Oncolytic viruses with expression control systems that provide optimal expression levels of the immune-inducing genes they carry, enabling effective treatment of even metastatic cancer while ensuring safety (oncolytic immunotherapy)

Abstract

To develop immuno-viral therapeutic vectors with optimal therapeutic efficacy while ensuring high safety based on the novel concept of finding the optimal expression level of a therapeutic gene for inducing a maximum therapeutic effect without side effects.SOLUTION: The present invention provides an oncolytic virus and the like that has an immunity-inducing gene functionally linked downstream of an E2F promoter or a promoter exhibiting activity equivalent thereto, in which a promoter of a nucleic acid encoding at least one factor essential for viral replication or assembly is replaced with the promoter of a factor whose expression is organ-specifically enhanced, or the promoter of a factor whose expression is enhanced cancer cell-specifically.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the field of cancer therapy using oncolytic viruses carrying immune-inducing genes, so-called oncolytic immunotherapy. [Background technology]

[0002] Oncolytic immunotherapy, which involves equipping an oncolytic virus, a genetically engineered virus that grows specifically in cancer cells and exhibits a killing effect, with an immune-inducing gene, is expected to be a leading candidate for an innovative cancer treatment worldwide, as shown by the approval of Amgen's T-Vec, which is equipped with a cytokine gene, as a first-in-class drug in Europe and the United States at the end of 2015 (Non-Patent Documents 1 to 3).

[0003] The present inventors pioneered the development of immune gene therapy by introducing immune genes using non-replicating viral vectors in the 1990s, the dawn of gene therapy (Non-Patent Documents 4 to 6). Since then, they have conducted their own research and development for many years, as evidenced by the publication of many papers on immune gene therapy.

[0004] On the other hand, the present inventors have developed a next-generation conditionally replicating adenovirus (m-CRA) that is a type of oncolytic virus, by modifying three elements, namely, the viral replication control part, therapeutic gene, and viral characteristics, and that ensures safety by controlling viral replication in a highly cancer cell-specific manner with multiple factors, and can also introduce therapeutic genes that can enhance therapeutic effects. We have developed an efficient method for producing limitatively replicating adenoviruses (Non-Patent Document 7, Patent Document 1). Although adenoviruses are medium-sized viruses with a genome size of 30-40 kb, genetic modification is not easy, and unlike non-replicating adenoviruses, there has not even been an efficient standardized production technique for limitatively replicating adenoviruses. Therefore, it has been difficult to develop m-CRAs with highly recombinant genes at multiple sites, and to produce multiple candidate m-CRAs and conduct comparative screening experiments. Therefore, the inventors first developed m-CRA technology, and by using this technology, they have been able to produce many candidate m-CRAs for the first time and verify them exploratory-ly, and have developed m-CRA drugs with pharmaceutical properties far superior to those of oncolytic viruses, which are a competing technology. First, the viral proliferation control section, which is the basic structure of m-CRA, As tumor-specific promoters, we have developed survivin-responsive m-CRA (Surv.m-CRA), which controls virus proliferation with the survivin gene promoter, and aurora kinase-responsive m-CRA (Patent Document 2), which controls virus proliferation with the aurora kinase gene promoter, both of which are used in the present invention. Surv.m-CRA has demonstrated superiority over competing technologies in terms of both safety and therapeutic effect (Non-Patent Document 9) compared to the best competing technology to date (CRA whose virus is propagated by the Tert promoter; one of the best competing technologies to date because the same basic structure has shown good results in clinical trials; Non-Patent Document 8), and has demonstrated superiority over conventional technologies in that it can effectively treat cancer stem cells for which existing treatment technologies (anticancer drugs, radiation therapy) are ineffective (Non-Patent Document 10).The Surv.m-CRA "not loaded with therapeutic genes" has been named Surv.m-CRA-1, and its safety has been confirmed in non-clinical trials. The inventors are currently conducting investigator-initiated clinical trials on human cancer patients, and good results are being confirmed in humans as well. Surv.m-CRA-1 is an oncolytic virus therapeutic drug that specifically (safely) and effectively treats cancer cells with the amplified viral proteins by specifically and efficiently replicating the virus only in cancer cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent No. 4478775 [Patent Document 2] Japanese Patent No. 5963363 [Non-patent literature]

[0006] [Non-Patent Document 1] Bilsland, AE et al. (2016) F1000Research.;5:2105. [Non-Patent Document 2] Liu BL et al. (2003) Gene Ther.;10(4):292-303. [Non-Patent Document 3] Chen DS et al. (2013) Immunity.;39(1):1-10. [Non-Patent Document 4] Chen SH et al. (1995) Proc Natl Acad Sci U S A.;92(7):2577-2581. [Non-Patent Document 5] Chen SH et al. (1996) Cancer Res.;56(16):3758-3762. [Non-Patent Document 6] Caruso M et al. (1996) Proc Natl Acad Sci US A.;93(21):11302-11306. [Non-Patent Document 7] Nagano S, et al. (2005) Gene Ther. 12(18):1385-1393 [Non-Patent Document 8] Nemunaitis J,et al.Mol Ther. 2010 18:429-34 [Non-Patent Document 9] Kamizono J, et al.: Survivin-responsive conditionally replicating adenovirus exhibits cancer-specific and efficient viral replication. Cancer Res. 65(12): 5284-5291, 2005 [Non-Patent Document 10] Tanoue K, et al. J Trans Med .12:27.doi: 10.1186 / 1479-5876-12-27, 2014 Summary of the Invention

[0007] Until now, in the field of oncolytic immunotherapy, while the focus has been on the search for immune-inducing genes that exert effective therapeutic effects, it has not been considered that "due to the property of oncolytic viruses that 'grow only in tumors,' it is necessary to ensure special safety." However, the present inventors have focused for the first time on the safety issue caused by the excessive production of immune-inducing factors, which is different from gene therapy using conventional non-replicating vectors (which often have one gene per cell, or even if multiple genes are introduced per cell, the number itself does not change from the time of introduction) because oncolytic viruses "grow" in cancer cells, and the immune-inducing genes carried by the oncolytic viruses are also amplified in correlation with this. In other words, the inventors have discovered that when using oncolytic immunotherapeutics, safety concerns arise due to the excessive production of immune-inducing factors from the immune-inducing genes carried by the propagated oncolytic viruses, and that, as a solution to this issue, the immune-inducing genes carried by oncolytic viruses have an optimal gene expression level specific to this system, and more specifically, that it is necessary to select a promoter that induces and controls the optimal expression level for the immune-inducing genes carried by oncolytic viruses.

[0008] The discovery of the problem that motivated the present invention was obtained from the inventor's unique professional knowledge and long-term experience, and has been accumulated as the researcher's undisclosed know-how. In other words, the present invention is based on the inventor's unique undisclosed knowledge about immune gene therapy accumulated over many years, and has led to the invention of this drug (the optimal oncolytic immunotherapeutic agent that overcomes the above-mentioned problems) and its technology by creating and analyzing multiple candidate Surv.m-CRA-2s that are equipped with immune-related genes using the independently developed m-CRA production technology in addition to the independently developed Surv.m-CRA-1.

[0009] Therefore, the present invention aims to develop a tumor-lytic immunotherapy virus that has optimal therapeutic effects while ensuring high safety, based on the unprecedented novel concept of "finding an expression control system that provides optimal expression levels of the immune-inducing gene carried by a tumor-lytic immunotherapy agent (a tumor-lytic virus carrying an immune-inducing gene), in order to induce powerful therapeutic effects even in metastatic cancers, while eliminating side effects and ensuring safety."

[0010] The present inventors have comprehensively compared and examined combinations of promoters with various strengths and various cytokine genes, assuming the incorporation of immune-inducing genes into Surv.m-CRA. As a result, they have found that a novel Surv.m-CRA (hereinafter, sometimes referred to as "the present recombinant virus") carrying an immune-inducing gene whose expression is controlled by a cell cycle-specific or tumor-specific E2F promoter (E2Fp) is a tumor-lytic immunotherapeutic agent with high safety and optimal therapeutic effect. In other words, although there have been several reports of so-called constitutively strong promoters that have been suggested to have strong ubiquitous transcription activity, the detailed differences in the characteristics of each promoter, especially the detailed differences in the expression levels, have not been made clear, and the detailed characteristics of various constitutively strong promoters, especially when used to control the expression of immune-inducing genes carried in tumor-lytic viruses, have not been analyzed. Our current study has revealed that, for the first time, "one of these promoters, the CA promoter (a modified chicken beta-actin promoter with human cytomegalovirus immediate-early enhancer), has extremely strong promoter activity, and the RSV promoter (Rous sarcoma virus long terminal repeat), one of the constitutively highly active promoters, has strong activity, although its activity is considerably lower than that of the CA promoter. In particular, the copy number of genes loaded onto oncolytic viruses in cancer cells is significantly amplified as the viruses grow. When loading onto oncolytic immunotherapy viruses to control the expression of immune-inducing genes, it is appropriate to position the E2F promoter as an "optimal (moderate)" transcriptional activity promoter." In other words, when the purpose is to express immune-inducing genes with oncolytic viruses, the use of the E2F promoter can dramatically improve safety compared to expression control with extremely strong promoters (such as the CA promoter) and strong promoters (the RSV promoter).The promoter (E2Fp) of the transcription factor E2F, whose main target gene is the tumor suppressor gene product pRB, controls expression in a cell cycle-specific or cancer-specific manner. This allows the expression of immune-inducing genes to be transiently expressed at low levels in normal cells other than cancer cells during the S phase, but is specifically regulated to be "optimal (moderate)" in cancer cells, demonstrating strong therapeutic effects while dramatically improving safety. Even more surprisingly, we found that in oncolytic viruses (oncolytic immunotherapeutic agents) that control the expression of immune-inducing genes with the E2F promoter, not only is safety improved dramatically, but the therapeutic effects of immune-inducing genes can also be enhanced and maximized.

[0011] In particular, the inventors have found that Surv.m-CRA / E2Fp-mGM-CSF efficiently infects and proliferates in hamster-derived cancer cells, induces high levels of GM-CSF expression in the cells, and exhibits a strong tumor-suppressing effect not only on the primary cancer site where the virus was injected, but also on metastatic sites, without causing any fatal side effects in a hamster allograft-bearing cancer model. This therapeutic effect is due to the direct induction of cell death by the oncolytic virus, as well as the specific (safe) and strong killing of cancer cells, which creates various cancer antigens locally, and at the same time, the sustained secretion of high concentrations of immune-inducing factors locally in the tumor, which can induce cancer-specific systemic anti-tumor immunity, mainly cellular immunity, very efficiently, and thus kills not only primary cancer sites but also metastatic cancer sites by inducing systemic anti-tumor immunity. In line with this, these in vivo therapeutic effects of the present invention, as an example, not only were the tumor disappearance effects at the administered tumor site significantly increased compared to Surv.m-CRA-1, which does not contain cytokines and is currently undergoing investigator-initiated clinical trials (the therapeutic effects are currently being demonstrated in human patients for whom standard treatment has been ineffective), but also dramatic prevention of distant metastasis was achieved by inducing systemic anti-tumor immunity through local treatment (since conventional treatment techniques cannot treat systemic metastatic cancer with local treatment, this therapeutic agent has significant differentiation and superiority), and therefore early clinical application is highly anticipated in terms of dramatic improvement in safety and revolutionarily powerful cancer therapeutic effects.

