Oncolytic viruses and cancer treatment using the same

Abstract

The present invention provides: an oncolytic virus such as a conditionally replicating adenovirus carrying the CXCL10 gene; the oncolytic virus additionally carrying the IL-2 gene and / or the GM-CSF gene on the same viral genome; a combination of the oncolytic virus and a separate oncolytic virus carrying the IL-2 gene and / or the GM-CSF gene; and a cancer treatment agent comprising any of the aforementioned oncolytic viruses or a combination thereof as an active ingredient.

Description

[Technical Field]

[0001] The present invention relates to oncolytic viruses carrying an immunostimulatory gene containing C-X-C motif chemokine ligand 10 (CXCL10) and their use in cancer therapy. More specifically, the present invention relates to oncolytic viruses carrying the CXCL10 gene, oncolytic viruses carrying the CXCL10 gene and interleukin-2 (IL-2) and / or granulocyte-macrophage colony-stimulating factor (GM-CSF) genes on the same or different oncolytic viruses, and oncolytic viruses carrying two or three factors including CXCL10 as immunostimulatory genes, as well as cancer treatments using them, particularly treatments that are effective even for invasive and metastatic intractable cancers. [Background technology]

[0002] In recent years, conditionally replicating adenoviruses (CRAs), which replicate and kill cancer cells specifically, have been shown to be promising for cancer treatment. These CRAs are engineered to differentiate viral replication in cancer cells and normal cells by modifying the E1 gene region, which is essential for adenovirus replication. There are two types of CRAs: (1) those that delete the Rb and p53 binding domains in the E1 region, which are essential for inducing the cellular environment necessary for adenovirus replication, thereby inhibiting their inactivation (i.e., blocking viral replication in normal cells); and (2) those that replace the endogenous promoter of the E1 gene with the promoter of a gene that is highly expressed in cancer cells, thereby enabling cancer-specific E1 gene expression (enhancing viral replication in cancer cells). The present inventors have developed a technology to efficiently generate "multifactorially controllable and treatable CRAs" (m-CRAs), which can be called next-generation CRAs, by using a variety of cancer-specific factors and therapeutic genes, such as deletions within the E1A and E1B gene regions, replacement of the endogenous promoters of these genes with exogenous promoters, and incorporation of other cancer therapeutic genes, thereby enabling precise control of viral proliferation and enhancing therapeutic effects (Patent Document 1, Non-Patent Document 1). Furthermore, using this technology, we have generated m-CRA (Surv.m-CRA-1) in which the virus replicates under the control of the promoter of survivin, a member of the inhibitor of apoptosis protein (IAP) family, and have reported its anticancer effects against various cancers that specifically express survivin (Patent Documents 2 and 3, Non-Patent Document 2).

[0003] What distinguishes virotherapy from chemotherapy and radiation therapy is that during the process of amplified virus destruction of cancer cells, the release of tumor-associated antigens and the adjuvant-like function of the virus induce antitumor immunity, primarily cellular immunity, potentially exerting a therapeutic effect throughout the body through immune responses. Specifically, during the immune system's elimination of the virus, tumor-associated antigens from destroyed cancer cells are processed and presented by antigen-presenting cells, potentially eliciting specific antitumor immunity. To maximize this antitumor immunity, oncolytic virus immunotherapy (OVI) using oncolytic viruses carrying immunostimulatory genes has been intensively investigated. For example, talimogene laherparepvec, the only OVI approved in Europe and the United States, is an oncolytic herpes simplex virus carrying the GM-CSF gene as an immunostimulatory gene downstream of the ubiquitous and powerful CMV promoter. The development of oncolytic adenoviruses containing cytokine genes is generally hindered by two technical limitations: vector construction and the need for animal models that permit viral replication. Using m-CRA technology, the inventors efficiently generated oncolytic adenoviruses carrying various candidate immunostimulatory genes under the control of various candidate promoters. They established an immune-retaining syngenic hamster cancer model in which human adenoviruses are replicable, and then established an evaluation system to systematically and accurately analyze their in vivo therapeutic efficacy (Non-Patent Document 3). Using this evaluation system, they tested m-CRA (Surv.m-CRA-2-G), in which the GM-CSF gene was tethered downstream of various promoters, and found that it could be safely administered while maintaining therapeutic efficacy (Patent Document 4). Furthermore, they demonstrated that m-CRA carrying the GM-CSF gene exhibited survival benefits comparable to or greater than those of anti-PD-1 antibodies, one of the existing promising cancer immunotherapeutic agents (Patent Document 4).

[0004] However, the effectiveness of oncolytic viruses carrying the GM-CSF gene in cancer treatment is not always sufficient. There is a need for further improvements in the therapeutic effect, particularly for refractory cancers that have infiltrated and metastasized. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2005-46101 A [Patent Document 2] WO 2005 / 115476 [Patent Document 3] WO 2022 / 244792 [Patent Document 4] WO 2019 / 093435 [Non-patent literature]

[0006] [Non-Patent Document 1] Nagano, S. et al., Gene Ther., 2005, 12(18): 1385-93 [Non-patent document 2] Kamizono, J. et al., Cancer Res., 2005, 65(12): 5284-91 [Non-patent document 3] Ijichi, N. et al., Mol. Ther., 2016, 24(S1): S164 Summary of the Invention [Problem to be solved by the invention]

[0007] The object of the present invention is to provide a novel OVI that not only has a cancer therapeutic effect, particularly a tumor growth inhibitory effect in primary lesions, but also further enhances systemic anti-tumor immunity, which is important for therapeutic effects in metastatic lesions. [Means for solving the problem]

[0008] As a result of extensive research to achieve the above-mentioned objective, the inventors have found that when the CXCL10 gene is used as an immunostimulatory gene, OVI carrying this gene can specifically damage various cancer cells more efficiently than oncolytic viruses that do not contain immune genes, and can significantly suppress tumor growth even when administered locally to a cancer-bearing hamster model. Although OVI carrying CXCL10 or GM-CSF genes alone may not exhibit significant tumor growth suppression due to differences in tumor system and dosage, the combination of OVI carrying CXCL10 genes with OVI carrying GM-CSF or IL-2 genes significantly suppressed tumor growth. Furthermore, when combined with CXCL10 and / or GM-CSF genes, OVI carrying IL-2 genes alone showed a tendency to suppress tumor growth compared to oncolytic viruses without immune genes. The dual combination of IL-2 and CXCL10 or GM-CSF genes further enhanced this effect, and the triple combination significantly suppressed tumor growth compared to oncolytic viruses without immune genes. Furthermore, challenge tests of primary cancer cells and heterologous cancer cells at distant sites showed the induction of primary cancer cell-specific systemic antitumor immunity, and the therapeutic effect at distant metastatic sites was higher with IL-2 gene alone, followed by two-drug combination and three-drug combination, in the same order as in the primary site. Furthermore, the poor weight gain caused by the introduction of immunostimulatory genes was mild in all cases, suggesting that the combined use of two or three factors did not result in any significant side effects.

[0009] Furthermore, the inventors designed and constructed a multi-factor-loaded OVI capable of expressing two or more immunostimulatory genes dicistronically or three factors tricistronically, aiming to increase the efficiency of co-transfection and co-expression of two or more immunostimulatory genes and reduce the viral dose. Compared with a cocktail of three OVIs loaded with the same total dose of each gene, the triple-factor-loaded OVI suppressed primary tumor growth to a similar extent in a tumor-bearing hamster model, and surprisingly, more potently prevented distant metastasis. OVI loaded with three therapeutic genes from human and mouse showed more significant suppression of both primary tumors and metastases than OVI loaded with no therapeutic genes.

[0010] The present inventors have conducted further research based on these findings and have completed the present invention, which provides the following.

[0011] [Section 1] An oncolytic virus comprising a nucleic acid encoding C-X-C motif chemokine ligand 10 (CXCL10) under the control of a promoter functional in cancer cells. [Section 2] Item 1. The oncolytic virus according to Item 1, wherein the promoter is a ubiquitous promoter, a cancer cell-specific promoter, or a promoter specific to an organ from which the cancer cells are derived. [Section 3] Item 1. The oncolytic virus according to Item 1, wherein the promoter is a CA promoter, a CMV promoter, an RSV promoter, or an E2F promoter. [Section 4] Item 4. The oncolytic virus according to any one of Items 1 to 3, wherein a promoter of a nucleic acid encoding at least one factor essential for viral replication or assembly is substituted with a cancer cell-specific promoter or a promoter specific to an organ from which the cancer cells are derived. [Section 5] Item 5. The oncolytic virus according to Item 4, wherein the cancer cell-specific promoter is a survivin promoter. [Section 6] Item 6. The oncolytic virus according to Item 4 or 5, wherein the virus is an adenovirus. [Section 7] Item 7. The oncolytic virus according to Item 6, wherein the factors essential for viral replication or assembly are E1A or E1AΔ24, and / or E1B or E1BΔ55K. [Section 8] Item 8. The oncolytic virus according to Item 7, wherein the promoter of the nucleic acid encoding E1A is replaced with a survivin promoter, and further wherein the promoter of the nucleic acid encoding E1BΔ55K is replaced with an exogenous promoter selected from a ubiquitous promoter, a cancer cell-specific promoter, and an organ-specific promoter from which the cancer cells are derived. [Section 9] The oncolytic virus of item 8, wherein the promoter of the nucleic acid encoding E1BΔ55K is substituted with a CMV promoter. [Section 10] Item 10. The oncolytic virus according to any one of Items 1 to 9, further comprising a nucleic acid encoding interleukin-2 (IL-2) under the control of a promoter functional in cancer cells, and / or a nucleic acid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) under the control of a promoter functional in cancer cells. [Section 11] Item 11. The oncolytic virus according to any one of Items 1 to 10, further comprising a nucleic acid encoding interleukin-2 (IL-2) under the control of a promoter functional in cancer cells, and a nucleic acid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) under the control of a promoter functional in cancer cells. [Section 12] Item 12. The oncolytic virus according to Item 10 or 11, wherein the promoter controlling the expression of CXCL10 and the promoter controlling the expression of IL-2 and / or the promoter controlling the expression of GM-CSF are a single promoter. [Section 13] A nucleic acid encoding CXCL10, a nucleic acid encoding IL-2 and / or a nucleic acid encoding GM-CSF are arranged in the 5'-directed manner. (i) CXCL10-IL-2; (ii) CXCL10-GM-CSF; (iii) IL-2-CXCL10; (iv) GM-CSF-CXCL10; (v) CXCL10-IL-2-GM-CSF; (vi) CXCL10-GM-CSF-IL-2; (vii) IL-2-CXCL10-GM-CSF; (viii) IL-2-GM-CSF-CXCL10; (ix) GM-CSF-CXCL10-IL-2; or (x) GM-CSF-IL-2-CXCL10 Item 13. The oncolytic virus according to Item 12, wherein the oncolytic virus is arranged in the order of: [Section 14] Item 14. The oncolytic virus according to Item 13, wherein the nucleic acids are linked via a 2A sequence or an IRES sequence. [Section 15] Item 15. The oncolytic virus according to Item 14, wherein the first nucleic acid and the second nucleic acid are linked from the 5' side via a P2A sequence, and the second nucleic acid and the third nucleic acid are linked via a T2A sequence. [Section 16] A combination of the oncolytic virus according to any one of Items 1 to 9 with the following oncolytic virus (a) and / or (b), or the following oncolytic virus (c): (a) an oncolytic virus comprising a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells; (b) an oncolytic virus comprising a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells; (c) an oncolytic virus comprising a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells and a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells; [Section 17] Item 17. The combination according to Item 16, wherein each of the promoters functional in the cancer cells is the same or different and is a ubiquitous promoter, a cancer cell-specific promoter, or a promoter specific to the organ from which the cancer cells are derived. [Section 18] Item 17. The combination according to Item 16, wherein each of the promoters functional in the cancer cells is the same or different and is a CA promoter, a CMV promoter, an RSV promoter, or an E2F promoter. [Section 19] Item 19. The combination according to any one of Items 16 to 18, wherein the oncolytic viruses are conditionally replicating adenoviruses, and the structures of the replication control regions of the oncolytic viruses are identical. [Section 20] 20. A cancer therapeutic agent comprising the oncolytic virus according to any one of Items 1 to 15 or the combination according to any one of Items 16 to 19 as an active ingredient. [Section 20a] A method for treating cancer, comprising administering to a subject having cancer an effective amount of the oncolytic virus according to any one of Items 1 to 15 or the combination according to any one of Items 16 to 19. [Section 20b] 20. The oncolytic virus according to any one of Items 1 to 15 or the combination according to any one of Items 16 to 19 for use in treating cancer. [Section 20c] 20. Use of the oncolytic virus according to any one of Items 1 to 15 or the combination according to any one of Items 16 to 19 for the manufacture of a cancer therapeutic agent. [Section 21] Item 21. The agent according to Item 20, which is administered locally to a primary cancer lesion. [Section 22] Item 22. The agent according to Item 20 or 21, which is for the treatment of invasive or metastatic cancer. [Section 23] Item 23. The agent according to any one of Items 20 to 22, which is administered multiple times. [Effects of the Invention]

