect of mouse IL-12 produced by T-mfIL12, a bilateral subcutaneous tumor model in nude mice was used (Clone M3 1.0 × 10⁻⁶ was subcutaneously injected into the left and right flanks of the nude mice). 6 A subcutaneous tumor treatment experiment was conducted using 50 μL DMEM per animal.

[0337] On the administration side, T-mfIL12 significantly inhibited the growth of subcutaneous tumors compared to moc (day 15, p<0.01; day 18, p<0.01). Figure 29 A). Furthermore, T-mfIL12 significantly inhibited subcutaneous tumor growth relative to T-01 (day 15, p<0.01; day 18, p<0.01). However, no significant differences were observed between any groups in the subcutaneous tumors on the untreated side, a result different from the experiment using DBA / 2 mice that observed tumor shrinkage on the untreated side. Figure 29 B).

[0338] Based on the above results, it can be concluded that T cells are required to enhance the anti-tumor effect of T-mfIL12 expression in mice against distant tumors.

[0339] 4. Analysis of IFNγ or IL-4-produced lymphocytes based on ELISpot assay

[0340] Based on the aforementioned immunohistochemical staining and treatment experiments using a nude mouse subcutaneous tumor model, T cells are crucial for enhancing the anti-tumor effect of T-mfIL12. Therefore, to evaluate the enhanced anti-tumor immune activity induced by T-mfIL12 in a DBA / 2 mouse Clone M3 subcutaneous tumor model, IFNγ-producing lymphocytes and IL-4-producing lymphocytes were analyzed using the ELISpot assay.

[0341] Results on the dosing side of the subcutaneous tumor treatment experiment ( Figure 10 In A), tumor shrinkage was observed in the T-mfIL12 administration group from days 13 to 17, suggesting that anti-tumor immunity may have been enhanced during this period. Day 14 was designated as the assay day. Statistical analysis of the results was performed using Student's t-test.

[0342] Compared with the mock group and the T-01 group, the T-mfIL12 group showed a significant increase in IFNγ-producing lymphocytes in response to Clone M3 cell stimulation (T-mfIL12-molck, p<0.01; T-mfIL12-T-01, p<0.05). Figure 30 A). On the other hand, no significant difference was observed in the number of IL-4-generated lymphocyte spots between the T-mfIL12 administration group and the T-01 administration group ( Figure 30 B).

[0343] [Example 7: Non-seminomatous germ cell tumor (NSGCT)]

[0344] Antitumor effects of T-01 and T-mfIL12 in the F9 model

[0345] The F9 model was selected (5.0 × 10⁶ F9 cells were injected into mouse NSGCT cells). 5 After adding 50 μL of LDE medium and 50 μL of BD matrix gel to a total volume of 100 μL, the solution was subcutaneously injected into the dorsal side of male 129 mice to obtain T-014.0 × 10⁻⁶. 4 PFU group, T-mfIL 124.0×10 4 The PFU group and the T-01-untreated mock group comprised a total of three groups (n=8 per group). Intratumoral administration of 20 μL solutions each of T-01, T-mfIL12, and mock was administered twice, on days 0 and 3. Tumor diameter was measured twice weekly, and the treatment was terminated on day 14 when the maximum tumor diameter in the mock group exceeded φ23 mm. After day 9, significant antitumor effects were observed in both the T-01 and T-mfIL12 groups compared to the mock group, and furthermore, the T-mfIL12 group showed a significant tumor-suppressive effect compared to the T-01 group. It should be noted that in the T-mfIL12 group, tumors in all individuals were suppressed to the same size as at the start of treatment, demonstrating a strong tumor-suppressive effect. Figure 31 (*P<0.01, ).

[0346] [The sequence recorded in the sequence list]

[0347] Sequence number 1: Base sequence of the inserted nucleotide of T-mfIL12; Sequence number 2: Base sequence of the inserted nucleotide of T-mfIL12; Encoded amino acid sequence; Sequence number 3: Base sequence of the inserted nucleotide of T-hIL12; Sequence number 4: Base sequence of the inserted nucleotide of T-hIL12; Encoded amino acid sequence; Sequence number 5: Primer.

[0348] Primer 6

[0349] Sequence number 7: Fusion polypeptide insertion site and its preceding and following base sequences. Sequence number 8: γ34.5 gene deletion site and its preceding and following base sequences (TR). L Within the region, the deletion site of gene sequence number 9α47 and the base sequences before and after it.