[0012] That is, the present invention relates to a recombinant virus having an E2F promoter (E2Fp) functionally linked to an immune-inducing gene. More preferably, the present invention relates to a multifactor-regulated tumor-specific proliferation (oncolytic) virus. Specifically, the present invention relates to the following inventions: (1) An oncolytic virus having an immune-inducible gene functionally linked downstream of the E2F promoter (E2Fp) or a promoter exhibiting equivalent activity. (2) The oncolytic virus described in (1), wherein the promoter exhibiting activity equivalent to that of the E2F promoter (E2Fp) is a survivin promoter, an Aurora kinase A gene promoter, or an Aurora kinase B gene promoter. (3) The oncolytic virus described in (1), wherein the promoter is an E2F promoter (E2Fp) or a survivin promoter. (4) The oncolytic virus described in (1), wherein the promoter is an E2F promoter (E2Fp). (5) The oncolytic virus according to any one of (1) to (4), wherein the immune-inducing gene is a cytokine gene. (6) The cytokine gene is Activin A, ANGPTL5, BAFF, BD-2 (β-Defensin-2), BD-3 (β-Defensin-3), BDNF, BMP-2, BMP-4, BMP-6, BMP-7, BMP-10, CCL1, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL6, CCL7 (MCP-3), CCL8 (MCP-2), CCL9 (MIP-1γ), CCL11 (Eotaxin-1), CCL12 (MCP-5), CCL13 (MCP-4), CCL14, CCL15 (MIP-1δ), CCL16, CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3β), CCL20 (MIP-3α), CCL21 (Exodus-2), CCL22, CCL23, CCL24 (Eotaxin-2), CCL25 (TECT), CCL26 (MIP-4α), CCL27, CCL28, CO40-Ligand (TRAP), CD137 (4-1BB)-Ligand, CNTF, CT-1, CX3CL1 (Fractalkine), CXCL1 (GRO1), CXCL2 (MIP-2α, GRO2), CXCL3 (MIP-2β, GRO3), CXCL4 (PF4), CXCL5, CXCL6, CXCL7, CXCL9, CXCL10, CXCL11, CXCL12 (SDF-1α), CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, DKK-1, DLL1, EGFs, EG-VEGF (Prokineticin 1), FasL, FGF-1 (acidic FGF), FGF-2 (basic FGF), FGF-3, FGF-4 (HGBF-4), FGF-5, FGF-6, FGF-7 (KGF, HBGF-7), FGF-8, FGF-9 (HBGF-9), FGF-10(KGF-2), FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, Flt3-Ligand, Galectin-1, Galectin-3, G -CSF, GDF-11, GDNF, GM-CSF, HB-EGF, HGF, IFN-α2a, IFN-α2b, IFN-β1a, IFN-β1b, IFN-γ1b, IGF-1, IGF-2, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, LIF, M-CSF, MIF, NGF-β, Noggin, NT-3 (NTF-3), NT-4 (NTF-4), Oncostatin The oncolytic virus according to (5), wherein the gene is a cytokine selected from the group consisting of M, OPG (TNFRSF11B), PDGF-AA, PDGF-AB, PDGF-BB, Pleiotrophin, Prolactin (Mammotropin), RANKL, R-Spondin-1, R-Spondin-2, R-Spondin-3, SCF (c-kit Ligand), SHH (C24II), TGF-α, TGF-β1, TGF-β3, TNF-α, TNF-β, TPO (MDGF), TRAIL, TSLP, VEGF, XCL1, and XCL2. (7) The oncolytic virus described in (6), wherein the cytokine gene is GM-CSF. (8) The oncolytic virus according to any one of (1) to (7), further characterized in that a promoter of a nucleic acid encoding at least one factor essential for viral replication or assembly is substituted with a promoter of a factor whose expression is enhanced in an organ-specific manner, or a promoter of a factor whose expression is enhanced in a cancer cell-specific manner. (9) The oncolytic virus described in (8), wherein the promoter of the factor whose expression is enhanced in an organ-specific manner is a promoter selected from the group consisting of an albumin promoter, an alpha-fetoprotein promoter, a prostate-specific antigen (PSA) promoter, a mitochondrial creatine kinase (MCK) promoter, a myelin basic protein (MB) promoter, a glial fibrillary acidic protein (GFAP) promoter, and a neurospecific enolase (NSE) promoter. (10) The oncolytic virus described in (8), wherein the promoter of the factor whose expression is specifically enhanced in cancer cells is a promoter selected from the group consisting of a telomerase reverse transcriptase (TERT) promoter, a carcinoembryonic antigen (CEA) promoter, a hypoxia responsive region (HRE) promoter, a Grp78 promoter, an L-plastin promoter, a hexokinase II promoter, a survivin promoter, and an Aurora kinase promoter. (11) The oncolytic virus according to (10), wherein the promoter of the factor whose expression is specifically enhanced in cancer cells is a survivin promoter, a human Aurora kinase A gene promoter, or a human Aurora kinase B gene promoter. (12) The oncolytic virus according to (10), wherein the promoter of the factor whose expression is specifically enhanced in cancer cells is a survivin promoter. (13) The oncolytic virus according to any one of (1) to (12), which is an adenovirus. (14) The oncolytic virus according to (13), wherein at least one factor essential for viral replication or assembly is a factor selected from E1A, E1AΔ24, E1B, and E1BΔ55K. (15) The oncolytic virus according to (13), wherein at least one factor essential for viral replication or assembly is E1A. (16) The oncolytic virus according to any one of (1) to (15), further comprising an expression cassette comprising an exogenous promoter operably linked to a nucleic acid encoding a cytotoxic factor or a therapeutic factor. (17) A cancer therapeutic agent comprising the oncolytic virus according to any one of (1) to (16). Effect of the Invention

[0013] The virus of the present invention can achieve a high anti-cancer effect without causing side effects by controlling the expression of an immunity-inducing gene by employing a promoter having an appropriately strong promoter activity. [Brief description of the drawings]

[0014] [Figure 1] This is a schematic diagram of GM-CSF-expressing Surv.m-CRA, in which an expression cassette containing mouse GM-CSF cDNA linked to the E2F promoter, RSV promoter, and CA promoter has been inserted into Surv.m-CRA, which has the Survivin promoter inserted upstream of E1A, an early gene essential for adenovirus proliferation. [Figure 2A] Hamster-derived cancer cells (HaK) were infected with the control Ad.CMV-EGFP (a "non-replicating" adenovirus in which the E1 region has been deleted and a genetic construct that expresses EGFP under the CMV promoter has been incorporated instead) at an MOI of 0.1 to 1000, and the photographs show the expression of EGFP 48 hours later. [Figure 2B] Hamster-derived cancer cells (HaP-T1) were infected with the control Ad.CMV-EGFP (a "non-replicating" adenovirus in which the E1 region has been deleted and a genetic construct that expresses EGFP under the CMV promoter has been incorporated instead) at an MOI of 0.1 to 1000, and the photographs show the expression of EGFP 48 hours later. [Figure 2C] Normal hamster-derived cells (BHK-21) were infected with the control Ad.CMV-EGFP (a "non-replicating" adenovirus in which the E1 region has been deleted and a gene construct that expresses EGFP under the CMV promoter has been incorporated instead) at an MOI of 0.1 to 1000, and the photographs show the expression of EGFP 48 hours later. [Diagram 3]3 is a graph showing EGFP expression for each MOI in FIG. 2. The vertical axis indicates the adenovirus infection multiplicity (AGTE), and the horizontal axis indicates the MOI (multiplicity of infection) at the time of viral infection. [Figure 4] This is a graph showing β-galactosidase activity 48 hours after infection and gene transfer with Ad.dE1.3 (no foreign LacZ gene expressed as a control), Ad.E2Fp-LacZ (expressing LacZ under the E2F promoter), Ad.RSVp-LacZ (expressing LacZ under the RSV promoter), and Ad.Survp-LacZ (expressing LacZ gene under the Survivin promoter), all of which are "non-" replicative adenovirus vectors that can only be used for gene transfer, in order to examine the activity of each promoter in hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21). The vertical axis shows the relative β-galactosidase activity compared to that after infection with the control virus Ad.dE1.3, and the horizontal axis shows each hamster-derived cell line. [Diagram 5] 1 is a graph showing the expression level of GM-CSF protein 48 hours after infection of hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21) with Ad.dE1.3 (Control), Surv.m-CRA, Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF. In the figure, the names are simply expressed as Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF, i.e., E2Fp-mGM-CSF, RSVp-mGM-CSF, and CAp-mGM-CSF, respectively, because the names are long. [Figure 6A]This graph shows the cytotoxicity 3 and 5 days after infection of hamster-derived cancer cells (HaK) with the ``non-'' replicating adenovirus vector Ad.dE1.3 as a control, and each of the oncolytic viruses Surv.m-CRA (no therapeutic gene), Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF at an MOI of 3 (top panel) or MOI of 30 (bottom panel). [Figure 6B] This graph shows the cytotoxicity 3 and 5 days after infection of hamster-derived cancer cells (HaP-T1) with the control ``non-replicating'' adenovirus vector Ad.dE1.3, and each of the oncolytic viruses Surv.m-CRA (no therapeutic gene), Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF at an MOI of 3 (top panel) or MOI of 30 (bottom panel). [Figure 6C] This graph shows the cytotoxicity 3 and 5 days after infection of normal hamster-derived cells (BHK-21) with the ``non-'' replicative adenovirus vector Ad.dE1.3 as a control, and each of the oncolytic viruses Surv.m-CRA (no therapeutic gene), Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF at an MOI of 3 (top panel) or MOI of 30 (bottom panel). [Figure 7] This is a graph showing the change in tumor diameter over time after a single administration of 1 x 109 PFU of each virus into the tumor of a 5-week-old female Syrian hamster in which 1 x 107 HaK cells were subcutaneously implanted on the dorsal side. [Figure 8] This is a graph showing the time course of survival rates after a single administration of 1 x 109 PFU of each virus into the tumor of 5-week-old female Syrian hamsters in which 1 x 107 HaK cells were subcutaneously implanted on the dorsal side. [Figure 9]1 is a graph showing the amount of mGM-CSF expression in tissues after infection with mGM-CSF-expressing Surv.m-CRA in a cancer-bearing hamster model. [Figure 10] FIG. 1 is a graph showing the survival curve after infection with mGM-CSF-expressing Surv.m-CRA in an orthotopic tumor-bearing hamster model. [Figure 11] FIG. 1 is a graph showing survival curves after infection with mGM-CSF-expressing Surv.m-CRA or administration of anti-PD-1 antibody in an orthotopic tumor-bearing hamster model. [Figure 12] This is a schematic diagram of IL-2 or IL-15 expressing Surv.m-CRA in which an expression cassette linking mouse or human IL-2 or IL-15 cDNA to the E2F promoter, RSV promoter, or CA promoter is inserted into Surv.m-CRA in which the Survivin promoter is incorporated upstream of E1A, an early gene essential for adenovirus proliferation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] This virus is characterized by the fact that the immune-inducing gene carried by the oncolytic virus is functionally linked downstream of the E2F promoter or a promoter with equivalent activity, thereby exhibiting a strong tumor-suppressing effect without causing lethal side effects.