[0012] According to the present invention, it is possible to induce cell-mediated systemic antitumor immunity specific to cancer antigens released from cancer cells killed by the proliferation of oncolytic viruses to a higher degree than before, which not only enhances the therapeutic effect on primary lesions but also may become an effective therapeutic means for refractory cancers with invasiveness and metastasis. [Brief explanation of the drawings]

[0013] [Figure 1-1] FIG. 1-1 is a schematic diagram of the genomic sequences of various Surv. m-CRAs having a mouse-derived CXCL10 gene unit downstream of various promoters prepared in Example 1. [Figure 1-2] FIG. 1-2 is a schematic diagram of the genomic sequences of various Surv. m-CRAs having a human-derived CXCL10 gene unit downstream of various promoters prepared in Example 1. [Figure 1-3]1-3 are schematic diagrams of the genomic sequences of the various Surv.m-CRAs prepared in Example 1, including those containing three therapeutic genes. [Figure 2-1] Figure 2-1 shows the results of ELISA quantification of mouse or human CXCL10 in the culture supernatant of HEK293 cells transfected with the P2 plasmid containing nucleic acid encoding mouse CXCL10. (A: Mouse CXCL10, B: Human CXCL10) [Figure 2-2] Figure 2-2 shows the results of quantification, by ELISA, of the expression of mouse CXCL10 in HaK cells, HaP-T1 cells, or BHK-21 cells infected with CRA carrying the mouse CXCL10 gene. *P<0.01 [Figure 2-3] Figure 2-3 shows the results of quantifying human CXCL10 expression in HaK cells infected with CRA carrying the human CXCL10 gene, using ELISA. *P<0.01 [Figure 3-1] Figure 3-1 shows the cytotoxic effects of various Surv.m-CRAs after in vitro infection in Example 3. *P<0.05 (vs. Ad.dE1.3); #P<0.05 (Surv.m-CRAs (No transgene)). [Figure 3-2] FIG. 3-2 shows representative phase-contrast images of HaK cells 3 or 5 days after infection in Example 3 at 40x (left) and 100x (right) magnifications. [Figure 3-3] FIG. 3-3 shows representative phase-contrast images of HaP-T1 cells 3 or 5 days after infection in Example 3 at 40x (left) and 100x (right) magnifications. [Figure 3-4] FIG. 3-4 shows representative phase-contrast images of BHK-21 cells 3 or 5 days after infection in Example 3 at 40x (left) and 100x (right) magnifications. [Figure 4] FIG. 4 is a diagram outlining the process of establishing a syngenic Syrian hamster cancer model in Example 4. [Figure 5]FIG. 5 shows the in vivo therapeutic effect of CXCL10-loaded Surv.m-CRA on subcutaneous tumors in syngenic Syrian hamsters in Example 5. [Figure 6] FIG. 6 shows the therapeutic effect of combined use of two types of cytokines on Surv.m-CRA-2 in Example 6. [Figure 7-1] FIG. 7-1 is a diagram showing an outline of the experimental protocol in Example 7. [Figure 7-2] FIG. 7-2 shows the therapeutic effect of three types of Surv.m-CRA-2 combined use on primary tumors in Example 7. [Figure 7-3] FIG. 7-3 is a diagram confirming the regression of HaK tumors at distant sites by combined treatment with three types of Surv.m-CRA-2 in Example 7. [Figure 7-4] FIG. 7-4 is a diagram confirming the regression of Hap-T1 tumor at a distant site by combined treatment with three types of Surv.m-CRA-2 in Example 7. [Figure 7-5] FIG. 7-5 shows the changes in body weight of mice over time during treatment in Example 7. [Figure 8] FIG. 8 shows the expression levels of cytokines in cells infected with Surv.m-CRA-2 carrying three types of cytokine genes. [Figure 9-1] FIG. 9-1 shows an outline of the experimental protocol in Example 10. [Figure 9-2] Figure 9-2 shows the cytotoxic effect of Surv.m-CRA-2 carrying three cytokine genes after in vitro infection (n=5). *P<0.05 (vs. Ad.dE1.3); #P<0.05 (vs. Surv.m-CRA (No transgene)). [Figure 10-1] FIG. 10-1 shows an outline of the experimental protocol in Example 11. [Figure 10-2]FIG. 10-2 shows the therapeutic effects on primary tumors of a combination of three types of Surv.m-CRA-2 and Surv.m-CRA-2 carrying three types of cytokine genes. [Figure 10-3] FIG. 10-3 shows that the combined administration of three types of Surv.m-CRA-2 and the administration of Surv.m-CRA-2 carrying three types of cytokine genes do not affect the body weight of recipients. [Figure 11-1] FIG. 11-1 shows an outline of the experimental protocol in Example 12. [Figure 11-2] FIG. 11-2 shows the therapeutic effects on primary tumors of two types of Surv.m-CRA-2 carrying three types of cytokine genes derived from mouse and human, and Surv.m-CRA carrying no therapeutic gene. [Figure 11-3] Figure 11-3 shows the therapeutic effect of two types of Surv.m-CRA-2 carrying three cytokine genes derived from mouse and human, and Surv.m-CRA carrying no therapeutic gene, on primary tumors in individual mice. The tumor formation rate 17 days after the initial virus administration is shown in the upper right corner of the graph. [Figure 11-4] Figure 11-4 shows the therapeutic effect on individual primary tumors of two administrations of Surv.m-CRA-2, which carries three cytokine genes derived from mouse and human, and Surv.m-CRA, which does not carry a therapeutic gene. The upper right corner of the graph shows the tumor formation rate 17 days after the second administration of the virus (35 days after the first administration). DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides an oncolytic virus carrying the CXCL10 gene as an immunostimulatory gene (hereinafter also referred to as the "oncolytic virus of the present invention"). The oncolytic virus of the present invention is surprisingly characterized by its high inhibitory effect on tumor growth, at least in primary lesions, when used alone, compared to a control oncolytic virus not carrying the CXCL10 gene. As used herein, "high therapeutic effect," such as tumor growth inhibition, preferably means that the effect is statistically significantly (e.g., p<0.05) higher than that of a control, but also encompasses cases where the therapeutic effect tends to be increased even though there is no significant difference.

[0015] CXCL10 is a chemokine that induces tumor infiltration and migration of immune cells, such as CXCR3-positive NK cells and T cells. However, previous studies have reported that CXCL10-loaded parvoviruses have a reduced therapeutic effect due to the suppression of NS1 protein expression, which is involved in viral replication (Dinsart et al., Hum Gene Ther, 2017, 28(3): 295-306). Furthermore, CXCL10-loaded adenoviruses increased immune cell infiltration into tumors but failed to suppress tumor growth alone (Li et al., Oncoimmunology, 2022, 11(1): 2118-210). Therefore, the fact that the oncolytic viruses of the present invention can suppress tumor growth more potently than oncolytic viruses not carrying a therapeutic gene alone is an exceptionally significant effect that would be unexpected even for those skilled in the art.

[0016] The oncolytic virus of the present invention comprises a nucleic acid encoding CXCL10 under the control of a promoter functional in cancer cells. Here, "functional in cancer cells" means that the promoter has transcriptional activity that induces CXCL10 expression at least in cancer cells, regardless of whether it has transcriptional activity in cells other than cancer cells. Examples of promoters functional in cancer cells include ubiquitous promoters, cancer cell-specific promoters, and promoters specific to organs from which cancer cells originate. As used herein, "cancer cell-specific" and "organ-specific" are not limited to promoters that exhibit no transcriptional activity in normal cells or other organs, but also encompass promoters that drive gene expression in normal cells and cells of other organs within a therapeutically acceptable range.

[0017] Examples of ubiquitous promoters include cytomegalovirus (CMV)-derived promoters (e.g., CMV immediate-early promoter; herein, also simply referred to as "CMV promoter"), Rous sarcoma virus (RSV)-derived promoters (e.g., RSV LTR; herein, also simply referred to as "RSV promoter"), chicken β-actin gene promoters to which a cytomegalovirus immediate-early gene enhancer has been added (herein, also referred to as "CA promoter"), human immunodeficiency virus (HIV)-derived promoters (e.g., HIV LTR), mouse mammary tumor virus (MMTV)-derived promoters (e.g., MMTV LTR), Moloney murine leukemia virus (MoMLV)-derived promoters (e.g., MoMLV LTR), herpes simplex virus (HSV)-derived promoters (e.g., HSV thymidine kinase (TK) promoter), SV40-derived promoters (e.g., SV40 early promoter), Epstein-Barr virus (EBV)-derived promoters, and adeno-associated virus (AAV)-derived promoters. (e.g., AAV p5 promoter), adenovirus (AdV)-derived promoters (Ad2 or Ad5 major late promoter), β-actin gene promoter, PGK gene promoter, transferrin gene promoter, etc. can be used.

[0018] Examples of cancer cell-specific promoters include the CEA (carcinoembryonic antigen) promoter (Mol. Cell. Biol., 10(6), 2738-2748, 1990), the E2F promoter (Neuman, E. et al., Mol. Cell. Biol., 14(10), 6607-6615, 1994), the OC (osteocalcin) promoter (Morrison, N.A. et al., Science, 246, 1158-1161, 1989), the FLK-1 promoter specific to malignant melanoma, fibrosarcoma, etc. (Xie, B. et al., Br. J. Cancer, 81, 1335-1343, 1999), and the VEGF promoter specific to lung cancer, etc. (Koshikawa, N. et al., Cancer Res., 60, 2936-2941, 2000), the c-Myc promoter specific to small cell lung cancer (Kumagai, T. et al., Cancer Res., 354-358, 1996), the SLPI promoter specific to lung cancer and ovarian cancer (Garver, RI et al., Gene Ther., 1, 46-50, 1994), the PSA promoter specific to prostate cancer (Latham, JP et al., Cancer Res., 60, 334-342, 2000), the tyrosinase promoter specific to malignant melanoma (Vile, RG et al., Cancer Res., 53, 962-967, 1993), and the AP-2 promoter specific to breast cancer (Pandha, HS et al., J. Clin. Oncol., 17, 2180-2189, 1999), and the telomerase reverse transcriptase (TERT) promoter, which is specific to many cancers including brain tumors (Takakura, M. et al., Cancer Res., 59, 551-557, 1999), hypoxia-responsive region (HRE) promoters specific to various cancers, Grp78 promoter, L-plastin promoter, hexokinase II promoter, survivin promoter, Aurora kinase A promoter, and Aurora kinase B promoter.

[0019] Organ-specific promoters from which cancer cells originate are appropriately selected depending on the organ from which the cancer to be treated originates. Examples include albumin and α-fetoprotein promoters, which are specific to the liver, etc.; prostate-specific antigen (PSA) promoters, which are specific to the prostate; mitochondrial creatine kinase (MCK) promoters, which are specific to various organs such as the muscle and brain; and myelin basic protein (MB), glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE) promoters, which are specific to the nervous system such as the brain.

[0020] In another embodiment, the oncolytic virus of the present invention may comprise a nucleic acid encoding CXCL10 under the control of an inducible promoter. Examples of inducible promoters that can be used include the metallothionein-1 gene promoter. When the metallothionein-1 gene promoter is used, CXCL10 can be expressed in cancer cells by administering an inducer, such as a heavy metal (e.g., gold, zinc, or cadmium), a steroid (e.g., dexamethasone), an alkylating agent, a chelating agent, or a cytokine, to the local area of ​​the cancer at a desired time.

[0021] It is desirable to select a promoter that can provide an appropriate gene expression level for CXCL10 expression, from the perspectives of both therapeutic efficacy and safety. Cytokine genes exhibit high physiological activity even at low expression levels, and their overexpression carries the risk of causing undesirable side effects such as cytokine storm. Furthermore, unlike non-replicating viruses, oncolytic viruses replicate abundantly in cancer cells, allowing therapeutic genes carried by viral vectors to be expressed in large amounts within cancer cells and released at high levels from destroyed cancer cells. Therefore, it is desirable to select a promoter that provides a CXCL10 expression level that does not cause undesirable side effects in the recipient, or that can cause tolerable side effects, within a range that suppresses tumor growth at the primary tumor site and induces cancer-specific systemic antitumor immunity sufficient to suppress cancer at invasive and distant metastatic sites. For example, when adenovirus is used as the oncolytic virus, such a suitable promoter can be selected by constructing a panel of m-CRAs in which a nucleic acid encoding CXCL10 is linked downstream of various candidate promoters using the m-CRA technology developed by the present inventors, and administering the m-CRAs to, for example, a cancer cell line or a hamster cancer model system established by the present inventors that allows the proliferation of adenovirus, and analyzing the expression levels, therapeutic effects, and side effects.