[0350] Serial number 10 primer

[0351] Primer Serial No. 11

[0352] Primer Serial Number 12

[0353] Primer Serial No. 13

Claims

1. A pharmaceutical composition comprising an IL-12-expressing recombinant herpes simplex virus (HSV), the IL-12-expressing recombinant herpes simplex virus (HSV) having a gene encoding a fusion polypeptide formed by linking a 35 kDa light chain (p35) of interleukin-12 (IL-12) with a 40 kDa heavy chain (p40) of interleukin-12 (IL-12) using two or more elastin motifs, and having the characteristics of (a) to (c) below. (a) ICP6 gene deletion or inactivation; (b) γ34.5 gene deletion or inactivation; (c) α47 gene deletion or inactivation.

2. The pharmaceutical composition according to claim 1, wherein, The fusion peptide is sequentially linked from the N-terminus to p40, two or more elastin motifs, and p35.

3. The pharmaceutical composition according to claim 1, wherein, The gene encoding the fusion polypeptide was inserted into the ICP6 locus.

4. The pharmaceutical composition according to claim 1, wherein, The gene encoding the fusion peptide is any of the following polynucleotides: (i) A polynucleotide consisting of the sequence of sequence number 1; (ii) A polynucleotide consisting of a sequence having more than 90% sequence identity with the sequence of sequence number 1 and encoding a polypeptide that can form active IL-12. (iii) A polynucleotide encoding a polypeptide consisting of the sequence of sequence number 2; (iv) A polynucleotide encoding a polypeptide that is composed of a sequence having more than 90% sequence identity with the sequence of sequence number 2 and can form an active IL-12. (v) A polynucleotide consisting of the sequence number 3; (vi) A polynucleotide consisting of a sequence having more than 90% sequence identity with the sequence of sequence number 3 and encoding a polypeptide that can form active IL-12. (vii) Encoding a polynucleotide of a polypeptide consisting of the sequence of sequence number 4; (viii) A polynucleotide that encodes a polypeptide consisting of a sequence that has more than 90% sequence identity with the sequence of sequence number 4 and can form an active IL-12.

5. The pharmaceutical composition according to claim 1, wherein, The recombinant HSV is derived from G47Δ.

6. The pharmaceutical composition according to any one of claims 1 to 5, which enhances the effect of IL-12 in the treatment of tumors.

7. The pharmaceutical composition according to claim 5, for local injection.

8. A method for enhancing the effect of IL-12 in the treatment of tumors, comprising the step of administering to a subject in need a pharmaceutical composition comprising a recombinant HSV expressing the IL-12 gene as described in any one of claims 1 to 5.

9. A pharmaceutical composition comprising a recombinant HSV expressing an IL-12 gene having a gene encoding IL-12 and possessing the characteristics described in (a) to (c) below, wherein the pharmaceutical composition induces the production of IFNγ through the expression of IL-12, and the replication capacity of the recombinant HSV expressing the IL-12 gene is not diminished. (a) ICP6 gene deletion or inactivation; (b) γ34.5 gene deletion or inactivation; (c) α47 gene deletion or inactivation.

10. A pharmaceutical composition for intravenous administration, comprising a recombinant HSV expressing an IL-12 gene having a gene encoding IL-12 and possessing the characteristics described in (a) to (c) below. (a) ICP6 gene deletion or inactivation; (b) γ34.5 gene deletion or inactivation; (c) α47 gene deletion or inactivation.

11. A pharmaceutical composition for intratumoral administration comprising a recombinant HSV expressing an IL-12 gene having a gene encoding IL-12 and having the characteristics described in (a) to (c) below, said pharmaceutical composition inducing systemic antitumor immunity, thereby achieving at least one of inhibiting metastasis and inhibiting the proliferation of untreated tumors. (a) ICP6 gene deletion or inactivation; (b) γ34.5 gene deletion or inactivation; (c) α47 gene deletion or inactivation.

12. A pharmaceutical composition for treating tumors requiring increased infiltration of immune cells, comprising a recombinant HSV expressing an IL-12 gene having a gene encoding IL-12 and possessing the characteristics described in (a) to (c) below. (a) ICP6 gene deletion or inactivation; (b) γ34.5 gene deletion or inactivation; (c) α47 gene deletion or inactivation.

13. The pharmaceutical composition according to any one of claims 9 to 12, wherein, The IL-12 expression-type recombinant HSV is the IL-12 expression-type recombinant HSV defined in any one of 1 to 5.

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