[0016] The "immunity-inducing gene" is not particularly limited as long as it is a gene encoding a protein having an immune-activating effect or a functional peptide fragment thereof, and is preferably a cytokine gene or a functional peptide fragment thereof, for example, Activin A, ANGPTL5, BAFF, BD-2 (β-Defensin-2), BD-3 (β-Defensin-3), BDNF, BMP-2, BMP-4, BMP-6, BMP-7, BMP-10, CCL1, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL6, CCL7 (MCP-3). , CCL8 (MCP-2), CCL9 (MIP-1γ), CCL11 (Eotaxin-1), CCL12 (MCP-5), CCL13 (MCP-4), CCL14, CCL15 (MIP-1δ), CCL16, CCL17(TARC), CCL18(PARC), CCL19(MIP-3β), CCL20(MIP-3α), CCL21(Exodus-2 ), CCL22, CCL23, CCL24 (Eotaxin-2), CCL25 (TECK), CCL26 (MIP-4α), CCL27, CCL28, CO40-Ligand (TRAP), CD137(4-1BB)-Ligand, CNTF, CT-1, CX3CL1(Fractalkine), CXCL1(GRO1), CXCL2(MIP-2α , GRO2), CXCL3 (MIP-2β, GRO3), CXCL4 (PF4), CXCL5, CXCL6, CXCL7, CXCL9, CXCL10, CXCL11, CXCL1 2 (SDF-1α), CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, DKK-1, DLL1, EGFs, EG-VEGF (Prokineticin 1), FasL, FGF-1 (acidic FGF), FGF-2 (basic FGF), FGF-3, FGF-4 (HGBF-4), FGF-5, FGF-6, FGF-7 (KGF, HBGF-7), FGF-8, FGF-9 (HBGF-9), FGF-10(KGF-2), FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, Flt3-Ligand, Galectin-1, Galectin-3, G -CSF, GDF-11, GDNF, GM-CSF, HB-EGF, HGF, IFN-α2a, IFN-α2b, IFN-β1a, IFN-β1b, IFN-γ1b, IGF-1, IGF-2, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, LIF, M-CSF, MIF, NGF-β, Noggin, NT-3 (NTF-3), NT-4 (NTF-4), Oncostatin M, OPG (TNFRSF11B), PDGF-AA, PDGF-AB, PDGF-BB, Pleiotrophin, Prolactin (Mammotropin), RANKL, R-Spondin-1, R-Spondin-2, R-Spondin-3, SCF (c-kit Ligand), SHH(C24II), TGF-α, TGF-β1, TGF-β3, TNF-α, TNF-β, TPO(MDGF), TRAIL, TSLP, VEGF, XCL1, and XCL2.

[0017] The "E2F promoter" is the promoter region of the E2F gene, which is the main target of a representative tumor suppressor gene product, pRB. For example, the E2F promoter may have the sequence set forth in SEQ ID NO: 2. The E2F promoter does not need to have the full length of the sequence set forth in SEQ ID NO: 2, and may consist of a part of the sequence of SEQ ID NO: 2 as long as the object of the present invention can be achieved.

[0018] A promoter that exhibits activity equivalent to that of the E2F promoter is not particularly limited as long as it is a promoter that exhibits promoter activity equivalent to that of the E2F promoter in tumor cells, and examples thereof include the survivin promoter, the Aurora kinase A gene promoter, and the Aurora kinase B gene promoter.

[0019] 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 present virus is a 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 a promoter of the survivin gene derived from human or mouse, more preferably a promoter of the human survivin gene.

[0020] The nucleotide sequence length of the survivin promoter is not particularly limited as long as it is specific to target cancer cells and can activate the transcription of a gene linked downstream to a degree that exerts sufficient therapeutic activity against cancer cells. For example, in the case of a mouse survivin gene promoter, the nucleotide sequence of positions -173 to -19 (nucleotide sequence of 1124 to 1278 in the nucleotide sequence shown in SEQ ID NO: 3) with the translation initiation point at +1, and in the case of a human survivin gene promoter, the nucleotide sequence of positions -173 to -1 (nucleotide sequence of 1296 to 1468 in the nucleotide sequence shown in SEQ ID NO: 4) with the translation initiation point at +1 can provide the desired specificity and transcription activity. Therefore, preferably, the survivin promoter used in the present recombinant virus contains at least the nucleotide sequence of positions 1124 to 1278 in the nucleotide sequence shown in SEQ ID NO: 3, or at least the nucleotide sequence of positions 1296 to 1468 in the nucleotide sequence shown in SEQ ID NO: 4. There is no particular upper limit on the nucleotide sequence length of the survivin promoter, but if the length of the 5' upstream region is too long, it may have an undesirable effect on the transcription activity and specificity of the promoter. For example, in the case of a human survivin gene promoter, a nucleotide sequence of about -6000 to -1 positions, with the translation initiation point at +1, can provide the desired specificity and transcription activity, but the 5' end of the promoter is preferably downstream of -3000 positions, more preferably downstream of -1500 positions. When using a survivin promoter derived from another mammal, a vector in which a reporter gene is linked downstream of a promoter of various lengths can be prepared, introduced into cancer cells, and the promoter activity can be evaluated using the expression of the reporter as an index, thereby determining the range of the suitable sequence length of the promoter.

[0021] The survivin promoter 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 (e.g., monkeys, cows, horses, pigs, dogs, cats, sheep, goats, rabbits, mice, rats, etc.) using a nucleic acid consisting of a known survivin gene promoter sequence (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) as a probe, cleaving the DNA fragments containing the desired partial promoter sequence using a DNase, for example, an appropriate restriction enzyme, separating the fragments by gel electrophoresis, recovering the desired band, and purifying the DNA. Alternatively, the survivin promoter partial sequence can be amplified and isolated by PCR using primers synthesized based on the known survivin gene promoter sequence, using a crude extract of the above cells or genomic DNA isolated therefrom as a template. For a mammal whose nucleotide sequence of the survivin promoter is unknown, the nucleotide sequence of the survivin promoter region of the animal can be obtained by performing a BLAST search on the genomic DNA of the animal using the survivin cDNA sequence of the animal as a query. The survivin promoter can also be obtained by chemically synthesizing a nucleic acid containing all or a part of a known survivin gene promoter sequence (e.g., the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4) using a commercially available automatic DNA / RNA synthesizer.

[0022] Promoters of human Aurora kinase A and Aurora kinase B genes have already been reported (Tanaka, M. et al., J. Biol. Chem., 277(12):10719-26, 2002; Kimura, M. et al., Biochem. Biophys. Res. Commun., 316:930-6, 2004). There are no particular limitations on the Aurora kinase promoter as long as it is a promoter 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 Drosophila Aurora-A, -B and -C genes. Promoters of Aurora kinase A gene or Aurora kinase B gene derived from human or other mammals are preferred, and promoters of human Aurora kinase A gene or human Aurora kinase B gene are more preferred.

[0023] The length of the nucleotide sequence of the Aurora kinase promoter is not particularly limited as long as it is specific to target disease cells (e.g., cancer cells) and can activate the transcription of a gene linked downstream to a degree that exerts sufficient therapeutic activity against a target disease. For example, in the case of a human Aurora kinase A gene promoter, the desired specificity and transcription activity can be obtained if it contains a nucleotide sequence from -124 to +354 positions (the nucleotide sequence shown in SEQ ID NO: 6; the nucleotide sequence from 1363 to 1840 positions in the nucleotide sequence shown in SEQ ID NO: 5) with the transcription start point at +1. In this specification, the human Aurora kinase A gene promoter includes not only the full-length human Aurora kinase A gene promoter but also a promoter containing the nucleotide sequence shown in SEQ ID NO: 6. In the case of a human Aurora kinase B gene promoter, the desired specificity and transcription activity can be obtained if it contains a nucleotide sequence from -185 to +361 positions (the nucleotide sequence shown in SEQ ID NO: 8; the nucleotide sequence from 1595 to 2140 positions in the nucleotide sequence shown in SEQ ID NO: 7) with the transcription start point at +1. In the present specification, the human Aurora kinase B gene promoter includes not only the full-length human Aurora kinase B gene promoter but also a promoter containing the nucleotide sequence shown in SEQ ID NO: 8. Preferably, the Aurora kinase promoter contains at least the nucleotide sequence of 1363 to 1840 in the nucleotide sequence shown in SEQ ID NO: 5, or at least the nucleotide sequence of 1595 to 2140 in the nucleotide sequence shown in SEQ ID NO: 7. There is no particular upper limit on the length of the nucleotide sequence of the Aurora kinase promoter, but if the length of the 5' upstream region is too long, it may have an undesirable effect on the transcription activity and specificity of the promoter. For example, in the case of the human Aurora kinase A gene promoter, the nucleotide sequence of positions -1486 to +354 (nucleotide sequence shown in SEQ ID NO: 5) with the transcription start point at +1, and in the case of the human Aurora kinase B gene promoter, the nucleotide sequence of positions -1779 to +361 (nucleotide sequence shown in SEQ ID NO: 7) with the transcription start point at +1 can provide the desired specificity and transcription activity.Therefore, in a preferred embodiment, the nucleic acid at the 5' end of the human Aurora kinase promoter used in the vector of the present invention is any one of nucleotides 1 to 1363 in the nucleotide sequence shown in SEQ ID NO: 5, or any one of nucleotides 1 to 1595 in the nucleotide sequence shown in SEQ ID NO: 7. When using Aurora kinase promoters derived from other mammals, preferred regions can be selected in a similar manner.

[0024] The Aurora kinase promoter can be prepared by cloning genomic DNA containing a known Aurora kinase gene promoter sequence from genomic DNA extracted from cells or tissues derived from humans or other mammals (e.g., monkeys, cows, horses, pigs, dogs, cats, sheep, goats, rabbits, mice, rats, etc.) using a nucleic acid consisting of the promoter sequence of the Aurora kinase gene as a probe (see, for example, Tanaka, M. et al., J. Biol. Chem., 277(12):10719-26, 2002; Kimura, M. et al., Biochem. Biophys. Res. Commun., 316:930-6, 2004), cleaving the genomic DNA with a DNase, for example an appropriate restriction enzyme, into DNA fragments containing a desired partial promoter sequence, separating the fragments by gel electrophoresis, and recovering the desired bands and purifying the DNA. Alternatively, the Aurora kinase promoter partial sequence can be amplified and isolated by PCR using primers synthesized based on the known Aurora kinase gene promoter sequence and a crude extract of the above cells or genomic DNA isolated therefrom as a template. For mammals whose Aurora kinase promoter nucleotide sequence is unknown, the nucleotide sequence of the Aurora kinase promoter region of the animal can be obtained by performing a BLAST search against the genomic DNA of the animal using the Aurora kinase cDNA sequence of the animal as a query.