[0022] In a preferred embodiment, a nucleic acid encoding CXCL10 can be placed under the control of a CA promoter, a CMV promoter, an RSV promoter, or an E2F promoter. As shown in the Examples below, these promoters exert high transcriptional activity in cancer cells in this order, and any of these promoters can kill various cancer cells in vitro with approximately equal success. Also, in a hamster model, administration of the CMV promoter, which has strong transcriptional activity, exerts a superior therapeutic effect compared to a control oncolytic virus lacking a therapeutic gene, without significant side effects.

[0023] The CA promoter, CMV promoter, RSV promoter, and E2F promoter used in the present invention include nucleic acids containing the nucleotide sequences set forth in SEQ ID NOS: 1, 2, 3, and 4, respectively, or nucleotide sequences that hybridize under stringent conditions with the complementary strand sequences of the respective nucleotide sequences and have cancer cell-specific transcription activity equivalent to that of promoters consisting of the respective nucleotide sequences. Examples of such nucleic acids include nucleic acids containing nucleotide sequences that share at least about 80%, preferably at least about 90%, more preferably at least about 95%, particularly preferably at least about 97%, and most preferably at least about 98% identity with the nucleotide sequences set forth in the respective SEQ ID NOS. The identity of the nucleotide sequences herein can be calculated, for example, using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions: expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3.

[0024] A promoter that controls the expression of CXCL10 can be prepared by cloning genomic DNA containing the promoter region from genomic DNA extracted from cells or tissues derived from humans or other mammals using a nucleic acid consisting of a known promoter sequence (e.g., a nucleotide sequence represented by any of SEQ ID NOS: 1 to 4) as a probe, cleaving the genomic DNA with a DNase, such as an appropriate restriction enzyme, into DNA fragments containing the desired partial promoter sequence, separating the fragments by gel electrophoresis, recovering the desired band, and purifying the DNA. Alternatively, the desired partial promoter sequence can be amplified and isolated by PCR using primers synthesized based on the known promoter sequence and a crude extract of the above cells or genomic DNA isolated therefrom as a template. In addition, a promoter that controls the expression of CXCL10 can also be obtained by chemically synthesizing a nucleic acid containing all or part of a known promoter sequence (e.g., a nucleotide sequence represented by any of SEQ ID NOs: 1 to 4) using a commercially available automatic DNA / RNA synthesizer.

[0025] Examples of the "CXCL10-encoding nucleic acid" used in the present invention include nucleic acids that contain the nucleotide sequence shown in SEQ ID NO: 5 (corresponding to the nucleotide sequence (CDS) from nucleotides 67 to 360 of the mRNA sequence of human CXCL10 registered in GenBank under accession number NM_001565) or a nucleotide sequence that hybridizes under stringent conditions with its complementary sequence, and encode a protein having activity equivalent to that of CXCL10 (e.g., the activity of inducing immune cell infiltration and migration into tumors). Examples of nucleic acids that hybridize under stringent conditions with the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 5 include nucleic acids containing a nucleotide sequence that shares at least about 60%, preferably at least about 70%, more preferably at least about 80%, particularly preferably at least about 90%, and most preferably at least about 95% identity with the nucleotide sequence shown in SEQ ID NO: 5. The nucleic acid encodes an amino acid sequence that has an identity of at least about 90%, preferably at least about 95%, more preferably at least about 97%, and particularly preferably at least about 98% to the amino acid sequence represented by SEQ ID NO: 6, such that a protein comprising the amino acid sequence has substantially the same activity (e.g., the activity of inducing immune cell infiltration and migration into tumors) as a protein comprising the amino acid sequence represented by SEQ ID NO: 6.

[0026] The nucleic acid encoding CXCL10 may be an ortholog in a non-human mammal of the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 5 (for example, a nucleic acid encoding mouse CXCL10 consists of the nucleotide sequence represented by SEQ ID NO: 7 (corresponding to the nucleotide sequence (CDS) from positions 76 to 369 of the mRNA sequence of mouse CXCL10 registered in GenBank under Accession No. NM_021274)). For example, it is desirable to use a nucleic acid encoding CXCL10 derived from the mammal to which the oncolytic virus of the present invention is to be administered. The animal to which the oncolytic virus of the present invention is to be administered is not particularly limited as long as it has cancer, and examples include humans, mice, rats, hamsters, rabbits, dogs, monkeys, etc., with humans being preferred. Thus, in a preferred embodiment, the nucleic acid encoding CXCL10 is a nucleic acid encoding human CXCL10 (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 6).

[0027] A nucleic acid encoding CXCL10 can be cloned by, for example, PCR amplification using synthetic DNA primers containing a portion of the nucleotide sequence of the CDS region of the CXCL10 gene, or by hybridization of the DNA incorporated into an appropriate expression vector with a labeled DNA fragment or synthetic DNA containing the nucleotide sequence of the CDS region of the CXCL10 gene. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989).

[0028] The nucleotide sequence of DNA can be converted using known kits, such as MutanTM-super Express Km (Takara Shuzo Co., Ltd.) and MutanTM-K (Takara Shuzo Co., Ltd.), according to known methods such as the ODA-LA PCR method, the Gapped duplex method, the Kunkel method, or methods similar thereto.

[0029] The cloned DNA can be used as is, or after digestion with a restriction enzyme or the addition of a linker, depending on the purpose. The DNA may have a translation initiation codon, ATG, at its 5'-end and a translation termination codon, TAA, TGA, or TAG, at its 3'-end. These translation initiation and termination codons can be added using an appropriate synthetic DNA adapter.

[0030] An expression vector containing a nucleic acid encoding CXCL10 can be produced, for example, by excising a desired fragment from a nucleic acid encoding the CDS region of the CXCL10 gene and ligating the fragment downstream of the promoter in the above-mentioned expression vector. The expression vector preferably contains a transcription termination signal, i.e., a terminator region, downstream of the nucleic acid encoding CXCL10. Furthermore, the expression vector may further contain a selectable marker gene for selecting transformed cells (e.g., a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, or phosphinothricin, or a gene that complements an auxotrophic mutation).

[0031] The oncolytic viruses of the present invention are not particularly limited in type, as long as they replicate specifically in cancer cells, killing (lysing) those cells, and the daughter viruses reinfect nearby cancer cells one after another, thereby exerting a tumor-suppressing effect. Cancer selectivity may be conferred by any mechanism, but they must be capable of carrying at least a CXCL10 expression cassette as an immunostimulatory gene. Oncolytic viruses that have been marketed or advanced to clinical trials include adenovirus, herpes simplex virus (HSV), vaccinia virus, measles virus, reovirus, Newcastle disease virus, coxsackievirus, and parvovirus. In the case of HSV, deletion of DNA polymerase genes such as ribonucleotide reductase and thymidine kinase eliminates pathogenicity in normal cells, allowing it to grow only in cancer cells with activated cell proliferation. Furthermore, the γ34.5 gene product prevents viral growth inhibition by host protein kinase R (PKR). Deletion of this gene reduces pathogenicity in normal cells and allows viral growth only in cancer cells expressing Ras, which inhibits PKR. For example, inactivating the thymidine kinase gene of vaccinia virus allows it to grow in a manner dependent on the thymidine kinase of the host cancer cells, while measles virus can grow in various cancer cells that highly express CD46, the receptor for the measles vaccine strain.

[0032] In a preferred embodiment, the oncolytic virus of the present invention has a promoter for a nucleic acid encoding at least one factor essential for viral replication or assembly replaced with a cancer cell-specific promoter or an organ-specific promoter from which the cancer cells are derived.

[0033] The term "factors essential for viral replication or assembly" refers to any protein essential for viral replication or assembly, such as viral proteins required for the transcription of various viral genes (for example, in adenovirus, transcription of the early genes E1A, E1B, E2, and E4 is required before transcription of viral structural proteins; E1A in particular is the first protein to be transcribed and translated after infection, and transcription of this E1A protein is essential for the initiation of subsequent transcription of various viral proteins), or viral structural proteins (for example, in adenovirus, late gene products L1, L2, L3, L4, L5, etc.). Such factors vary depending on the virus species used, but for example, in the case of adenovirus, they include E1A, E1B, E2, and E4, preferably E1A and E1B, and more preferably E1A; in the case of adeno-associated virus, they include Rep78 and Rep68 under the control of the p5 promoter, and Rep52 and Rep40 under the control of the p19 promoter; in the case of herpes simplex virus, they include early gene products such as ICP0, ICP4, ICP22, and ICP27, and thymidine kinase; and in the case of Sendai virus, they include N protein, P protein, and L protein.

[0034] Factors essential for viral replication may be deleted for regions that are essential for inducing the cellular environment necessary for viral replication in normal cells but are not necessary for viral replication in cancer cells. For example, viral replication in normal cells requires inactivation of Rb and p53 to initiate the cell cycle. However, cancer cells are already undergoing the cell cycle, and therefore, in the case of adenovirus, the Rb-binding region of E1A and the p53-binding region of E1B are not required for viral replication in cancer cells. Therefore, the conditionally replicating adenovirus (CRA) of the present invention can be deleted for cancer cell-specific viral replication by deleting the E1A 24KDa region (E1AΔ24), the E1B 55KDa region (E1BΔ55K), or the E1B 19KDa region (E1BΔ19).

[0035] At least one endogenous promoter of a gene encoding a factor essential for viral replication or assembly is replaced with a cancer cell-specific promoter or a promoter specific to the organ from which the cancer cells are derived. As the "cancer cell-specific promoter" and the "cancer cell-derived organ-specific promoter," those exemplified above as promoters controlling the expression of CXCL10 can also be preferably used. Preferred examples of the cancer cell-specific promoter include the survivin promoter, Aurora kinase A promoter, and Aurora kinase B promoter, and more preferably the survivin promoter.

[0036] The promoters of mouse and human survivin genes have been isolated, and their sequence information has been disclosed (see, for example, Li, F. and Altieri, DC, Cancer Res., 59: 3143-3151, 1999; Li, F. and Altieri, DC, Biochem. J., 344: 305-311, 1999). The survivin promoter used in the oncolytic viruses of the present invention is the promoter of the human survivin gene or its orthologous gene in other mammals (e.g., monkeys, cows, horses, pigs, dogs, cats, sheep, goats, rabbits, mice, rats, etc.), preferably the promoter of the survivin gene derived from human or mouse (comprising the nucleotide sequences set forth in SEQ ID NOS: 9 and 10, respectively, or a partial sequence thereof). Depending on the mammal to be treated, it is preferable to use a survivin promoter of the same species; however, a heterologous promoter may also be used as long as it can exert promoter activity sufficient to infect cancer cells efficiently and achieve killing effects. For example, an oncolytic virus containing the mouse survivin gene promoter can be used as a vector for treating human cancer.

[0037] The Aurora kinase promoter used in the oncolytic virus of the present invention is not particularly limited as long as it is derived from a gene belonging to the Aurora kinase family, and examples thereof include mammalian (e.g., human, monkey, cow, horse, pig, dog, cat, sheep, goat, rabbit, mouse, rat, etc.) orthologs of the Drosophila Aurora-A, -B, and -C genes. A promoter of the Aurora kinase A gene or Aurora kinase B gene derived from human or other mammals is preferred, and a human Aurora kinase A or human Aurora kinase B promoter is more preferred. Depending on the mammal to be treated, it is preferable to use an Aurora kinase promoter of the same species; however, a heterologous promoter may also be used as long as it can exert promoter activity sufficient to infect cancer cells efficiently and provide a killing effect.

[0038] The nucleotide sequence lengths of the survivin promoter and Aurora kinase promoter are not particularly limited as long as they are specific to target cancer cells and can activate the transcription of a gene linked downstream to an extent that sufficient therapeutic activity against cancer is exerted, and for example, those having the sequence lengths described in WO 2019 / 093435 can be used. More specifically, for example, in the case of a mouse survivin promoter, the nucleotide sequence from positions −173 to −19, relative to the translation initiation point +1 (the nucleotide sequence from positions 1124 to 1278 in the nucleotide sequence shown in SEQ ID NO: 10), and in the case of a human survivin promoter, the nucleotide sequence from positions −173 to −1, relative to the translation initiation point +1 (the nucleotide sequence from positions 1296 to 1468 in the nucleotide sequence shown in SEQ ID NO: 9), can be used to obtain the desired specificity and transcriptional activity. Therefore, the survivin promoter used in the present invention preferably comprises at least a partial nucleotide sequence from positions 1124 to 1278 of the nucleotide sequence shown in SEQ ID NO: 10, or at least a partial nucleotide sequence from positions 1296 to 1468 of the nucleotide sequence shown in SEQ ID NO: 9, and in a preferred embodiment, the survivin promoter essentially consists of such a partial nucleotide sequence. WO 2019 / 093435 may also be referenced for suitable nucleotide sequences of Aurora kinase promoters.