[0025] The immune-inducing gene can be isolated as a cDNA from cells or tissues that produce it by a method known per se, and functionally linked downstream of an E2F promoter or a promoter exhibiting an activity equivalent thereto. An expression cassette containing a nucleic acid encoding an immune-inducing gene under the control of such a promoter preferably contains a suitable polyadenylation sequence downstream of the nucleic acid or gene. As described below, a recombinant virus containing an E2F promoter functionally linked to an immune-inducing gene or a promoter exhibiting an activity equivalent thereto may have a nucleic acid encoding a protein required for viral replication or assembly under the control of a promoter of a factor whose expression is enhanced in an organ-specific manner, or a promoter of a factor whose expression is enhanced in a cancer cell-specific manner. In addition, a recombinant virus containing an E2F promoter functionally linked to an immune-inducing gene or a promoter exhibiting an activity equivalent thereto may be a recombinant virus in which a region contained in a viral protein that is essential for inducing a cellular environment required for viral proliferation in normal cells but is not necessary for viral proliferation in cancer cells is deleted. Furthermore, a recombinant virus containing an E2F promoter functionally linked to an immune-inducing gene, or a promoter exhibiting activity equivalent thereto, may be a recombinant virus in which a nucleic acid encoding a protein necessary for viral replication or assembly is functionally linked to a promoter of a factor whose expression is enhanced in an organ-specific manner, or a promoter of a factor whose expression is enhanced in a cancer cell-specific manner, and which is deficient in a region that is essential for inducing a cellular environment necessary for viral proliferation in normal cells but is not necessary for viral proliferation in cancer cells.

[0026] As used herein, the term "operably linked" means that a promoter is linked in such a way that the promoter exerts its activity to enable expression of a downstream gene.

[0027] In one aspect, the recombinant virus of the present invention is an oncolytic virus, in other words, a conditionally replicating virus (CRV), characterized in that the promoter of a nucleic acid encoding at least one factor essential for viral replication or assembly is replaced with a promoter of a factor whose expression is enhanced in an organ-specific manner or a promoter of a factor whose expression is enhanced in a cancer cell-specific manner. The recombinant virus specifically promotes viral proliferation (predominantly over normal cells) in target disease cells such as cancer cells in which the promoter activity is high, thereby exhibiting a cytotoxic activity specific to the cell and expressing other genes incorporated into the viral gene in a cell-specific manner. That is, the present invention relates to a vector characterized by proliferation. Furthermore, the recombinant virus not only specifically causes viral proliferation in the infected target cells and kills (lyses) the cells, but also repeats the step of daughter viruses released from the lysed cells infecting surrounding target cells not infected with the virus and killing the cells, thereby ultimately introducing the recombinant virus into many target cancer cells in the primary focus at the injection site, thereby achieving a therapeutic effect. Furthermore, it brings about specific (safe) and powerful killing of cancer cells, shrinking the tumor, and furthermore, the cell death creates various cancer antigens locally, and at the same time, it can induce cancer-specific systemic anti-tumor immunity, mainly cellular immunity, very efficiently by continuously secreting high concentrations of immune-inducing factors locally in the tumor. This induced anti-tumor immunity can effectively treat not only cancer cells in the primary lesion that have not been transfected with the gene, but also cancer cells in distant metastases that have not been administered the virus.

[0028] "Factors essential for viral replication or assembly" refers to genes encoding any of the proteins essential for the virus to replicate itself, such as viral structural proteins, or genes encoding any of the proteins essential for the virus to assemble. Factors essential for viral replication or assembly vary depending on the virus species used. For example, in the case of adenovirus, early genes E1A, E1B, E2, and E4, which act to control the transcription of viral proteins after transcription has begun at the early stage of infection, or Rb-binding region-deficient E1A (E1AΔ24) and p53-binding region-deficient E1B (E1BΔ55K), which will be described later, can be mentioned. In particular, E1A is a gene that is highly suitable for controlling viral proliferation specifically in target cells such as cancer cells, since it is transcribed first after adenovirus infection and subsequent viral replication does not occur without the expression of E1A. Similar effects can also be obtained by controlling other early genes essential for viral replication. Furthermore, the late genes L1, L2, L3, L4, and L5 that code for the structural genes of adenovirus are proteins that are transcribed at the late stage when cell division occurs after infection and that constitute the viral structure, and the viral proliferation can also be controlled specifically in target cells such as cancer cells by controlling the expression of these late genes. A proliferation-regulated virus can be obtained by replacing the endogenous promoter of a gene that codes for a protein required for viral replication or assembly with the promoter of a factor whose expression is enhanced in an organ-specific manner, or the promoter of a factor whose expression is enhanced in a cancer cell-specific manner.

[0029] Examples of "promoters of factors whose expression is enhanced in an organ-specific manner" (organ-specific promoters) include, for example, the promoters of albumin and α-fetoprotein, which are specific to the liver, the promoter of prostate-specific antigen (PSA), which is specific to the prostate, the promoter of mitochondrial creatine kinase (MCK), which is specific to various organs such as muscle and brain, and the promoters of myelin basic protein (MB), glial fibrillary acidic protein (GFAP), and neuronal specific enolase (NSE), which are specific to the nervous system such as the brain.

[0030] Examples of "promoters of factors whose expression is specifically enhanced in cancer cells" (cancer cell-specific promoters) include the CEA (carcinoembryonic antigen) promoter, which is specifically expressed only in cancer cells (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, NA et al., Science, 246, 1158-1161, 1989), the FLK-1 promoter, which is specific to malignant melanoma, fibrosarcoma, etc. (Xie, B. et al., Br. J. Cancer, 81, 1335-1343, 1999), the VEGF promoter, which is specific to lung cancer, etc. (Koshikawa, N. et al., Cancer Res.,60,2936-2941,2000), c-Myc promoter specific to small cell lung cancer (Kumagai,T.et al.,Cancer Res.,354-358,1996), SLPI promoter specific to lung cancer, ovarian cancer, etc. (Garver,RIet al.,Gene Ther.,1,46-50,1994), PSA promoter specific to prostate cancer (Latham,JPet al.,Cancer Res.,60,334-342,2000), Tyrosinase promoter specific to malignant melanoma, etc. (Vile,RG et al.,Cancer Res.,53,962-967,1993), AP-2 promoter specific to breast cancer (Pandha,HS et al.,J. Examples include 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), as well as the hypoxia responsive region (HRE) promoter, Grp78 promoter, L-plastin promoter, hexokinase II promoter, and survivin promoter, which are specific to various cancers.

[0031] In addition, the various promoters contained in the present vector can also be obtained by chemically synthesizing a nucleic acid containing all or part of the nucleotide sequence of a known promoter sequence using a commercially available automatic DNA / RNA synthesizer.

[0032] If at least one of the nucleic acids encoding proteins necessary for viral replication or assembly is under the control of a promoter of a factor whose expression is enhanced in an organ-specific manner or a promoter of a factor whose expression is enhanced in a cancer cell-specific manner, viral proliferation or assembly will be limited to an environment in which the promoter is activated, and therefore nucleic acids encoding proteins necessary for the replication or assembly of other viruses may be under the control of any exogenous promoter. For example, when a promoter capable of constitutive expression in a mammal is used as an exogenous promoter, a cytomegalovirus (CMV)-derived promoter (e.g., CMV immediate early promoter), a human immunodeficiency virus (HIV)-derived promoter (e.g., HIV LTR), a Rous sarcoma virus (RSV)-derived promoter (e.g., RSV LTR), a mouse mammary tumor virus (MMTV)-derived promoter (e.g., MMTV LTR), a Moloney murine leukemia virus (MoMLV)-derived promoter (e.g., MoMLV LTR), a herpes simplex virus (HSV)-derived promoter (e.g., HSV thymidine kinase (TK) promoter), a SV40-derived promoter (e.g., SV40 early promoter), an Epstein-Barr virus (EBV)-derived promoter, or an adeno-associated virus (AAV)-derived promoter (e.g., AAV Constitutive promoters such as p5 promoter, adenovirus (AdV)-derived promoters (Ad2 or Ad5 tumor late promoters), and gene promoters of mammalian structural proteins such as the β-actin gene promoter, PGK gene promoter, and transferrin gene promoter can be used.

[0033] Alternatively, an inducible promoter can be used as any exogenous promoter, and an example of such an inducible promoter is the metallothionein-1 gene promoter. When the metallothionein-1 gene promoter is used, expression of a viral protein can be induced specifically in a target disease cell at any time by locally administering an inducer such as heavy metals such as gold, zinc, cadmium, steroids such as dexamethasone, alkylating agents, chelating agents, or cytokines to the location of the target disease cell at a desired time.

[0034] Alternatively, genes for proteins required for replication or assembly of two or more viruses can be placed under the control of the promoter of a factor whose expression is enhanced in the same or different organs, or the promoter of a factor whose expression is enhanced in a cancer cell-specific manner.

[0035] The recombinant virus may be a recombinant virus in which a region contained in the viral protein that is essential for inducing a cellular environment necessary for the proliferation of the virus in normal cells but is not necessary for the proliferation of the virus in cancer cells is deleted. For example, in order for the adenovirus to proliferate in normal cells, it is necessary to inactivate Rb or p53 to turn the cell cycle, but since the cell cycle is already turning in cancer cells, the Rb binding region of E1A and the p53 binding region of E1B are not essential for the proliferation of the adenovirus in cancer cells. Therefore, in the case of adenovirus, by deleting the E1A24KDa region (E1AΔ24), the E1B55KDa region (E1BΔ55K), or the E1B19KDa region (E1BΔ19), the virus will proliferate in cancer cells but not in normal cells, i.e., the virus will proliferate in cancer cells specifically. In the case of this type of recombinant virus, even if the gene of the protein necessary for viral replication is not under the control of a cancer cell-specific promoter, the recombinant virus of the present invention can cause cancer cell-specific proliferation. The recombinant virus of the present invention may be a recombinant virus in which the gene of the protein necessary for such viral replication or assembly is not under the control of a tissue-specific promoter or a cancer cell-specific promoter, and in which at least one of the regions (e.g., E1A24KDa region, E1B55KDa region, and E1B19KDa region) that is essential for inducing the cellular environment necessary for viral proliferation in normal cells but not necessary for viral proliferation in cancer cells is deleted.