[0039] A conditionally replicating virus (CRV) that is dependent on a cancer cell-specific or organ-specific promoter from which the cancer cell originates and that is introduced into a cell cannot replicate in an environment in which the promoter is not activated (e.g., normal cells), and therefore does not harm the cell. On the other hand, when a cancer cell-specific or organ-specific promoter-dependent CRV enters an environment in which the promoter is activated (e.g., cancer cells), the virus replicates there and damages the cell due to the cytotoxicity of the viral proteins. The virus released from the lysed cell successively infects surrounding cells that have not been transfected with the vector, and the same process is repeated. In this way, theoretically, the CRV can ultimately be introduced into all cancer cells within the lesion.

[0040] Since at least one of the nucleic acids encoding factors essential for viral replication or assembly is under the control of a cancer cell-specific or organ-specific promoter, viral growth or assembly is limited to an environment in which the promoter is activated, nucleic acids encoding other factors essential for viral replication or assembly may be under the control of any exogenous promoter different from the promoter. For example, the ubiquitous promoter, cancer cell-specific promoter, cancer cell-derived organ-specific promoter, inducible promoter, etc., exemplified above as promoters controlling the expression of CXCL10, can be similarly preferably used. Furthermore, when nucleic acids encoding factors essential for the replication or assembly of two or more viruses are placed under the control of the same cancer cell-specific or organ-specific promoter, they may be arranged polycistronically under the control of a single promoter, or monocistronically under the control of separate promoters.

[0041] Alternatively, as a nucleic acid encoding a factor essential for viral replication that is controlled by a promoter other than a cancer cell-specific or organ-specific promoter, a nucleic acid encoding the above-mentioned mutant viral protein (e.g., E1AΔ24, E1BΔ55K) that has been deleted for a region that is essential for inducing the cellular environment necessary for viral proliferation in normal cells but is not necessary for viral proliferation in target cancer cells can also be used.

[0042] In a preferred embodiment, the CRA of the present invention has the promoter of the nucleic acid encoding E1A replaced with a survivin promoter, and the promoter of the nucleic acid encoding E1BΔ55K replaced with an exogenous promoter selected from a ubiquitous promoter, a cancer cell-specific promoter, and a promoter specific to an organ from which the cancer cells are derived. In a particularly preferred embodiment, the exogenous promoter is a CMV promoter.

[0043] In a preferred embodiment of the present invention, the multifactorial cancer-specific growth-regulated recombinant adenovirus system (m-CRA; JP 2005-046101 A and WO 2005 / 012536 A) developed by the present inventors is used. Examples of plasmid vectors suitable for use in constructing m-CRA are illustrated in the above-mentioned patent documents. In the figure, the survivin promoter or the like is used as promoter A and / or promoter B in plasmid vector P1, and any of the promoters controlling the expression of CXCL10 can be used as promoter C in plasmid vector P2. In a specific embodiment shown in the examples below, there are provided a plasmid vector P1 comprising an E1A gene (which may lack the 24 KDa region) operably linked to a survivin promoter and an E1B gene (which may lack the 19 KDa or 55 KDa region) operably linked to a ubiquitous promoter (such as a CMV promoter); a plasmid vector P2 comprising a nucleic acid encoding CXCL10 operably linked to a ubiquitous promoter (such as a CA promoter, CMV promoter, or RSV promoter) or a cancer cell-specific promoter (such as an E2F promoter); and a backbone plasmid P3 comprising an adenovirus genome lacking the E1 region (which may have a target cell-specific mutation in the fiber gene). These three plasmids are combined appropriately, and then plasmid fusion is performed using the Cre recombinase-loxP system. The target plasmid is selected using the drug resistance gene and ori carried by each plasmid. This generates a cancer cell-specific replication-competent adenovirus (CRA) vector plasmid carrying a survivin promoter-E1A expression cassette, a ubiquitous promoter-E1B expression cassette, and a ubiquitous or cancer cell-specific promoter-CXCL10 expression cassette. This vector can then be used to transfect an E1A-complementing cell line (e.g., 293 cells) to produce a CRA vector.

[0044] The present invention also provides an oncolytic virus that combines the CXCL10 gene with an IL-2 and / or GM-CSF gene as an immunostimulatory gene, wherein the nucleic acid encoding IL-2 and / or the nucleic acid encoding GM-CSF may be contained in a single oncolytic virus together with the nucleic acid encoding CXCL10, or may be contained in an oncolytic virus separate from the nucleic acid encoding CXCL10.

[0045] When a nucleic acid encoding IL-2 and a nucleic acid encoding GM-CSF are contained in separate oncolytic viruses, the present invention provides a method for producing an oncolytic virus comprising: (a) an oncolytic virus comprising a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells; and / or (b) an oncolytic virus comprising a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells; Provide a combination with.

[0046] The promoters functional in cancer cells in the oncolytic viruses (a) and (b) above can each independently be a ubiquitous promoter, a cancer cell-specific promoter, or a promoter specific to the organ from which the cancer cells originate. The ubiquitous promoters, cancer cell-specific promoters, and organ-specific promoters from which the cancer cells originate, exemplified as promoters controlling CXCL10 expression for the oncolytic viruses of the present invention, can also be preferably used as these promoters. The promoters functional in cancer cells in the oncolytic viruses (a) and / or (b) can be the same as or different from the promoter controlling CXCL10 expression in the oncolytic viruses of the present invention. In a preferred embodiment, the promoter controlling IL-2 expression and the promoter controlling GM-CSF expression are each independently a CA promoter, a CMV promoter, an RSV promoter, or an E2F promoter.

[0047] In a preferred embodiment, the oncolytic viruses (a) and (b) can be oncolytic viruses having the same structure as the oncolytic viruses containing the nucleic acid encoding CXCL10, except that the nucleic acid encoding CXCL10 is replaced with a nucleic acid encoding IL-2 or GM-CSF. Thus, in a particularly preferred embodiment, the oncolytic viruses (a) and (b) are CRAs, like the oncolytic viruses of the present invention, and the structure of the proliferation control region of each oncolytic virus is also identical to that of the oncolytic viruses of the present invention. In particular, in the oncolytic viruses of the present invention and the oncolytic viruses (a) and (b), the promoter of the nucleic acid encoding E1A is preferably substituted with a survivin promoter, and the promoter of the nucleic acid encoding E1BΔ55K is preferably substituted with an exogenous promoter selected from a ubiquitous promoter, a cancer cell-specific promoter, and an organ-specific promoter from which the cancer cells are derived, preferably a CMV promoter.

[0048] Examples of the "nucleic acid encoding IL-2" used in the present invention include a nucleic acid that contains a nucleotide sequence represented by SEQ ID NO: 11 (corresponding to the nucleotide sequence (CDS) from positions 286 to 744 of the mRNA sequence of human IL-2 registered in GenBank under Accession No. NM_000586) or a nucleotide sequence that hybridizes under stringent conditions with its complementary sequence, and encodes a protein having activity equivalent to that of IL-2 (e.g., T cell stimulating activity). Examples of nucleic acids that hybridize under stringent conditions with the complementary sequence of the nucleotide sequence represented by SEQ ID NO: 11 include nucleic acids containing a nucleotide sequence that has about 60% or more, preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more identity to the nucleotide sequence represented by SEQ ID NO: 11. The nucleic acid encodes an amino acid sequence that has an identity of at least about 90%, preferably at least about 95%, more preferably at least about 97%, and particularly preferably at least about 98% to the amino acid sequence represented by SEQ ID NO: 12, such that a protein comprising the amino acid sequence has activity (e.g., T cell stimulating activity) substantially equivalent to that of a protein comprising the amino acid sequence represented by SEQ ID NO: 12.

[0049] The nucleic acid encoding IL-2 may be an ortholog in a non-human mammal of the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 11 (for example, a nucleic acid encoding mouse IL-2 consists of the nucleotide sequence represented by SEQ ID NO: 13 (corresponding to the nucleotide sequence (CDS) from positions 49 to 555 of the mRNA sequence of mouse IL-2 registered in GenBank under Accession No. NM_008366)). For example, it is desirable to use a nucleic acid encoding IL-2 derived from the mammal to be administered. The animal to which the oncolytic virus of the present invention is administered is not particularly limited as long as it has cancer, and examples include humans, mice, rats, hamsters, rabbits, dogs, monkeys, etc., with humans being preferred. Thus, in a preferred embodiment, the nucleic acid encoding IL-2 is a nucleic acid encoding human IL-2 (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 12).

[0050] Examples of the "nucleic acid encoding GM-CSF" used in the present invention include nucleic acids that contain the nucleotide sequence set forth in SEQ ID NO: 15 (corresponding to the nucleotide sequence (CDS) from positions 36 to 467 of the mRNA sequence of human GM-CSF registered in GenBank under accession number NM_000758) or a nucleotide sequence that hybridizes under stringent conditions with its complementary sequence, and encode a protein with activity equivalent to that of GM-CSF (e.g., antigen presentation enhancing activity). Examples of nucleic acids that hybridize under stringent conditions with the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 15 include nucleic acids containing a nucleotide sequence that shares at least about 60%, preferably at least about 70%, more preferably at least about 80%, particularly preferably at least about 90%, and most preferably at least about 95% identity with the nucleotide sequence set forth in SEQ ID NO: 15. The nucleic acid encodes an amino acid sequence that has an identity of at least about 90%, preferably at least about 95%, more preferably at least about 97%, and particularly preferably at least about 98% to the amino acid sequence represented by SEQ ID NO: 16, such that a protein comprising the amino acid sequence has substantially the same activity (e.g., antigen presentation enhancing activity) as a protein comprising the amino acid sequence represented by SEQ ID NO: 16.

[0051] The nucleic acid encoding GM-CSF may be an ortholog in a non-human mammal of the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 15 (for example, a nucleic acid encoding mouse GM-CSF consists of the nucleotide sequence represented by SEQ ID NO: 17 (corresponding to the nucleotide sequence (CDS) from positions 290 to 712 of the mRNA sequence of mouse GM-CSF registered in GenBank under Accession No. NM_009969)). For example, it is desirable to use a nucleic acid encoding GM-CSF derived from the mammal to which the virus is to be administered. The animal to which the oncolytic virus of the present invention is to be administered is not particularly limited as long as it has cancer, and examples include humans, mice, rats, hamsters, rabbits, dogs, monkeys, etc., with humans being preferred. Thus, in a preferred embodiment, the nucleic acid encoding GM-CSF is a nucleic acid encoding human GM-CSF (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 18).

[0052] Nucleic acids encoding IL-2 or GM-CSF can be cloned by, for example, PCR amplification using synthetic DNA primers containing a portion of the nucleotide sequence of the CDS region of the IL-2 or GM-CSF gene, or by hybridization of the DNA incorporated into an appropriate expression vector with a labeled DNA fragment or synthetic DNA containing the nucleotide sequence of the CDS region of the IL-2 or GM-CSF gene. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd ed. (supra).

[0053] The nucleotide sequence of DNA can be converted using known kits, such as MutanTM-super Express Km (Takara Shuzo Co., Ltd.) and MutanTM-K (Takara Shuzo Co., Ltd.), according to known methods such as the ODA-LA PCR method, the Gapped duplex method, the Kunkel method, or methods similar thereto.

[0054] The cloned DNA can be used as is, or after digestion with a restriction enzyme or the addition of a linker, depending on the purpose. The DNA may have a translation initiation codon, ATG, at its 5'-end and a translation termination codon, TAA, TGA, or TAG, at its 3'-end. These translation initiation and termination codons can be added using an appropriate synthetic DNA adapter.

[0055] An expression vector containing a nucleic acid encoding IL-2 or GM-CSF can be produced, for example, by excising a desired fragment from a nucleic acid encoding the CDS region of the IL-2 or GM-CSF gene and ligating the fragment downstream of the promoter in the above-mentioned expression vector. The expression vector preferably contains a transcription termination signal, i.e., a terminator region, downstream of the nucleic acid encoding IL-2 or GM-CSF. Furthermore, the expression vector may further contain a selectable marker gene for selecting transformed cells (e.g., a gene conferring resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, or phosphinothricin, or a gene complementing an auxotrophic mutation).

[0056] In another preferred embodiment, the oncolytic virus of the present invention comprises a nucleic acid encoding IL-2 and / or a nucleic acid encoding GM-CSF together with a nucleic acid encoding CXCL10 in a single oncolytic virus. Accordingly, the present invention also provides the oncolytic virus of the present invention, further comprising a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells and / or a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells. Here, the promoter controlling the expression of CXCL10 and the promoter controlling the expression of IL-2 and / or the promoter controlling the expression of GM-CSF may be a single promoter or separate promoters. When the promoter controlling the expression of CXCL10 and the promoter controlling the expression of IL-2 and / or the promoter controlling the expression of GM-CSF are a single promoter, the nucleic acid encoding CXCL10 and the nucleic acid encoding IL-2 and / or the nucleic acid encoding GM-CSF are linked via a sequence enabling polycistronic expression (e.g., an IRES sequence or a 2A sequence (P2A, T2A, E2A, F2A)).