[0036] In particular, a recombinant virus in which a gene lacking a region essential for inducing a cellular environment necessary for viral proliferation in normal cells but not necessary for viral proliferation in cancer cells is functionally linked downstream of an organ-specific promoter or a cancer cell-specific promoter is called a conditionally replicating virus regulated with multiple factors (m-CRV) (JP Patent Publication 2005-046101 and WO 2005 / 012536). Preferably, the present invention relates to such a conditionally replicating virus regulated with multiple factors.

[0037] This multifactor-regulated cancer-specific replication virus can be prepared as follows: For example, a plasmid vector P1 containing an E1A gene (which may lack the 24 KDa region) functionally linked to a tissue-specific promoter or a cancer-specific promoter, and an E1B gene (which may lack the 19 KDa or 55 KDa region) functionally linked to a constitutive promoter (such as a CMV promoter), a plasmid vector P2 containing an immune-inducing gene functionally linked to any one promoter selected from the RSV promoter, the E2F promoter, and promoters exhibiting activity equivalent thereto, and a backbone plasmid P3 containing an adenovirus genome lacking the E1 region (which may have a target cell-specific mutation in the fiber gene), are prepared, and these three types of plasmids are appropriately combined and plasmid-fused using the Cre recombinase loxP system. By selecting the target plasmid using the drug resistance gene and ori carried by each plasmid, a cancer cell-specific replicating adenovirus (CRA) vector plasmid carrying a tissue-specific promoter or cancer-specific promoter-E1A expression cassette, a constitutive promoter-E1B expression cassette, and any one of promoter-immune induction gene expression cassettes selected from the RSV promoter, E2F promoter, and promoters exhibiting activity equivalent thereto can be prepared. Then, a CRA vector can be prepared by transfecting the vector into a cell line (e.g., 293 cells) that complements E1A.

[0038] This recombinant virus can further contain a replication origin for autonomous amplification in host cells and a selection marker gene for selecting transformed cells (such as a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin, or a gene that complements auxotrophic mutations).

[0039] The disease for which the recombinant virus can be used as a therapeutic vector is cancer. In the present specification, examples of "cancer" include renal cell carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, 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, liver cancer, bile duct cancer, and the like. These include, but are not limited to, carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, 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, and multiple myeloma.

[0040] "Oncolytic virus" means a recombinant virus that grows in an infected tumor cell, lyses the infected cell, and releases daughter viruses outside the cell. In this way, the oncolytic virus kills the infected tumor cell, and the daughter viruses infect surrounding uninfected tumor cells and kill the cells in the same way. By repeating this process, the oncolytic virus has a high tumor cell killing efficiency. Oncolytic viruses do not usually cause cell lysis in normal cells. Various methods are already known in the art as such a tumor-selective cell lysis mechanism. Examples of recombinant viruses used as oncolytic viruses include adenoviruses, retroviruses, adeno-associated viruses, herpes viruses, herpes simplex viruses, lentiviruses, vaccinia viruses, pox viruses, polio viruses, Sindbis viruses, and Sendai viruses. Adenoviruses have the advantages of extremely high gene transfer efficiency, being able to be introduced into non-dividing cells, and extremely rare integration of introduced genes into host chromosomes.

[0041] In one embodiment, the recombinant virus may comprise an organ-specific promoter or a cancer-specific promoter operably linked to a nucleic acid encoding a cytotoxic or therapeutic factor. The nucleic acid encoding a cytotoxic factor may, for example, encode a protein or RNA that, when transcribed (and translated), directly or indirectly induces cell death or at least growth inhibition. The therapeutic factor may encode a protein (or RNA) that, when transcribed (and translated), directly or indirectly induces a therapeutic effect against a target disease by an action other than a cytotoxic action. For example, cytotoxic or therapeutic factors include tumor suppressor genes (p53, p21, etc.), cytokine genes (GM-CSF, IL-2, IL-4, IFN, etc.), apoptosis-inducing genes (Fas, etc.), genes encoding proteins constituting ion channels (sodium channels, etc.), genes for proteins that can damage cells by converting prodrugs into poisons (suicide genes) (HSV-thymidine kinase, cytosine deaminase, etc.), antisense nucleic acids against cancer-causing genes (antisense nucleic acids against TGF-β, antisense nucleic acids against survivin, etc.), angiogenesis-suppressing genes (platelet factor IV, angiostatin, endostatin, soluble VEGF receptor, etc.), miRNAs or mimics thereof that have cancer-suppressing effects, or antisense nucleic acids of miRNAs that have cancer-promoting effects, aptamers, ribozymes, etc. In addition, when the proliferation of the recombinant virus can be sufficiently controlled by employing the promoter and / or gene that controls the proliferation of the virus as already described, i.e., when the recombinant virus hardly proliferates in normal cells but proliferates sufficiently in cancer cells, the nucleic acid encoding the above-mentioned cytotoxic factor or therapeutic factor may be functionally linked to any of the above-mentioned exogenous promoters.

[0042] A nucleic acid encoding a cytotoxic or therapeutic factor can be isolated as a cDNA from a cell or tissue that produces the cytotoxic or therapeutic factor by a method known per se, and can be operably linked to the downstream of an Aurora kinase promoter. An expression cassette containing a nucleic acid encoding a cytotoxic or therapeutic factor under the control of an Aurora kinase promoter preferably contains a suitable polyadenylation sequence downstream of the nucleic acid or gene.

[0043] Although it is preferable to use a promoter of the same species as the mammalian subject of treatment, a heterologous promoter may also be used as long as it can exert promoter activity to a degree that provides sufficient infection efficiency and killing effect. For example, a mouse gene promoter can be used as a vector for human treatment.

[0044] As used herein, being "specific" to cancer cells is not limited to the case where the compound exhibits absolutely no activity in normal cells, but also includes the case where the compound drives gene expression in normal cells within a therapeutically acceptable range.

[0045] Since the recombinant virus can grow specifically in target cancer cells, exert cytotoxicity specifically in the cells, and express an immune-inducing gene at an optimal expression level, it can be mixed with a pharmacologically acceptable carrier as necessary to prepare various formulations such as injections, and then used as a cancer treatment drug with reduced cytokine-dependent side effects. As the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as formulation materials are used, and are incorporated as excipients, lubricants, binders, and disintegrants in solid formulations, and solvents, solubilizing agents, suspending agents, isotonic agents, buffers, and soothing agents in liquid formulations. In addition, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as necessary.

[0046] In one aspect, the present invention relates to a method for treating cancer with less damage to non-target cells, comprising administering the recombinant virus to tumor cells. In the method for treating cancer using the recombinant virus, the immune-inducing protein expressed from the immune-inducing gene introduced by the recombinant virus is quickly cleared from the serum and spleen, which are directly related to side effects throughout the body. This is therefore advantageous in suppressing side effects caused by excessive proteins of the immune-inducing gene circulating throughout the body. Although the immune-inducing protein expressed from the immune-inducing gene introduced by the recombinant virus disappears in a short time, the recombinant virus exhibits a tumor suppressing (tumor cell killing) effect equal to or greater than that of a therapeutic virus that produces a large amount of immune-inducing gene expression under the control of a highly active promoter such as CA. Surprisingly, in the determination of the final and most important therapeutic effect, namely the extension of survival rate, the recombinant virus produces a stronger therapeutic effect than a therapeutic virus that produces a large amount of immune-inducing protein due to the expression of a large amount of immune-inducing gene under the control of a highly active promoter such as CA. That is, the recombinant virus produces high concentrations of immune-inducing proteins in tumors for a short period of time, i.e., only for the period required to induce antitumor immunity, and such a high concentration only in the tumor local area, i.e., an optimal amount of immune-inducing proteins for an optimal period of time, efficiently induces specific systemic antitumor immunity against tumors, and as a result, has the effect of sufficiently suppressing tumor growth by inducing cell death in tumor cells. Thus, the present invention has demonstrated for the first time that a high concentration only in the tumor local area, i.e., an optimal amount of immune-inducing proteins for an optimal period of time, is extremely safe and has a sufficient therapeutic effect of suppressing tumor growth, and can efficiently induce systemic specific (safe) antitumor immunity through tumor local treatment, completely prevents and treats metastasis in untreated sites, and is extremely effective in extending the survival rate, which is the final therapeutic effect.Thus, a cancer treatment method comprising administering the present recombinant virus to a patient has low toxicity and damage to non-target cells (particularly normal cells) and specifically damages or kills tumor cells, due to the low amount of unnecessary and side-effect-causing immune-inducing protein in organs or tissues other than the tumor and / or the immune-inducing protein, which is the final product of expression of the immune-inducing gene, disappearing in a short period of time. As an example, the present invention is a cancer treatment method using an oncolytic virus expressing an immune-inducing gene, comprising administering the present recombinant virus to tumor cells, characterized in that the amount of the immune-inducing protein in the spleen or serum, which are representative organs or tissues clearly suggesting the induction of side effects of the immune-inducing protein and its risk, is low in a short period of time (an optimal amount is supplied for an optimal period of time) compared to the case of administering an oncolytic virus having an immune-inducing gene operably linked downstream of a CA promoter or an RSV promoter. Alternatively, in another embodiment, the present invention may be a method for treating cancer using an oncolytic virus expressing an immune-inducing gene, comprising administering the recombinant virus to tumor cells, characterized in that unnecessary circulation and supply of the protein expressed from the immune-inducing gene to the entire body, such as the spleen or serum, is dramatically reduced in amount and for a shorter period of time compared to the case of administering an oncolytic virus having an immune-inducing gene operably linked downstream of an RSV promoter (i.e., the therapeutic effect is not reduced but rather enhanced, and the possibility of side effects is extremely low).

[0047] The disease therapeutic agent containing this recombinant virus is administered either by the ex vivo method, in which appropriate cells of the animal to be treated (or the cells of an animal of the same or different species as the animal to be treated) are removed from the body, cultured, and then introduced and returned (or transplanted) to the body, or by the in vivo method, in which the vector is directly administered into the body of the subject to be administered for introduction, with the in vivo method being preferred. In the case of the in vivo method, the preparation can be administered, for example, by injection, catheter, balloon catheter, local injection, etc. The site of administration can be into the blood, into a tumor, into the peritoneal cavity, or into muscle, with administration into a tumor being preferred.

[0048] The dosage of the disease therapeutic agent containing the present recombinant virus varies depending on the type of recombinant virus, promoter activity in target cells, type of therapeutic factor, administration route, severity of disease, animal species to be administered, drug tolerance, body weight, age, etc. of the administered subject. For example, when a cancer-specific replication-type adenovirus is used as the recombinant virus, the dosage of the therapeutic agent containing the present recombinant virus is 1×10 virus particles in conventional clinical trials of cancer gene therapy. 10 ~10 12 Since safety has been confirmed using particles / tumor, the same amount is the standard for administration (Molecular Therapy, 18: 429-434, 2010). In fact, the inventor has been conducting investigator-initiated clinical trials of Surv.m-CRA-1 (not carrying a therapeutic gene) for 10 years. 10 ~10 12 Testing is currently being conducted on the amount of particles / tumor, and therapeutic efficacy and safety have been confirmed in all cases to date. EXAMPLES

[0049] The present invention will be described in more detail below based on examples. However, the present invention is not limited to these examples. All documents cited throughout this application are incorporated herein by reference in their entirety. This application also claims priority from Japanese Patent Application No. 2017-215579, filed on November 8, 2017, and Japanese Patent Application No. 2018-050722, filed on March 19, 2018. All contents of these Japanese patent applications from which this application claims priority are incorporated herein by reference in their entirety.