[0057] When the oncolytic virus of the present invention is an adenovirus, the size of the foreign gene that can be carried is limited. Therefore, placing the foreign genes under the control of a single promoter is advantageous, particularly when carrying a nucleic acid encoding IL-2 and a nucleic acid encoding GM-CSF in addition to a nucleic acid encoding CXCL10. On the other hand, when the oncolytic virus used allows for the insertion of larger foreign genes, placing each nucleic acid under the control of a separate promoter may be preferable, as this allows for highly controlled expression of each immunostimulatory gene. When each nucleic acid is placed under the control of a separate promoter, each promoter may be the same or different and may be an exogenous promoter selected from a ubiquitous promoter, a cancer cell-specific promoter, and a promoter specific to the organ from which the cancer cells are derived. Polycistronic expression may result in differences in the expression levels of each gene. Therefore, to ensure equal expression levels for each gene, each nucleic acid may be linked downstream of the same separate promoter.

[0058] One advantage of incorporating a nucleic acid encoding CXCL10, a nucleic acid encoding IL-2, and / or a nucleic acid encoding GM-CSF into a single oncolytic virus is improved infection and expression efficiency and reduced dosage. If each gene is incorporated into a separate oncolytic virus, simultaneous infection of cancer cells may result in reduced expression efficiency, requiring two or three times the dosage to introduce the same number of copies.

[0059] An oncolytic virus comprising a nucleic acid encoding CXCL10 and a nucleic acid encoding IL-2 and / or a nucleic acid encoding GM-CSF in a polycistronic expression arrangement, (i) CXCL10-IL-2; (ii) CXCL10-GM-CSF; (iii) IL-2-CXCL10; (iv) GM-CSF-CXCL10; (v) CXCL10-IL-2-GM-CSF; (vi) CXCL10-GM-CSF-IL-2; (vii) IL-2-CXCL10-GM-CSF; (viii) IL-2-GM-CSF-CXCL10; (ix) GM-CSF-CXCL10-IL-2; or (x) GM-CSF-IL-2-CXCL10 Examples of such viruses include oncolytic viruses, which are arranged in the order of

[0060] In a preferred embodiment, in an oncolytic virus comprising a nucleic acid encoding CXCL10, a nucleic acid encoding IL-2, and a nucleic acid encoding GM-CSF in a polycistronic expression-enabling arrangement, the first nucleic acid and the second nucleic acid are linked from the 5' side via a P2A sequence, and the second nucleic acid and the third nucleic acid are linked via a T2A sequence.

[0061] The present invention provides oncolytic virus immunotherapy (OVI) using two immunostimulatory genes, the CXCL10 gene and either the IL-2 gene or the GM-CSF gene, in combination, or three immunostimulatory genes, the CXCL10 gene, the IL-2 gene, and the GM-CSF gene. While the two-factor combination enhances the therapeutic effect compared to the CXCL10 gene alone, the three-factor combination can further enhance the therapeutic effect.

[0062] When three factors are used in combination, the three factors may be contained in separate oncolytic viruses, two of the factors may be contained in a single oncolytic virus and the other factor may be contained in a separate oncolytic virus, or all three factors may be contained in a single oncolytic virus, and any of these embodiments may be used. Thus, in a preferred embodiment, an oncolytic virus of the present invention containing only a nucleic acid encoding CXCL10 as an immunostimulatory gene, and (c) an oncolytic virus comprising a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells and a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells; A combination of is provided. The oncolytic virus (c) above can be produced in the same manner as the oncolytic virus of the present invention containing two or more immunostimulatory genes.

[0063] The present invention also provides an oncolytic virus immunotherapy agent (OVI), i.e., a cancer therapeutic agent (hereinafter also referred to as "the OVI of the present invention" or "the therapeutic agent of the present invention"), which contains any of the oncolytic viruses of the present invention or a combination of any of the oncolytic viruses as an active ingredient.

[0064] The cancers targeted by the therapeutic agent of the present invention are not particularly limited, and examples thereof include renal cell carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, and liver cancer. Cancers that can be treated with the present invention include, but are not limited to, cholangiocarcinoma, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic leukemia, polycythemia vera, lymphoma, multiple myeloma, etc. Preferred target cancers include refractory cancers with invasiveness and metastasis that are ineffective against conventional OVI and other existing cancer treatments.

[0065] In the therapeutic agent of the present invention, the oncolytic virus as the active ingredient can be mixed with a pharmacologically acceptable carrier as needed to prepare various formulations, such as injections, and then used as a therapeutic agent for cancer. As the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical ingredients are used, and are incorporated as excipients, lubricants, binders, disintegrants in solid formulations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid formulations. Furthermore, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as needed.

[0066] Suitable examples of excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate. Suitable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica, and the like. Suitable examples of binders include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Suitable examples of disintegrants include lactose, sucrose, starch, carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, sodium carboxymethylstarch, light anhydrous silicic acid, and low-substituted hydroxypropylcellulose. Suitable examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil, and the like. Suitable examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate. Suitable examples of suspending agents include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates; and polyoxyethylene hydrogenated castor oil. Suitable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, and the like. Suitable examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate. Suitable examples of soothing agents include benzyl alcohol. Suitable examples of the preservative include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid. Suitable examples of antioxidants include sulfites and ascorbic acid salts. Suitable examples of coloring agents include water-soluble food tar dyes (e.g., food dyes such as Food Red Nos. 2 and 3, Food Yellow Nos. 4 and 5, and Food Blue Nos. 1 and 2), water-insoluble lake dyes (e.g., aluminum salts of the above-mentioned water-soluble food tar dyes), and natural dyes (e.g., β-carotene, chlorophyll, red iron oxide, etc.). Suitable examples of sweeteners include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia, and the like.

[0067] The therapeutic agents of the present invention can be administered either by the ex vivo method, in which the cancer cells of the animal to be treated are removed from the body, cultured, and then introduced back into the body (or transplanted), or by the in vivo method, in which the vector is directly administered into the body of the recipient. In the ex vivo method, the vector can be introduced into the target cells by microinjection, calcium phosphate coprecipitation, PEG, electroporation, or the like. In the in vivo method, the formulation can be administered, for example, by injection, catheter, balloon catheter, local injection, or implantation of an implant incorporating the oncolytic virus of the present invention into the lesion.

[0068] The dose of the therapeutic agent of the present invention varies depending on the type of oncolytic virus, promoter activity in the target cancer cells, the type of immunostimulatory gene to be combined, the administration route, the severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc., of the administered subject. For example, when a cancer-specific replication-competent adenovirus is used as the oncolytic virus, the dose of the therapeutic agent of the present invention is 1 x 10 viral particles (vp) in clinical trials of conventional cancer gene therapy. 10 ~10 12 Since safety has been confirmed using vp / tumor, the same amount is used as a guide for administration (Molecular Therapy, 18: 429-434, 2010).

[0069] The therapeutic agent of the present invention can be administered once or multiple times, and the number of doses and the interval between doses can be selected appropriately.

[0070] The therapeutic agent of the present invention can be used in combination with other cancer therapeutic agents or treatment methods. Examples of other cancer therapeutic agents or treatment methods include immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, etc.), CAR-T cell therapy, chemotherapeutic agents (e.g., 5-fluorouracil, cisplatin, etc.), and radiation therapy. Immune checkpoint inhibitors are known to be less effective against cancers with low immune cell infiltration. However, the therapeutic agent of the present invention activates immune cells and promotes their infiltration into cancer tissue, and is therefore expected to have a strong synergistic effect. Furthermore, CAR-T cell therapy has yet to achieve sufficient therapeutic efficacy against solid cancers. Possible reasons for this include the difficulty of CAR-T cells infiltrating into solid cancer tissues, the presence of cancer cells that do not express the target cancer antigen, and the immunosuppressive environment within cancer tissues, which inhibits CAR-T cell function. Administration of the OVI of the present invention is expected to induce the expression of cytokines and chemokines, promote tumor infiltration of CAR-T cells, and improve the immunosuppressive environment within cancer tissue, preventing the suppression of CAR-T cell function.

[0071] Other cancer therapeutic agents or treatment methods used in combination with the therapeutic agent of the present invention can be administered or carried out in accordance with the dosage and administration of the therapeutic agent or treatment method when used alone.

[0072] The present invention will be described in more detail using the following examples, but the scope of the present invention is not limited to these examples in any way. [Example]

[0073] Materials and Methods [Cell culture] The HaK (Syrian hamster renal cancer cell line) cell line was provided by Dr. William SM Wold (Saint Louis University School of Medicine) [Thomas, MA, et al., Syrian hamster as a permissive immunocompetent animal model for the study of oncolytic adenovirus vectors. Cancer Res, 2006. 66(3): pp. 1270-1276.]. HaP-T1 (Syrian hamster pancreatic adenocarcinoma) cells were obtained from the Riken Cell Bank (Ibaraki, Japan), and BHK-21 (Baby hamster kidney) cells were obtained from the JCRB Cell Bank (Osaka, Japan). HaK cells were cultured in Dulbecco's modified Eagle's medium (Nacalai Tesque, Kyoto, Japan). HaP-T1 cells were cultured in MEM supplemented with 1% non-essential amino acids (Sigma-Aldrich, St. Louis, MO) and 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA). BHK-21 cells were cultured in MEM supplemented with 1% NEAA. All media were supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaille, France), 100 units / mL penicillin, and 100 μg / mL streptomycin (Thermo Fisher Scientific).

[0074] [Animal experiments] All animal experiments were conducted in accordance with the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and with the approval of the Laboratory Animal Research Institute, Research Support Center, Kagoshima University.

[0075] [Remote Site Challenge Test] Hamsters were challenged with tumorigenic doses of parental (HaK) or heterologous (HaP-T1) cancer cells 2 weeks after virus administration. Briefly, hamsters bearing subcutaneous tumors were administered Surv.m-CRA (no transgene) (n = 6), Surv.m-CRA / RSVp-mIL-2 (n = 6), Surv.m-CRA / RSVp-mIL-2 + Surv.m-CRA / CAp-mCXCL10 (n = 6), Surv.m-CRA / RSVp-mIL-2 + Surv.m-CRA / E2Fp-mGM-CSF (n = 6), or Surv.m-CRA / RSVp-mIL-2 + Surv.m-CRA / CAp-mCXCL10 + Surv.m-CRA / E2Fp-mGM-CSF (n = 6), as described below. After 14 days, the hamsters were inoculated with HaK cells and HaP-T1 cells (1 × 10 per group). 7 The animals were then macroscopically observed for the presence of tumor nodules after various viral treatments for an additional 74 days.

[0076] [Statistical analysis] Data are expressed as mean ± standard error. Data were evaluated by two-tailed Student's t-test. A value of P < 0.05 was considered to indicate statistical significance.

[0077] [Example 1] Preparation of Surv.m-CRA-2 carrying cytokine genes E1-deleted replication-deficient adenovirus (Ad.dE1.3) was prepared as previously reported [Murofushi, Y., et al., Cell cycle-specific changes in hTERT promoter activity in normal and cancerous cells in adenoviral gene therapy: a promising implication of telomerase-dependent targeted cancer gene therapy. Int J Oncol, 2006. 29(3): pp. 681-8; Kamizono, J., et al., Survivin-responsive conditionally replicating adenovirus exhibits cancer-specific and efficient viral replication. Cancer Res, 2005. 65(12): pp. 5284-91; Kamizono, J., et al., Survivin-responsive conditionally replicating adenovirus exhibits cancer-specific and efficient viral replication. Cancer Res, 2005. 65(12): pp. 5284-91; Terazaki, Y., et al., An optimal therapeutic expression level is crucial for suicide gene therapy for hepatic metastatic cancer in mice. Hepatology, 2003. 37(1): p. 155-63., Ushikoshi, H., et al., Local overexpression of HB-EGF aggravates remodeling following myocardial infarction by activating noncardiomyocytes. Lab Invest, 2005. 85(7): p. 862-73., Sakamoto, K., et al., Heparin-binding epidermal growth factor-like growth factor and hepatocyte growth factor inhibit cholestatic liver injury in mice through different mechanisms. Int J Mol Med, 2016. 38(6): p. 1673-1682., Khai, N.C., et al., In vivo hepatic HB-EGF gene transduction inhibits Fas-induced liver injury and induces liver regeneration in mice: a comparative study to HGF. J Hepatol, 2006. 44(6): p. 1046-54., Horikawa, Y., et al., Assessment of an altered E1B promoter on the specificity and potency of triple-regulated conditionally replicating adenoviruses: implications for the generation of ideal m-CRAs. Cancer Gene Ther, 2***1. 18(10): p. 724-33.]。尚、Ad.dE1.3は導入遺伝子を含まない。.