[0050] Example 1: Structure of GM-CSF-expressing Surv.m-CRA The Surv.m-CRA had the Survivin promoter inserted upstream of E1A, an early gene essential for adenovirus proliferation, and expression cassettes were inserted that linked mouse GM-CSF cDNA to the E2F promoter, RSV promoter, and CA promoter to construct the following three types of GM-CSF-expressing Surv.m-CRA (Figure 1). Adenovirus construction was performed according to the method described by Nagano et al., Gene Therapy (2005) 12,1385-1393.

[0051] (1) Construction of P1+3 plasmid As the first step of the construction, P1+3 was prepared by restriction enzyme digestion from the replication-control plasmid P1 and the adenovirus backbone plasmid P3 (pAd.HM4, pAd.HM10; Mizuguchi and Kay, Hum. Gene Ther. 1999).

[0052] The adenovirus backbone plasmid P3 is equipped with human adenovirus type 5 genomic DNA, but the E1 gene region necessary for viral proliferation is deleted. A proliferation control cassette (Survpr-E1A-CMVpr-E1B19K) of about 3 kb prepared by using I-Ceu I and PI-Sce I of the proliferation control plasmid P1 (pHM5-Survpr-E1A-CMVpr-E1B19K) was inserted into this E1 deletion region. This proliferation control cassette has a structure in which the viral proliferation factors E1A and E1B genes are linked downstream of the cancer-specific Survivin promoter and the constitutive CMV promoter, respectively. The obtained P1+3 plasmids were named pAd.HM4-Survpr-E1A-CMVpr-E1B19K and pAd.HM10-Survpr-E1A-CMVpr-E1B19K, respectively.

[0053] (2) Preparation of P2 plasmid In the second step, a therapeutic gene transfer plasmid P2 was prepared by ligating mouse GM-CSF cDNA as a therapeutic gene downstream of each of three promoters (CA promoter, E2F promoter, and RSV promoter). (2)-1:pUni / CApr-mGM-CSF The construction procedure was as follows: first, the CA promoter was inserted into the plasmid pUni / V5-HisC-tet(c) (hereafter referred to as pUni), which has a LoxP sequence and a BGH poly A sequence, and then the coding sequence (CDS) of mGM-CSF was inserted downstream of that as a therapeutic gene.

[0054] First, the vector pUni was cleaved with Stu I (blunt end) and dephosphorylated to prevent self-ligation. The CA promoter to be inserted was cleaved at BgI II on the 5' side and BamH I on the 3' side of pHM-CAGpr-mKate2, and the CA promoter sequence containing a Sma I site at the rear was excised and blunt-ended. These were ligated to create pUni / CApr.

[0055] Next, pUni / CApr was digested at Sma I (blunt end) located downstream of the CA promoter, and dephosphorylated to prevent self-ligation. Meanwhile, the mGM-CSF CDS to be inserted was prepared by digesting the 5' EcoR I and 3' BamH I sites of the plasmid pBluescript SKII+mGM-CSF (RDB01469; RIKEN), and the mGM-CSF CDS was excised and blunt-ended. These were then ligated together to produce pUni / CApr-mGM-CSF.

[0056] (2)-2:pUni / E2Fpr-mGM-CSF Using pABS4-E2Fp-GFP containing the E2F promoter sequence as a template, PCR was performed using a sense primer with Xho I and Nco I sites added to the 5' end; 5'-TCAGTCCTCGAGCCATGGGGTACCATCCGGACAAAGCC-3' (SEQ ID NO: 10) and an antisense primer with Age I, Sal I and Spe I sites added to the 3' end; 5'-GGACGTACCGGTGTCGACACTAGTCGAGGGCTCGATCCCGCTCC-3' (SEQ ID NO: 11) to amplify the E2F promoter sequence. After cleavage with Xho I and Age I, the resulting product was inserted into the Xho I and Age I sites of pUni to prepare pUni / E2Fpr. On the other hand, PCR was performed using pBluescript SKII+mGM-CSF as a template, a sense primer with an Age I site added to the 5' end; 5'-TCAGTCACCGGTAGGAGGATGTGGCTGCAGAATTTACT-3' (SEQ ID NO: 12), and an antisense primer with an Apa I site added to the 3' end; 5'-GGACGTGGGCCCTCATTTTTGGCCTGGTTTTT-3' (SEQ ID NO: 13), to amplify the mGM-CSF cDNA sequence. After cleavage with Age I and Stu I, the cDNA was inserted into the Age I and Stu I sites of pUni / E2Fpr to prepare pUni / E2Fpr-mGM-CSF.

[0057] (2)-3:pUni / RSVpr-mGM-CSF Using pGEM-RSV-S containing the RSV promoter sequence as a template, PCR was performed using a sense primer with Xho I and Nco I sites added to the 5' end; 5'-TCAGTCCTCGAGCCATGGGCTTCGCGATGTACGGGCCA-3' (SEQ ID NO: 14) and an antisense primer with Age I, Sal I and Spe I sites added to the 3' end; 5'-GGACGTACCGGTGTCGACACTAGTACACCAATGTGGTGAATGGT-3' (SEQ ID NO: 15) to amplify the RSV promoter sequence. After cleavage with Xho I and Age I, the resulting product was inserted into the Xho I and Age I sites of pUni to prepare pUni / RSVpr. On the other hand, PCR was performed using pBluescript SKII+mGM-CSF as a template, a sense primer with an Age I site added to the 5' end; 5'-TCAGTCACCGGTAGGAGGATGTGGCTGCAGAATTTACT-3' (SEQ ID NO: 12), and an antisense primer with an Apa I site added to the 3' end; 5'-GACGTGGGCCCTCATTTTTGGCCTGGTTTTT-3' (SEQ ID NO: 13), to amplify the mGM-CSF cDNA sequence. After cleavage with Age I and Stu I, the cDNA was inserted into the Age I and Stu I sites of pUni / RSVpr to prepare pUni / RSVpr-mGM-CSF.

[0058] (3) Construction of P1+2+3 plasmid As the final step, the P1+2+3 plasmid was prepared from the P1+3 and P2 plasmids described above by homologous recombination between specific sequences (LoxP) using the sequence-specific recombinase Cre. First, the P1+3 plasmid (pAd.HM4-Surv.m-CRA) and the P2 plasmid (pUni / CApr-mGM-CSF) were subjected to Cre / LoxP homologous recombination at a molar ratio of 1:0.3 to prepare the P1+2+3 plasmid (pAd.HM4-Surv.m-CRA / CApr-mGM-CSF). Similarly, the P1+3 plasmid (pAd.HM10.Surv.m-CRA) and pUni / E2Fpr-mGM-CSF or pUni / RSVpr-mGM-CSF) were subjected to Cre / LoxP homologous recombination at a molar ratio of 1:0.3 to generate the P1+2+3 plasmids (pAd.HM10-Surv.m-CRA / E2Fpr-mGM-CSF and pAd.HM10-Surv.m-CRA / RSVpr-mGM-CSF), respectively.

[0059] (4) Preparation of adenovirus The plasmids pAd.Surv.E1A-CMV.E1B19K, pAd.HM4-Surv.m-CRA / CApr-mGM-CSF, pAd.HM10-Surv.m-CRA / E2Fpr-mGM-CSF, and pAd.HM10-Surv.m-CRA / RSVpr-mGM-CSF were each cleaved with the restriction enzyme Pac I and transfected into adenovirus-producing HEK293 cells. Viral plaques were then isolated and used as seed viruses.

[0060] (5) Amplification of adenovirus Each seed virus was infected into HEK293 cells seeded in a 24-well plate, and the cells and culture medium were harvested when 90% cytopathic effect (CPE) was observed. These virus-infected cell suspensions were frozen and thawed three times, and then infected into HEK293 cells seeded in a 10 cm dish. This virus infection and recovery procedure was gradually scaled up, and finally amplified to 40 15 cm dishes, and the cells and culture medium were harvested and frozen for storage.

[0061] (6) Purification of adenovirus Each adenovirus was purified from the collected cell fluid by cesium chloride density gradient ultracentrifugation. First, primary purification was performed by ultracentrifugation at 35,000 rpm, 10°C, for 1 hour, and the virus band was collected. Then, secondary purification was performed by ultracentrifugation at 35,000 rpm, 10°C, for 18 hours, and the virus band was collected. The collected virus fluid was fractionated using an Econo-pac 10DG desalting column (Bio-Rad Laboratories) to obtain a purified adenovirus fluid. Then, OD 260 After estimating the number of virus particles by this method, glycerol was added to a final concentration of 10%, and the mixture was frozen and stored.

[0062] (7) Preparation of Surv.m-CRA expressing IL-2 and IL-15 (7-1) Preparation of Surv.m-CRA expressing human IL-2 First, a therapeutic gene introduction plasmid P2 (pUni / E2Fpr-hIL-2) was prepared by linking human IL-2 cDNA downstream of the E2F promoter. At that time, PCR was performed using pBluescript SKII+hIL-2 as a template, a sense primer with an Age I site added to the 5' side; 5'-TCAGTCACCGGTGCCACAATGTACAGGATGCAACTCCT-3' (SEQ ID NO: 16), and an antisense primer with an Apa I site added to the 3' side; 5'-GGACGTGGGCCCTCAAGTCAGTGTTGAGATGA-3' (SEQ ID NO: 17), to amplify the hIL-2 cDNA sequence. After that, after cleavage with Age I and Stu I, it was inserted into the Age I and Stu I sites of pUni / E2Fpr to prepare pUni / E2Fpr-hIL-2. Then, the P1+2+3 plasmid (pAd.HM4-Surv.m-CRA / E2Fpr-hIL-2) was generated.

[0063] (7-2) Preparation of mouse IL-2 expressing Surv.m-CRA First, a therapeutic gene introduction plasmid P2 (pUni / E2Fpr-mIL-2) was prepared by linking mouse IL-2 cDNA downstream of the E2F promoter. At that time, PCR was performed using pBluescript SKII+mIL-2 as a template, a sense primer with an Age I site added to the 5' side; 5'-TCAGTCACCGGTGCAGGCATGTACAGCATGCAGCTCGC-3' (SEQ ID NO: 18), and an antisense primer with an Apa I site added to the 3' side; 5'-GGACGTGGGCCCTTATTGAGGGCTTGTTGAGA-3' (SEQ ID NO: 19), to amplify the mIL-2 cDNA sequence. After that, after cleavage with Age I and Stu I, it was inserted into the Age I and Stu I sites of pUni / E2Fpr to prepare pUni / E2Fpr-mIL-2. Then, the P1+2+3 plasmid (pAd.HM4-Surv.m-CRA / E2Fpr-mIL-2) was generated.

[0064] (7-3) Preparation of Surv.m-CRA expressing human IL-15 First, a therapeutic gene introduction plasmid P2 (pUni / E2Fpr-hIL-15) was prepared by linking human IL-15 cDNA downstream of the E2F promoter. At that time, PCR was performed using hIL-15 cDNA as a template, a sense primer with an Age I site added to the 5' end; 5'-TCAGTCACCGGTTGAGTAATGAGAATTTCGAAACCACA-3' (SEQ ID NO: 20), and an antisense primer with an Apa I site added to the 3' end; 5'-GGACGTGGGCCCTCAAGAAGTGTTGATGAACA-3' (SEQ ID NO: 21), to amplify the hIL-15 cDNA sequence. After that, after cleavage with Age I and Stu I, it was inserted into the Age I and Stu I sites of pUni / E2Fpr to prepare pUni / E2Fpr-hIL-15. Then, the P1+2+3 plasmid (pAd.HM4-Surv.m-CRA / E2Fpr-hIL-15) was generated.

[0065] (7-4) Preparation of mouse IL-15 expressing Surv.m-CRA First, a therapeutic gene introduction plasmid P2 (pUni / E2Fpr-mIL-15) was prepared by linking mouse IL-15 cDNA downstream of the E2F promoter. At that time, PCR was performed using mIL-15 cDNA as a template, a sense primer with an Age I site added to the 5' side; 5'-TCAGTCACCGGTTAAGTAATGAAAATTTTGAAACCATA-3' (SEQ ID NO: 22), and an antisense primer with an Apa I site added to the 3' side; 5'-GGACGTGGGCCCTCAGGACGTGTTGATGAACA-3' (SEQ ID NO: 23), to amplify the mIL-15 cDNA sequence. After that, after cleavage with Age I and Stu I, it was inserted into the Age I and Stu I sites of pUni / E2Fpr to prepare pUni / E2Fpr-mIL-15. Then, the P1+2+3 plasmid (pAd.HM4-Surv.m-CRA / E2Fpr-mIL-15) was generated.

[0066] Thereafter, the adenovirus was produced, amplified, and purified in the same manner. Finally, after preparing DNA derived from each adenovirus, PCR was performed using specific primers to confirm specific amplification. Of the viruses whose viral titers could be determined, Surv.m-CRA / E2Fpr-mL-2 had a titer of 1.5 × 10 10 PFU / mL, Surv.m-CRA / E2Fpr-hIL-15 was 2.8 × 10 10 The PFU / mL.

[0067] Example 2: Adenovirus infection efficiency in hamster-derived cells The infection efficiency of adenovirus in hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21) was examined. Syrian hamster kidney tumor-derived HaK cells were kindly provided by Prof. M. Wold of Saint Louis University School of Medicine. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Nacalai Tesque) containing 10% Fetal Bovine Serum (FBS; Biowest) and 1% penicillin-streptomycin (Nacalai Tesque) at 37°C under 5% CO2. Syrian hamster pancreatic tumor-derived HaP-T1 cells were purchased from the RIKEN BRC cell bank (RBRC-RCB0411). The cells were cultured in MEM (Minimum Essential Medium; SIGMA) containing 10% FBS, 1% non-essential amino acids (NEAA; SIGMA), 1 mM sodium pyruvate (Thermo Fisher Scientific), and 1% penicillin-streptomycin under 37°C under 5% CO2. Syrian hamster neonatal kidney-derived BHK-21 cells were purchased from the JCRB Cell Bank of the National Institutes of Biomedical Innovation, Health and Nutrition (JCRB9020). Culture was performed at 37°C under 5% CO2 conditions in MEM containing 10% FBS, 1% NEAA, and 1% penicillin-streptomycin (Nacalai Tesque).

[0068] The cells were seeded into a 12-well plate the day before and counted on the day of infection. HaK: 6.45 × 10 5 cells / well, HaP-T1: 6.75×10 5 Cells / well, BHK-21: 6.75×10 5 The cells were then infected with a non-replicating adenovirus (Ad.CMV-EGFP) expressing EGFP under the control of a constitutive and strong CMV (cytomegalovirus) promoter at a multiplicity of infection (MOI) of 0, 0.1, 0.3, 1, 3, 10, 30, 100, 300, or 1000 for 1 hour (MOI 0 was the non-virus-infected control). After 48 hours of culture, the cells were photographed under a fluorescent microscope at the magnifications shown in the figure. The cells were then detached with a trypsin / EDTA solution and subjected to flow cytometry (FACS) analysis using a flow cytometer SH800Z (Sony) to examine the EGFP positivity rate (adenovirus infection efficiency) under each condition.

[0069] As a result, an MOI-dependent increase in EGFP-positive rate was observed (Figures 2 and 3). The adenovirus infection efficiency (AGTE) at MOI 30 was 95% or more in HaK and HaP-T1, and 70% or more in BHK-21, indicating efficient adenovirus infection in all cells. On the other hand, at MOI 300 or more, strong cytotoxicity was observed in all cells.

[0070] Example 3: Promoter activity in hamster-derived cells The promoter activity of E2F, RSV, and Survivin was examined in hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21). The cells were seeded on a 6-well plate the day before and counted on the day of infection. HaK: 1.29 × 10 6 cells / well, HaP-T1: 1.0×10 6 Cells / well, BHK-21: 1.7×10 6cells / well. Then, Ad.dE1.3 (Control), which lacks the genes E1 and E3 important for viral proliferation, and Ad.E2Fp-LacZ, Ad.RSVp-LacZ, and Ad.Survp-LacZ, which express the β-galactosidase gene (LacZ) under the control of each promoter, were infected at MOI30 for 1 hour and then cultured for 48 hours. Then, the β-galactosidase activity in the cell lysate was examined using Beta-Glo (registered trademark) Assay System (Promega). The experiment was performed in 3 wells under each condition, and the data were shown as the mean ± standard error.

[0071] The results are shown in Figure 4. In cancer cells (HaK cells), the RSV and CMV promoters showed strong activity, while the E2F and survivin promoters showed moderate activity. In HaP-T1 cells, the survivin, E2F, and RSV promoters showed moderate activity. On the other hand, the Survivin promoter was almost not active in normal cells. The other promoters also tended to be less active in normal cells compared to cancer cells, but the E2F promoter showed even lower activity in normal cells than the RSV and CMV promoters. From this, it was revealed that the E2F and survivin promoters showed moderate promoter activity in two different types of cancer cells and much lower activity than the RSV and CMV promoters in normal cells, that is, they showed the characteristics of cancer cell-specific (dominant) promoters.

[0072] Example 4: Expression of mouse GM-CSF in mGM-CSF-expressing Surv.m-CRA-infected hamster cells Since the gene sequence of hamster GM-CSF has not been reported, mouse GM-CSF (mGM-CSF) was used. The protein expression levels of mGM-CSF in hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21) infected with each mGM-CSF-expressing Surv.m-CRA were examined by ELISA (Enzyme-Linked ImmunoSorbent Assay). The cells were seeded on a 6-well plate the day before and counted on the day of infection. HaK: 1.4×10 6 cells / well, HaP-T1: 1.47×10 6 Cells / well, BHK-21: 2.2 × 10 6 The number of cells / well was 100. The cells were infected with Ad.dE1.3 (control), Surv.m-CRA, Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF at an MOI of 1, 10, or 100 for 1 hour, and then cultured for 48 hours. The culture supernatants were then collected, and the expression levels of mGM-CSF protein were examined using a Mouse GM-CSF Quantikine ELISA Kit (R&D systems). ELISA measurements were performed on two wells of each cell supernatant, and the data are shown as the mean ± standard error. Statistical differences between conditions were examined using Student's t test (*, P<0.05). In addition, a similar study was performed on each Surv.m-CRA expressing human GM-CSF (hGM-CSF) (Surv.m-CRA / E2Fp-hGM-CSF, Surv.h-CRA / RSVp-mGM-CSF, Surv.m-CRA / CAp-hGM-CSF).

[0073] The results are shown in Figure 5. An MOI-dependent increase in mouse GM-CSF expression was observed in all cells (Figure 5A-C). Comparison between the promoters showed that the CA promoter induced the strongest expression, resulting in very high levels of cytokine secretion, followed by the RSV promoter, which induced the strongest expression (and therefore high levels of cytokine secretion), while E2F induced moderate levels of cytokine expression and secretion. No significant difference in expression levels was observed between the cells. A similar tendency was also observed for human GM-CSF expression (Figure 5D). These results (Figures 4 and 5) indicate that the RSV and CMV promoters are ubiquitous (regardless of cell type) and relatively strongly activated, and that the E2F promoter has slightly lower or equal activity to the Survivin promoter in cancer but lower activity than RSV or CMV, and in normal cells, its activity is lower than RSV or CMV but not as low as Survivin, and although weaker than the Survivin promoter, it shows cancer specificity. These results are the first to clarify the promoter characteristics of each promoter in hamster cells, or specifically, the "specific" differences in relative activity levels and the degree of cancer-specific activity of these four promoters in cancer cells and normal cells.

[0074] Example 5 Cytotoxic effect of mGM-CSF-expressing Surv.m-CRA-infected hamster cells The cytotoxicity of each mGM-CSF-expressing Surv.m-CRA against hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21) was examined by measuring the number of viable cells. 2 Cells were seeded at 1000 cells / well. Ad.dE1.3 (control), Surv.m-CRA, Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF were infected for 1 hour at MOIs of 3 and 30, and then cultured. Cytotoxicity was evaluated by WST-8 assay using the viable cell counting reagent SF (Nacalai Tesque) at 3 and 5 days after infection.

[0075] As a result, in HaK and HaP-T1, Surv.m-CRA and the three types of GM-CSF-expressing Surv.m-CRA all showed significant cytotoxicity compared to the control (Figure 6). In addition, the three types of GM-CSF-expressing Surv.m-CRA all showed significantly higher cytotoxicity compared to Surv.m-CRA. In particular, the CA promoter was the strongest, followed by the RSV promoter, and E2F was weaker than CA. Stronger cytotoxicity was observed at MOI30 compared to MOI3. On the other hand, in BHK-21, although a moderate cytotoxic effect was observed in the mGM-CSF-expressing virus at MOI30, no significant cytotoxicity was observed in any virus at MOI3. In other words, the favorable characteristics of the Surv.m-CRA, which is the CRA that controls viral proliferation with the Survivin promoter, and which serves as the base (backbone), such as safety due to its high cancer specificity, were demonstrated.