[0078] It should be noted that there seems to be an error in the original text where "2***1" is likely a typo. It should probably be "2011". The translation would be more accurate with the corrected text.Suv.m-CRA, which contains wild-type E1A downstream of the Survivin promoter, E1BΔ55K downstream of the CMV promoter, and a therapeutic gene downstream of a specific promoter, was constructed using a previously reported m-CRA construction method [Watanabe, M., et al., Adenovirus Biology, Recombinant Adenovirus, and Adenovirus Usage in Gene Therapy. Viruses, 2021. 13(12)., Kamizono, J., et al., Survivin-responsive conditionally replicating adenovirus exhibits cancer-specific and efficient viral replication. Cancer Res, 2005. 65(12): p. 5284-91., Tanoue, K., et al., Survivin-responsive conditionally replicating adenovirus kills rhabdomyosarcoma stem cells more efficiently than their progeny. J Transl Med, 2014. 12: p. 27., Horikawa, Y., et al., Assessment of an altered E1B promoter on the specificity and potency of triple-regulated conditionally replicating adenoviruses: implications for the generation of ideal m-CRAs. Cancer Gene Ther, 2011. 18(10): p. 724-33.].

[0079] We constructed CRAs carrying nucleic acids encoding CXCL10, GM-CSF, and / or IL-2 as therapeutic genes. Specifically, we constructed the following 16 types of CRAs: (1) Surv.m-CRA / E2Fp-mCXCL10 (2) Surv.m-CRA / RSVp-mCXCL10 (3) Surv.m-CRA / CMVp-mCXCL10 (4) Surv.m-CRA / CAp-mCXCL10 (5) Surv.m-CRA / E2Fp-hCXCL10 (6) Surv.m-CRA / RSVp-hCXCL10 (7) Surv.m-CRA / CMVp-hCXCL10 (8) Surv.m-CRA / CAp-hCXCL10 (9) Surv.m-CRA / E2Fp-mGM-CSF (10) Surv.m-CRA / RSVp-mIL-2 (11) Surv.m-CRA / CMV-mCXCL10-mIL2-mGM (12) Surv.m-CRA / CMV-mCXCL10-mGM-mIL2 (13) Surv.m-CRA / CMV-hCXCL10-hIL2-hGM (14) Surv.m-CRA / CMV-hCXCL10-hGM-hIL2 (15) Surv.m-CRA(No transgene) (16) Surv.m-CRA / CMVp-EGFP

[0080] E2Fp refers to the E2F-1 promoter, RSVp refers to the Rous sarcoma virus long terminal repeat (RSV) promoter, CMVp refers to the human cytomegalovirus (CMV) promoter, and Cap refers to the CMV enhancer and beta-actin (CA) promoter. The "m" or "h" prefixed to each cytokine name indicates mouse or human origin, respectively. (15) Surv.m-CRA (No transgene) does not express a therapeutic gene, and (16) Surv.m-CRA / CMVp-EGFP expresses the enhanced green fluorescent protein (EGFP) gene under the control of the CMV promoter. For reference, the gene sequences of mouse and human CXCL10-single CRAs are shown in Figure 1-1 (mouse) and Figure 1-2 (human). The gene sequences of the CRA carrying three therapeutic genes are shown in Figure 1-3 (mouse and human). In Figure 1-3, P2A represents the porcine teschovirus-1-derived 2A sequence, and T2A represents the Thosea asigna virus-derived 2A sequence.

[0081] [Example 2] In vitro functional verification of the prepared Surv.m-CRA-2 Of the various CRAs prepared in Example 1, the protein expression of CXCL10 in cells was examined. Specifically, HEK293 cells seeded on 6-well plates were transfected with various P2 plasmids expressing the CXCL10 gene. Additionally, various Surv.m-CRAs expressing the CXCL10 gene were infected into HaK, HaP-T1, or BHK-21 cells at an MOI of 1, 10, or 100 (only HaK cells were infected with CRAs carrying the human CXCL10 gene). All cells were cultured for 48 hours, after which the supernatants were collected and frozen at -80°C. CXCL10 protein expression analysis in HEK293 cells and in HaK, HaP-T1, or BHK-21 cells was performed as follows. Mouse CXCL10 was measured using the Mouse CXCL10 DuoSet ELISA (DY466, R&D Systems, Minneapolis, MN), and human CXCL10 was measured using the Human CXCL10 Quantikine ELISA Kit (DIP100, R&D Systems) according to the manufacturer's protocol. The total protein concentration of each sample was determined by Bradford assay (Bio-Rad, Hercules, CA).

[0082] The results are shown in Figure 2-1 (A: Expression of mouse CXCL10 in HEK293 cells transfected with a P2 plasmid containing nucleic acid encoding mouse CXCL10; B: Expression of human CXCL10 in HEK293 cells transfected with a P2 plasmid containing nucleic acid encoding human CXCL10), Figure 2-2 (Expression of mouse CXCL10 in HaK cells, HaP-T1 cells, or BHK-21 cells infected with a CRA carrying the mouse CXCL10 gene), and Figure 2-3 (Expression of human CXCL10 in HaK cells infected with a CRA carrying the human CXCL10 gene).

[0083] As shown in Figures 2-1 to 2-3, mouse CXCL10 protein was not detected in cells infected with Surv.m-CRA (no transgene) or without infection. In contrast, infection with Surv.m-CRA / E2Fp-mCXCL10, Surv.m-CRA / RSVp-mCXCL10, Surv.m-CRA / CMVp-mCXCL10, or Surv.m-CRA / CAp-mCXCL10 resulted in a low, mild, moderate, or high increase in mouse CXCL10 protein secretion in a virus dose-dependent manner in all cell lines (approximately 1-fold, 10-fold, and 100-fold differences, respectively). Furthermore, similar trends were observed with each Surv.m-CRA containing the human CXCL10 gene.

[0084] [Example 3] Examination of in vitro cytotoxicity after virus infection (WST assay) The cytotoxic effect after viral infection in vitro was examined using Surv.m-CRA carrying the mouse CXCL10 gene prepared in Example 1. Specifically, the experiment was carried out as follows: HaK cells (800 cells / well), HaP-T1 cells, or BHK-21 cells (500 cells / well) seeded in a 24-well plate were infected with Surv.m-CRA or Ad.dE1.3 at an MOI of 0.3. Cell viability was measured 3 and 5 days after infection by WST-8 assay using Cell Count Reagent SF (registered trademark) (Nacalai Tesque) according to the manufacturer's protocol and previously reported methods [Tanoue, K., et al., Survivin-responsive conditionally replicating adenovirus kills rhabdomyosarcoma stem cells more efficiently than their progeny. J Transl Med, 2014. 12: p. 27., Terazaki, Y., et al., An optimal therapeutic expression level is crucial for suicide gene therapy for hepatic metastatic cancer in mice. Hepatology, 2003. 37(1): pp. 155-63., Horikawa, Y., et al., Assessment of an altered E1B promoter on the specificity and potency of triple-regulated conditionally replicating adenoviruses: implications for the generation of ideal m-CRAs. Cancer Gene Ther, 2011. 18(10): p. 724-33., Mitsui, K., et al., Conditionally replicating adenovirus prevents pluripotent stem cell-derived teratoma by specifically eliminating undifferentiated cells. Mol Ther Methods Clin Dev, 2015. 2: p. 15026., Yuge, K., et al., Adenoviral gene transduction of hepatocyte growth factor elicits inhibitory effects for hepatoma. Int J Oncol, 2005. 27(1): p. 77-85.]. .

[0085] In short, it is as follows: (1) The viruses used were Ad.dE1.3, Surv.m-CRA (no transgene), Surv.m-CRA / CMV-EGFP, Surv.m-CRA / E2F-mCXCL10, Surv.m-CRA / RSV-mCXCL10, Surv.m-CRA / CMV-mCXCL10, and Surv.m-CRA / CA-mCXCL10 (MOI: 0.3). (2) Hak cells were seeded in a 24-well plate at 800 cells / well, and Hap-T1 cells or BHK-21 cells were seeded at 500 cells / well. (3) Culture for 24 hours. (4) Virus infection and incubation (3 to 5 days). (5) Replace the medium with 500 μl of medium containing 50 μl of Cell Count Reagent SF. (6) Incubate (1 to 4 hours). (7) Measure the absorbance at 450 nm using a microplate reader.

[0086] The results are shown in Figure 3-1. Figures 3-2 to 3-4 show representative phase-contrast images of HaK cells, HaP-T1 cells, and BHK-21 cells 3 and 5 days after virus infection, at 40x (left) and 100x (right) magnifications, respectively.

[0087] As shown in Figure 3-1, Surv.m-CRA (No transgene) induced significant cell death in cancer cells, but not in normal cells. This indicates that Surv.m-CRA replicates strictly in hamster cancer cells, as previously reported in human cells [Watanabe, M., et al., Adenovirus Biology, Recombinant Adenovirus, and Adenovirus Usage in Gene Therapy. Viruses, 2021. 13(12)., Kamizono, J., et al., Survivin-responsive conditionally replicating adenovirus exhibits cancer-specific and efficient viral replication. Cancer Res, 2005. 65(12): p. 5284-91., Tanoue, K., et al., Survivin-responsive conditionally replicating adenovirus kills rhabdomyosarcoma stem cells more efficiently than their progeny. J Transl Med, 2014. 12: p. 27., Horikawa, Y., et al., Assessment of an altered E1B promoter on the specificity and potency of triple-regulated conditionally Replicating adenoviruses: implications for the generation of ideal m-CRAs. Cancer Gene Ther, 2011. 18(10): p. 724-33., Mitsui, K., et al., Conditionally replicating adenovirus prevents pluripotent stem cell-derived teratoma by specifically eliminating undifferentiated cells. Mol Ther Methods Clin Dev, 2015. 2: p. 15026., Ide, K., et al., A Novel Construction of Lentiviral Vectors for Eliminating Tumorigenic Cells from Pluripotent Stem Cells. Stem Cells, 2018. 36(2): p. 230-239.] Expression of transgenes containing mCXCL10 and EGFP showed varying degrees of cytotoxicity. Because EGFP remains intracellularly and non-immune cells do not express the CXCL10 receptor, this transgene-dependent cytotoxicity is presumed to be due to the nonspecific action of the transgene-derived amplified protein within the infected cells.

[0088] [Example 4] Establishment of a syngenic Syrian hamster cancer model To fully investigate the in vivo therapeutic effects, such as replication-dependent virus-dependent cytotoxicity and cytokine-induced immune responses, we established a syngenic hamster cancer model by subcutaneously transplanting HaK cells into Syrian hamsters that are permissive for human serotype 5 adenovirus replication. Figure 4 shows an overview of the establishment process.

[0089] Human adenovirus does not grow in mice, but can partially grow in Syrian hamsters, so this model is thought to be useful as a refractory cancer model for Surv.m-CRA treatment.

[0090] [Example 5] Examination of the therapeutic effect of CXCL10-loaded Surv.m-CRA-2 Using the Syrian hamster subcutaneous tumor model established in Example 4, the therapeutic effect of CXCL10-loaded Surv.m-CRA-2 was examined.

[0091] Hamster models of subcutaneous tumors were grown using 2 × 10 cells suspended in 200 μL of DMEM containing 50% Matrigel (BD Biosciences, Franklin Lakes, NJ). 7 HaK cells were transplanted into the dorsal flank of 5- to 6-week-old female Syrian hamsters (Japan SLC, Shizuoka, Japan). Tumor volumes were 200 to 580 mm. 3 After reaching this age, the hamsters were randomly divided into 5 to 6 groups.

[0092] For monotherapy with each Surv.m-CRAs expressing CXCL10, 1.0 × 10 9 A single injection of 100 μL of a buffer solution (10 mmol / L Tris-HCl pH 7.4, 1 mmol / L MgCl, 10% glycerol, and 20 μg / ml hexadimethrine bromide) containing 1.0 × 10 pfu (plaque-forming unit) was administered into the tumor. 9 pfu of Ad.dE1.3 (n=7), Surv.m-CRA (No transgene) (n=8), Surv.m-CRA / E2Fp-hCXCL10 (n=8), Surv.m-CRA / CMVp-hCXCL10 (n=7), or Surv.m-CRA / CMVp-mCXCL10 (n=8) were administered.

[0093] Thereafter, tumor size was measured twice a week, and tumor volume was calculated using the following formula: Volume = major axis x (minor axis) 2 ×0.5(mm 3 ). Body weight was monitored twice weekly using a digital balance. Histopathological analysis was performed as previously reported [Kamizono, J., et al., Survivin-responsive conditionally replicating adenovirus exhibits cancer-specific and efficient viral replication. Cancer Res, 2005. 65(12): pp. 5284-91; Chen, SH, et al., Combination gene therapy for liver metastasis of colon carcinoma in vivo. Proc Natl Acad Sci USA, 1995. 92(7): pp. 2577-81; Terazaki, Y., et al., An optimal therapeutic expression level is crucial for suicide gene therapy for hepatic metastatic cancer in mice. Hepatology, 2003. 37(1): pp. 155-63; Horikawa, Y., et al., Assessment of an altered E1B promoter on the specificity and potency of triple-regulated conditionally replicating adenoviruses: implications for the generation of ideal m-CRAs. Cancer Gene Ther, 2011. 18(10): p. 724-33.].

[0094] The results are shown in Figure 5. As shown in Figure 5, a single intratumoral injection of Surv.m-CRA (no transgene) significantly suppressed tumor growth compared to the control replication-deficient Ad.dE1.3-treated group, although the difference was only significant at some time points. Three types of Surv.m-CRA expressing CXCL10 under the control of different promoters (Surv.m-CRA / E2Fp-hCXCL10, Surv.m-CRA / CMVp-hCXCL10, and Surv.m-CRA / CMVp-mCXCL10) all further enhanced tumor growth suppression compared to Surv.m-CRA (no transgene). This effect was most pronounced in hamsters treated with Surv.m-CRA / CMVp-hCXCL10. However, no significant difference was observed in hamsters treated with Surv.m-CRA / CMVp-mCXCL10.

[0095] [Example 6] Examination of the therapeutic effect of Surv.m-CRA-2 by combining two types of cytokines We investigated whether combining the two cytokines would enhance the therapeutic effect.

[0096] On day 0, one hamster from each group was given 2.0 × 10 9 A single intratumor injection of 100 μL of buffer containing pfu Ad.dE1.3 (n=7), Surv.m-CRA (no transgene) (n=7), Surv.m-CRA / E2Fp-mGM-CSF (n=7), Surv.m-CRA / CAp-mCXCL10 (n=7), a combination of Surv.m-CRA / CAp-mCXCL10 and Surv.m-CRA / E2Fp-mGM-CSF (n=8), or a combination of Surv.m-CRA / CAp-mCXCL10 and Surv.m-CRA / RSVp-mIL-2 (n=8) was performed. Tumor size was measured as in Example 5.

[0097] The results are shown in Figure 6. As shown in Figure 6, monotherapy with Surv.m-CRA (no transgene) or Surv.m-CRA expressing a transgene (CXCL10 or GM-CSF) did not significantly suppress tumor growth compared to the control group. On the other hand, when Surv.m-CRA / CAp-mCXCL10 was combined with Surv.m-CRA / E2Fp-mGM-CSF or Surv.m-CRA / RSVp-mIL-2, the tumor growth inhibitory effect was dramatically enhanced compared to the control. Therefore, it was suggested that combination therapy using a combination of therapeutic genes provides a synergistic therapeutic effect compared to monotherapy.

[0098] [Example 7] Examination of the therapeutic effect of Surv.m-CRA-2 by combining three types of cytokines We investigated whether the combined use of three cytokines enhances the therapeutic effect. In this example, we also investigated the therapeutic effect on the primary tumor and the induction of primary tumor-specific systemic antitumor immunity in a distant site challenge test. An overview of the experiment is shown in Figure 7-1.

[0099] On day 0, one hamster from each group received 1.5 × 10 9 A single intratumor injection of 100 μL of buffer containing pfu of Surv.m-CRA (no transgene) (n = 6), Surv.m-CRA / RSVp-mIL-2 (n = 6), a combination of Surv.m-CRA / RSVp-mIL-2 and Surv.m-CRA / CAp-mCXCL10 (n = 6), a combination of Surv.m-CRA / RSVp-mIL-2 and Surv.m-CRA / E2Fp-mGM-CSF (n = 6), or a combination of Surv.m-CRA / RSVp-mIL-2, Surv.m-CRA / CAp-mCXCL10, and Surv.m-CRA / E2Fp-mGM-CSF (n = 6) was administered.

[0100] In addition, a distant site challenge test was performed as follows: Two weeks after virus administration, hamsters were challenged with a tumorigenic dose of parental cancer cells (HaK) or heterologous cancer cells (HaP-T1). Specifically, hamsters bearing subcutaneous tumors were treated with Surv.m-CRA (no transgene) (n = 6), Surv.m-CRA / RSVp-mIL-2 (n = 6), a combination of Surv.m-CRA / RSVp-mIL-2 and Surv.m-CRA / CAp-mCXCL10 (n = 6), a combination of Surv.m-CRA / RSVp-mIL-2 and Surv.m-CRA / E2Fp-mGM-CSF (n = 6), or a combination of Surv.m-CRA / RSVp-mIL-2, Surv.m-CRA / CAp-mCXCL10, and Surv.m-CRA / E2Fp-mGM-CSF (n = 6) in the same manner as described above. After 14 days, the hamsters were inoculated with HaK cells and HaP-T1 cells (1 × 10 per group). 7 The animals were inoculated with 1000 mAbs of 1000 mAbs each into the left and right dorsal regions, respectively. Treated animals were macroscopically observed for the presence of tumor nodules after various viral treatments for an additional 74 days. Furthermore, body weight was monitored as an indicator of the side effects of these treatments.

[0101] The results are shown in Figure 7-2 (therapeutic effect of combined treatment with three types of Surv.m-CRA-2 on primary tumors), Figure 7-3 (regression of distant HaK tumors by combined treatment with three types of Surv.m-CRA-2 (challenge test)), Figure 7-4 (regression of distant Hap-T1 tumors by combined treatment with three types of Surv.m-CRA-2 (challenge test)), and Figure 7-5 (changes in body weight over time).

[0102] This example demonstrated that the use of Surv.m-CRA, which controls the expression of three cytokines with different mechanisms of action (CXCL10, GM-CSF, and IL-2), can more effectively suppress tumor growth than monotherapy or combination therapy with two cytokines. Additionally, this example demonstrated that the therapeutic strategy of the present invention can induce systemic antitumor immunity. Furthermore, when using the therapeutic strategy of the present invention, the poor weight gain was mild between treatment groups, suggesting no significant side effects.

[0103] [Example 8] Confirmation of the expression of each gene in the P2 plasmid carrying three types of therapeutic genes Surv.m-CRA-2 carrying the three therapeutic genes prepared in Example 1 (i.e., (12) Surv.m-CRA / CMV-mCXCL10-mIL2-mGM (hereinafter, sometimes referred to as "Surv.m-CRA / CMVp-mCIG"), (13) Surv.m-CRA / CMV-mCXCL10-mGM-mIL2 (hereinafter, sometimes referred to as "Surv.m-CRA / CMVp-mCGI"), (14) Surv.m-CRA / CMV-hCXCL10-hIL2-hGM (hereinafter, sometimes referred to as "Surv.m-CRA / CMVp-hCIG"), and (15) Each P2 plasmid used in the construction of Surv.m-CRA / CMV-hCXCL10-hGM-hIL2 (hereinafter sometimes referred to as "Surv.m-CRA / CMVp-hCGI") was transfected into HEK293 cells using the same method as in Example 2, and the expression levels of each protein were confirmed. To detect the transgene proteins (mCXCL10, mIL-2, mGM-CSF, hCXCL10, hIL-2, and hGM-CSF) in vitro, MILLIPLEX® Mouse Cytokine / Chemokine Magnetic Bead Panel (MCYTOMAG-70K, Millipore, Burlington, MA) and MILLIPLEX® Human Cytokine / Chemokine / Growth Factor Panel A (HCYTA-60K, Millipore) were used with the Luminex-MAGPIX multiplex immunoassay system according to the manufacturer's instructions. Data were analyzed using xPONENT Software (Millipore).

[0104] As a result, the expression of mouse or human CXCL10, IL-2, and GM-CSF was confirmed by transfection of P2 plasmid containing nucleic acids encoding mouse or human CXCL10, IL-2, and GM-CSF.

[0105] [Example 9] Confirmation of gene expression in Surv.m-CRA-2 carrying three types of therapeutic genes Surv.m-CRA / CMVp-mCIG, Surv.m-CRA / CMVp-mCGI, Surv.m-CRA / CMVp-hCIG, and Surv.m-CRA / CMVp-hCGI carrying the three therapeutic genes prepared in Example 1 were infected into HEK293 cells, and the expression levels of the proteins encoded by each therapeutic gene were confirmed by ELISA. Culture supernatants were collected 48 hours after virus infection, and the expression levels of mouse genes were examined using the MILLIPLEX® Mouse Cytokine / Chemokine panel. Expression levels of human genes were measured using the Human CXCL10 Quantikine ELISA Kit (R&D Systems), Human GM-CSF DuoSet ELISA (R&D Systems), and Human IL-2 Quantikine ELISA Kit (R&D Systems). HEK293 cells were transfected with the P2 plasmids (pUni / CMVp-mCIG, pUni / CMVp-mCGI, pUni / CMVp-hCIG, and pUni / CMVp-hCGI) used to construct each virus, and the supernatants collected 96 hours later were used as positive controls.

[0106] As a result, in both mice and humans, the expression levels of CXCL10, GM-CSF, and IL-2 were almost equal in CGI (Figure 8). On the other hand, higher levels of GM-CSF were detected in CIG compared to CXCL10 and IL-2 (Figure 8, right). In any case, expression of all three factors was confirmed in both constructs.

[0107] [Example 10] In vitro cytotoxicity of Surv.m-CRA-2 carrying three therapeutic genes (WST assay) The cytotoxicity of Surv.m-CRA / CMVp-mCGI or Surv.m-CRA / CMVp-mCIG against hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21) was examined by measuring the number of viable cells. On the previous day, 5 × 10 cells of each type were added to a 96-well plate. 3Cells were seeded at 1000 cells / well. Cells were infected with Ad.dE1.3 (control), Surv.m-CRA (no transgene), Surv.m-CRA / CMVp-mCGI, or Surv.m-CRA / CMVp-mCIG at an MOI of 3 for 1 hour and then cultured. Cytotoxicity was assessed 3 and 5 days after infection by WST-8 assay using the viable cell counting reagent SF (Nacalai Tesque). The experimental protocol is shown in Figure 9-1.

[0108] As a result, in HaK and HaP-T1 cells, Surv.m-CRA (no transgene), Surv.m-CRA / CMVp-mCGI, and Surv.m-CRA / CMVp-mCIG all showed significant cytotoxic effects compared to the control (Figure 9-2).On the other hand, in BHK-21 cells, Surv.m-CRA (no transgene) and Surv.m-CRA / CMVp-mCGI did not show significant cytotoxic effects, but Surv.m-CRA / CMVp-mCIG did show cytotoxic effects (Figure 9-2).

[0109] [Example 11] Examination of the therapeutic effect of Surv.m-CRA-2 carrying three types of therapeutic genes In this example, we confirm the in vivo therapeutic effect of Surv.m-CRA-2 carrying three therapeutic genes. Using the same method as in Example 7 above, except for using Surv.m-CRA-2 carrying three therapeutic genes, we investigate the therapeutic effect on the primary tumor in a Syrian hamster subcutaneous tumor model and the induction of primary tumor-specific systemic antitumor immunity in a distant site challenge test. When three types of OVIs carrying different genes are administered, it is not guaranteed that all three genes will be fully introduced into a single cell. In contrast, when one type of OVI carrying all three genes is administered, all three genes are reliably introduced into a single cell. Therefore, administering one virus carrying all three genes may enhance the therapeutic effect compared to administering three different types of OVIs. Furthermore, administering one virus carrying all three genes simultaneously allows for a lower dosage compared to administering three types of OVIs, which is expected to improve safety. Example 9 confirmed that Surv.m-CRA-2 carrying all three genes correctly expressed the proteins encoded by each therapeutic gene, and Example 10 confirmed its in vitro cytotoxic effect on cancer cells. Therefore, treatment with Surv.m-CRA-2 carrying all three genes simultaneously is expected to achieve therapeutic efficacy and safety comparable to or superior to the therapeutic efficacy shown in Example 7.

[0110] (1) Inhibition of tumor growth at the primary site 1 x 10 7 HaK cells were subcutaneously transplanted into a single dorsal site at a density of 1 × 10 cells in 50% Matrigel. 7 Approximately 14 days later, when the diameter of the transplanted tumor reached 6-10 mm, the cells were treated with either Surv.m-CRA (no transgene) or Surv.m-CRA / CMVp-mCGI viruses (both in PBS at 1.5 × 10 9 PFU / 100 μl), or a mixture of Surv.m-CRA / CAp-mCXCL10, Surv.m-CRA / E2Fp-mGM-CSF, and Surv.m-CRA / RSVp-mIL-2 viruses (5 × 10 8 PFU / 100 μl, total 1.5×10 9A single injection of Surv.m-CRA (adjusted to PFU / 100 μl) was administered into the tumor, and changes in tumor diameter over time were evaluated. The numbers of mice in each group administered Surv.m-CRA (no transgene), Surv.m-CRA / CAp-mCXCL10 + Surv.m-CRA / E2Fp-mGM-CSF + Surv.m-CRA / RSVp-mIL-2, and Surv.m-CRA / CMVp-mCGI were 8, 8, and 7, respectively. The experimental protocol is shown in Figure 10-1. Tumor size was measured twice weekly using digital calipers. Tumor volume was calculated as long axis (mm) × short axis (mm) × short axis (mm) × 0.5 (mm). 3 The data are shown as mean ± standard error, and statistically significant differences between groups were tested using Student's T-test.

[0111] As a result, Surv.m-CRA / CAp-mCXCL10 + Surv.m-CRA / E2Fp-mGM-CSF + Surv.m-CRA / RSVp-mIL-2 and Surv.m-CRA / CMVp-mCGI showed significantly enhanced therapeutic effects (tumor-suppressing effects) in the treated primary cancer cells (nodules) compared to Surv.m-CRA (No transgene), which does not carry a therapeutic gene (#, P<0.05 vs Surv.m-CRA (No transgene)) (Figure 10-2).

[0112] (2) Antitumor immune effects against distant metastasis For each hamster in (1) above, 7.5 × 10 6 7.5 × 10 Hak and HaP-T1 cells were each grown in 50% Matrigel. 6 After adjusting the concentration to cells / 100 μl, the cells were subcutaneously transplanted into the dorsal side, and 17 days later, the formation of secondary transplanted tumors was evaluated (FIG. 10-1).

[0113] The results are shown in Table 1. The tumor formation rates after HaK retransplantation in the Surv.m-CRA (no transgene), Surv.m-CRA / CAp-mCXCL10 + Surv.m-CRA / E2Fp-mGM-CSF + Surv.m-CRA / RSVp-mIL-2, and Surv.m-CRA / CMVp-mCGI treatment groups were 88%, 50%, and 29%, respectively. In contrast, tumor formation was observed in all HaP-T1 transplants. These results demonstrate that the Surv.m-CRA / CAp-mCXCL10 + Surv.m-CRA / E2Fp-mGM-CSF + Surv.m-CRA / RSVp-mIL-2 and Surv.m-CRA / CMVp-mCGI treatment groups induce specific systemic antitumor immunity, potently inhibiting (treating) metastatic cancer cells. By carrying three therapeutic genes in one virus, a stronger anti-tumor immune effect was achieved compared to when each gene was carried in a separate virus and administered as a cocktail.

[0114] [Table 1]

[0115] Furthermore, as shown in Figure 10-3, no weight loss was observed in any of the treatment groups after viral therapy, demonstrating high safety. Furthermore, appetite was maintained in the treatment group, confirming that there were no safety issues.

[0116] [Example 12] Examination of the therapeutic effect of Surv.m-CRA-2 carrying three therapeutic genes derived from mice or humans (1) Inhibition of tumor growth at the primary site 1 x 10 7 HaK cells were subcutaneously transplanted into a single dorsal site at a density of 1 × 10 cells in 50% Matrigel. 7Approximately 10 days later, when the diameter of the transplanted tumors reached 6-10 mm, each of the Ad.dE1.3 (control), Surv.m-CRA (no transgene), Surv.m-CRA / CMVp-mCGI, and Surv.m-CRA / CMVp-hCGI viruses was added to the tumors at a concentration of 1.5 × 10 cells / 200 μl in PBS. 9 The virus was adjusted to PFU / 100 μl and injected into the tumor for the first time, and changes in tumor diameter were evaluated over time. Seventeen days after the first injection, the same dose of virus was administered. The numbers of mice in each group administered Ad.dE1.3 (control), Surv.m-CRA (no transgene), Surv.m-CRA / CMVp-mCGI, and Surv.m-CRA / CMVp-hCGI were n = 8, 8, 7, and 8, respectively. The experimental protocol is shown in Figure 11-1. Tumor size was measured twice weekly using digital calipers. Tumor volume was calculated as long axis (mm) × short axis (mm) × short axis (mm) × 0.5 (mm). 3 The data are shown as mean ± standard error, and statistically significant differences between groups were tested using Student's T-test.

[0117] As a result, Surv.m-CRA (No transgene), Surv.m-CRA / CMVp-mCGI, and Surv.m-CRA / CMVp-hCGI all showed significant therapeutic effects (tumor-suppressing effects) compared to the control (Ad.dE1.3) (*, p<0.05 vs. Ad.dE1.3 (Control)) (Figure 11-2). The two types, Surv.m-CRA / CMVp-mCGI and Surv.m-CRA / CMVp-hCGI, showed a tendency for further enhanced therapeutic effects (tumor-suppressing effects) compared to Surv.m-CRA (No transgene), which does not carry a therapeutic gene. By day 17 after the first administration, tumor regrowth was observed in all animals in the Surv.m-CRA (no transgene) group, whereas complete tumor regression was observed in 13.5% of the Surv.m-CRA / CMVp-hCGI group and 29% of the Surv.m-CRA / CMVp-mCGI group (Figure 11-3). Furthermore, by day 17 after the second administration (35 days after the first administration), the tumor regrowth rates were 62.5%, 37.5%, and 28.6% in the Surv.m-CRA (no transgene), Surv.m-CRA / CMVp-hCGI, and Surv.m-CRA / CMVp-mCGI groups, respectively. Compared with Surv.m-CRA without a therapeutic gene, Surv.m-CRA carrying three therapeutic genes derived from mouse or human demonstrated significantly enhanced therapeutic efficacy (Figure 11-4). Without being bound by any particular theory, one possible reason why Surv.m-CRA, which carries a mouse gene, had a higher therapeutic effect than a human gene is that there is a higher homology between mice and hamsters than between humans and hamsters, especially for GM-CSF.

[0118] (2) Antitumor immune effects against distant metastasis For each hamster in (1) above, 7.5 × 10 6 7.5 × 10 Hak and HaP-T1 cells were each grown in 50% Matrigel. 6After adjusting the concentration to 1.5 × 10 cells / 100 μl, the mice were subcutaneously implanted into the dorsal side. As described above, 3 days later (17 days after the initial virus administration), each of the Ad.dE1.3 (control), Surv.m-CRA (no transgene), Surv.m-CRA / CMVp-mCGI, and Surv.m-CRA / CMVp-hCGI viruses was injected in PBS at a concentration of 1.5 × 10 cells / 100 μl. 9 The solution was adjusted to PFU / 100 μl and injected into the primary tumor (second administration), and 14 days later, the formation of secondary transplanted tumors was evaluated (FIG. 11-1).

[0119] The results are shown in Table 2. The tumor formation rates after HaK re-implantation in the Surv.m-CRA (no transgene), Surv.m-CRA / CMVp-mCGI, and Surv.m-CRA / CMVp-hCGI treatment groups were 50%, 29%, and 25%, respectively. In contrast, tumor formation was observed in all HaP-T1 transplants. These results demonstrate that in the therapeutic gene-carrying virus group, two doses of the virus enhanced the release of tumor-associated antigens and immune stimulatory factors from lysed primary cancer cells and the adjuvant-like function of the virus compared to a single dose, resulting in more potent inhibition (treatment) of metastatic cancer cells than in the group treated with a virus not carrying a therapeutic gene.

[0120] [Table 2] [Industrial Applicability]

[0121] The OVI of the present invention not only has an enhanced tumor growth inhibitory effect at the primary lesion compared to conventional OVI, but also has a significantly enhanced ability to induce systemic primary cancer-specific anti-tumor immunity, making it possible for it to exert excellent therapeutic effects even against refractory invasive and metastatic cancers that are ineffective against conventional cancer virotherapy, including OVI, and other existing cancer treatments.Therefore, the present invention is extremely useful.

[0122] This application is based on patent application No. 2024-7682 filed in Japan (filing date: January 22, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. a nucleic acid encoding C-X-C motif chemokine ligand 10 (CXCL10) under the control of a promoter functional in cancer cells, a nucleic acid encoding interleukin-2 (IL-2) under the control of a promoter functional in cancer cells, and / or a nucleic acid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) under the control of a promoter functional in cancer cells; Oncolytic viruses, including

2. The oncolytic virus of claim 1 , comprising a nucleic acid encoding CXCL10, a nucleic acid encoding IL-2, and a nucleic acid encoding GM-CSF.

3. The oncolytic virus according to claim 1, wherein the promoter controlling the expression of CXCL10 and the promoter controlling the expression of IL-2 and / or the promoter controlling the expression of GM-CSF are a single promoter.

4. A nucleic acid encoding CXCL10, a nucleic acid encoding IL-2 and / or a nucleic acid encoding GM-CSF are arranged in the 5'-directed manner. (i) CXCL10-IL-2; (ii) CXCL10-GM-CSF; (iii) IL-2-CXCL10; (iv) GM-CSF-CXCL10; (v) CXCL10-IL-2-GM-CSF; (vi) CXCL10-GM-CSF-IL-2; (vii) IL-2-CXCL10-GM-CSF; (viii) IL-2-GM-CSF-CXCL10; (ix) GM-CSF-CXCL10-IL-2; or (x) GM-CSF-IL-2-CXCL10 The oncolytic virus of claim 3 , wherein the nucleotides are arranged in the order:

5. The oncolytic virus of claim 4, wherein each of the nucleic acids is linked via a 2A sequence or an IRES sequence.

6. The oncolytic virus of claim 5, wherein the first nucleic acid and the second nucleic acid are linked from the 5' side via a P2A sequence, and the second nucleic acid and the third nucleic acid are linked via a T2A sequence.

7. A nucleic acid encoding CXCL10, a nucleic acid encoding IL-2, and a nucleic acid encoding GM-CSF are (v) CXCL10-IL-2-GM-CSF; or (vi) CXCL10-GM-CSF-IL-2 The oncolytic virus according to any one of claims 4 to 6, wherein the oncolytic virus is arranged in the order of:

8. The oncolytic virus according to claim 3 , wherein the promoter is a ubiquitous promoter, a cancer cell-specific promoter, or a promoter specific to an organ from which the cancer cells are derived.

9. The oncolytic virus of claim 3 , wherein the promoter is a CA promoter, a CMV promoter, an RSV promoter, or an E2F promoter.

10. The oncolytic virus of claim 1, wherein the promoter of a nucleic acid encoding at least one factor essential for viral replication or assembly is replaced with a cancer cell-specific promoter or an organ-specific promoter from which the cancer cells are derived.

11. The oncolytic virus of claim 10 , wherein the cancer cell-specific promoter is a survivin promoter.

12. The oncolytic virus of claim 10 , wherein the virus is an adenovirus.

13. The oncolytic virus of claim 12, wherein the factors essential for viral replication or assembly are E1A or E1AΔ24, and / or E1B or E1BΔ55K.

14. The oncolytic virus described in claim 13, wherein the promoter of the nucleic acid encoding E1A is replaced with a survivin promoter, and further, the promoter of the nucleic acid encoding E1BΔ55K is replaced with an exogenous promoter selected from a ubiquitous promoter, a cancer cell-specific promoter, and an organ-specific promoter from which the cancer cells are derived.

15. The oncolytic virus of claim 14, wherein the promoter of the nucleic acid encoding E1BΔ55K is replaced with a CMV promoter.

16. (a) A combination of an oncolytic virus containing a nucleic acid encoding CXCL10 under the control of a promoter functional in cancer cells and the oncolytic virus of (b) and / or (c) below, or the oncolytic virus of (d). (b) an oncolytic virus comprising a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells; (c) an oncolytic virus comprising a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells; (d) an oncolytic virus comprising a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells and a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells;

17. The combination product according to claim 16 , wherein each of the promoters functional in the cancer cells is the same or different and is a ubiquitous promoter, a cancer cell-specific promoter, or an organ-specific promoter from which the cancer cells are derived.

18. The combination according to claim 16, wherein each of the promoters functional in the cancer cells is the same or different and is a CA promoter, a CMV promoter, an RSV promoter, or an E2F promoter.

19. The combination according to claim 16, wherein the oncolytic viruses are conditionally replicating adenoviruses and the structures of the replication control regions of each oncolytic virus are identical.

20. A cancer therapeutic agent comprising the oncolytic virus according to any one of claims 1 to 6 and 8 to 15 as an active ingredient.

21. The agent according to claim 20, which is administered locally to a primary cancer lesion.

22. The agent according to claim 20, which is for the treatment of invasive / metastatic cancer.

23. The method of claim 20, which is administered multiple times.

24. A cancer therapeutic agent comprising the oncolytic virus according to claim 7 as an active ingredient.

25. A cancer therapeutic agent comprising the combination according to claim 16 as an active ingredient.

26. The agent according to claim 25, which is administered locally to a primary cancer lesion.

27. The agent according to claim 25, which is for the treatment of invasive / metastatic cancer.

28. The method of claim 25, which is administered multiple times.

Images