[0076] Example 6: Tumor-suppressing effect of mGM-CSF-expressing Surv.m-CRA in a tumor-bearing hamster model 1 × 10 7 HaK cells were subcutaneously transplanted at one site on the dorsum of the mouse. The HaK cells were transplanted at a concentration of 1 × 10 7 After about 33 days, when the diameter of the transplanted tumor reached 6-10 mm, each of the viruses Ad.dE1.3 (Control), Surv.m-CRA, Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF was added to PBS at a concentration of 1 × 10 9After adjusting the concentration to PFU / 100 μl, a single injection was performed into the tumor, and changes in tumor diameter over time were evaluated (Figure 7). The numbers of mice in each group administered with Ad.dE1.3 (control), Surv.m-CRA, Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF viruses were n=12, 9, 11, 11, and 12, respectively. Tumor size was measured twice a week using a digital caliper. Tumor volume was measured as major axis (mm) × minor axis (mm) × minor axis (mm) × 0.5 (mm 3 The data are shown as mean ± standard error, and statistically significant differences between groups were tested by Student's t test (*, P<0.05 vs. Ad.dE1.3; #, P<0.05 vs. Surv.m-CRA).

[0077] As a result, Surv.m-CRA and the three types of GM-CSF-expressing Surv.m-CRA all showed significant (strong) therapeutic effects (tumor-suppressing effects) against the treated primary cancer cells (nodules) compared to the control (Ad.dE1.3) (Figure 7). In addition, when compared to Surv.m-CRA not carrying a therapeutic gene, all three types of GM-CSF-expressing Surv.m-CRA showed a significant further "enhancement" of the therapeutic effect (tumor-suppressing effect).

[0078] Example 7 Survival curve after infection with mGM-CSF-expressing Surv.m-CRA in a tumor-bearing hamster model The effect of a single injection of each of the above viruses on the survival of HaK-transplanted hamsters was evaluated by survival curve analysis using the Kaplan-Meier method. Statistical differences between groups were examined using the log-rank test.

[0079] As a result, no significant difference was observed between any of the groups over the entire period (Figure 8). However, in the Surv.m-CRA / CAp-mGM-CSF-infected group, deaths occurred between 10 and 20 days after infection (Figure 8). Autopsies of these animals revealed pulmonary edema, spleen enlargement, dark kidneys, and white spots on the liver, and pathological analysis confirmed that the cause of death was systemic fatal side effects due to immune system abnormalities associated with cytokine overexpression. These experimental results reaffirmed the need for optimal expression levels of GM-CSF. In other words, this experiment revealed for the first time that, among promoters such as CA, E2F, and RSV, which were previously vaguely considered to be "strongly active promoters," CA has much stronger promoter activity than E2F, and that, particularly in the control of immune-inducing genes loaded onto oncolytic viruses, the CA promoter is an unsuitable promoter that brings about lethal side effects, while the E2F promoter, despite inducing moderate expression, is an "optimal (moderate)" promoter that shows strong therapeutic effects while ensuring high safety. In other words, this experiment created the new concept that "there exists an optimal (moderate) promoter for controlling immune-inducing genes loaded onto oncolytic viruses," and also created a specific invention that the expression level induced by the E2F promoter is optimal for this system.

[0080] Example 8 Rechallenge test in hamsters after treatment with mGM-CSF-expressing Surv.m-CRA 1 × 10 7 HaK were subcutaneously transplanted at a single site on the dorsum of the mouse. The cells were implanted at a concentration of 1 × 10 7 After about 28 days, when the diameter of the transplanted tumor reached 6-10 mm, each of the viruses Ad.dE1.3 (Control), Surv.m-CRA, Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF was added to PBS at a concentration of 1 × 109 After adjusting the concentration to PFU / 100 μl, the virus was injected once into the tumor. The number of mice in each group administered with Ad.dE1.3 (control), Surv.m-CRA, Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF viruses was 4, 5, 4, 3, and 3, respectively. 14 days after virus injection, 7.5 × 10 6 HaK and HaP-T1 cells were each cultured at 7.5 × 10 6 After preparing the cells at a concentration of 200 μl per 200 μl, the cells were subcutaneously transplanted onto the dorsum of the mouse. 15 days later, the formation of a secondary transplanted tumor was evaluated.

[0081] The results are shown in Table 1 below. In the GM-CSF-expressing Surv.m-CRA triple-treatment group, no tumor formation was observed in the re-implantation of HaK, whereas tumor formation was observed in all cases of HaP-T1 transplantation. In the control (Ad.dE1.3) and Surv.m-CRA treatment groups, tumor formation was observed in all cases of both HaK and HaP-T1 secondary transplantation. From the above results, it was revealed that in the mGM-CSF-expressing Surv.m-CRA treatment group, even when a moderate promoter such as the E2F promoter was used, systemic anti-tumor immunity specific to HaK (treated cancer cells) was induced, as in the case of a strong promoter such as CA, and metastatic cancer cells could be potently inhibited (treated).

[0082] [Table 1]

[0083] (Example 9) Expression of mGM-CSF in a tumor-bearing hamster model Examination of the amount of mGM-CSF expression in tissues after infection with Surv.m-CRA 1 × 10 7 HaK were subcutaneously transplanted at a single site on the dorsum of the mouse. The cells were implanted at a concentration of 1 × 10 7After about 42 days, when the diameter of the transplanted tumor reached 6-10 mm, each of the viruses Ad.dE1.3 (Control), Surv.m-CRA, Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF was added to PBS at a concentration of 1 × 10 9 After adjusting the concentration to PFU / 100μl, the virus was injected once into the tumor. Two and seven days later, serum, tumor, and spleen were isolated, and the expression level of mGM-CSF in the tissues was examined by ELISA. The number of mice in each group administered the virus was n=3 for each day. Data are shown as mean ± standard error, and statistically significant differences between groups were tested by Student's t test (*, P<0.05 vs Surv.m-CRA / E2Fpr-mGM-CSF; #, P<0.05 vs Surv.m-CRA / RSVpr-mGM-CSF).

[0084] As a result, all three types of GM-CSF-expressing Surv.m-CRA showed significantly higher mGM-CSF expression levels than the control (Ad.dE1.3) (Figure 9). In particular, the mGM-CSF expression level in the serum on Day 2 with the CA promoter was more than 1000 times higher than that with the other two promoters. Therefore, this result further clearly demonstrated that the cause of the early deaths in the Surv.m-CRA / CAp-mGM-CSF-infected group observed in Figure 8 was abnormal pathological levels of systemic immune activation due to cytokine overexpression. Surprisingly, the E2F promoter was found to exert high antitumor activity and survival time extension effects as described above, even though it induced expression of GM-CSF at a level below the detection limit in serum, tumor, and spleen on Day 5.

[0085] Example 10: Survival curve after infection with mGM-CSF-expressing Surv.m-CRA in an orthotopic tumor-bearing hamster model 1×10 7 Hamster renal cell carcinoma-derived HaK cells were transplanted into one kidney of a 5-week-old female Syrian hamster. The cells were transplanted at 1 × 10 7 After about 14 days, each of the viruses, Ad.dE1.3 (Control), Surv.m-CRA, Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF, was added to PBS at a concentration of 1 × 10 9 After adjusting the concentration to PFU / 100μl, the virus was injected once into the kidney of the tumor-injected side. The effect of a single injection of each virus on survival in orthotopic tumor-bearing hamster models was evaluated by survival curve analysis using the Kaplan-Meier method. Statistical differences between groups were examined using the log-rank test.

[0086] As a result, in the Surv.m-CRA / CAp-mGM-CSF group, as in the subcutaneous tumor model, deaths occurred due to side effects of systemic pathological (abnormal) immune activity associated with cytokine overexpression between 5 and 10 days after infection (early treatment period), and survival rates were significantly shortened, i.e., mortality rates were increased, compared with the Ad.dE1.3 (control) and Surv.m-CRA groups. Interestingly, the groups administered Surv.m-CRA / E2Fp-mGM-CSF and Surv.m-CRA / RSVp-mGM-CSF showed a very marked and significant increase in survival not only compared with the Surv.m-CRA / CAp-mGM-CSF group, but also compared with the Ad.dE1.3 (control: non-proliferating adeno-luis) and Surv.m-CRA (no therapeutic gene) groups. However, no significant differences were observed between the Surv.m-CRA / E2Fp-mGM-CSF group and the Surv.m-CRA / RSVp-mGM-CSF group. It is particularly noteworthy that while there is no significant difference in the therapeutic effects of Surv.m-CRA / E2Fp-mGM-CSF, Surv.m-CRA / RSVp-mGM-CSF, and Surv.m-CRA / CAp-mGM-CSF in terms of the inhibitory effect on tumor growth in a subcutaneous tumor model (commonly used in simple and easy experimental systems), the present invention has revealed for the first time that, in an analysis of long-term survival rates in an orthotopic tumor model that reflects the pathological condition and allows the ultimate usefulness of cancer therapeutic drugs to be evaluated in actual human clinical practice, Surv.m-CRA / E2Fp-mGM-CSF and Surv.m-CRA / RSVp-mGM-CSF showed very high efficacy, while Surv.m-CRA / CAp-mGM-CSF showed poor efficacy (lethal and dangerous). Furthermore, as shown in Figure 9, although the RSV promoter expressed lower levels of GM-CSF than the CA promoter, it expressed GM-CSF at levels detected in the spleen on day 5, which may have caused side effects not reflected in survival. Therefore, this experiment revealed for the first time that when immune-inducing genes are carried in oncolytic viruses, the optimal expression level not only dramatically improves safety, but also enhances the therapeutic effect itself.Furthermore, it was revealed for the first time that the promoter capable of achieving the optimal expression level is the E2F promoter and a promoter which brings about an expression level equivalent to that of the E2F promoter.

[0087] Example 11 Comparison of survival curves after infection with mGM-CSF-expressing Surv.m-CRA and administration of anti-mouse PD-1 antibody in orthotopic tumor-bearing hamster models 1×10 7 Hamster renal cell carcinoma-derived HaK cells were transplanted into one kidney of a 5-week-old female Syrian hamster. The cells were transplanted at 1 × 10 7 The cells were prepared in Corning® Matrigel Basement Membrane Matrix (Corning) at 1×109 PFU / 100μL. After about 14 days, Ad.dE1.3 (Control) or Surv.m-CRA / E2Fp-mGM-CSF was adjusted to 1×109 PFU / 100μL with PBS and injected once into the kidney of the tumor-implanted side. In addition, 500μg of anti-mouse PD-1 antibody or isotype control antibody was administered intraperitoneally on the same day. This day was designated Day 0, and the same amount of the antibody was administered on Days 2, 4, 6, and 8 in the same manner. The effect on survival was then analyzed in the same manner.

[0088] As a result, the survival rate was significantly longer in the Surv.m-CRA / E2Fpr-mGM-CSF group (N=14), i.e., the mortality rate was lower, than in the Ad.dE1.3 (control) group (N=17).Furthermore, the survival rate tended to be equal to or greater than that of the anti-mouse PD-1 antibody-administered group (N=17).

[0089] To summarize the results of all these examples, in the present invention, the concept itself was created for the first time that "in oncolytic immunotherapeutic agents (oncolytic viruses carrying immune-inducing genes), the immune-inducing genes carried along with the virus are also amplified, so that precise control of the optimal expression level of the immune-inducing factor is essential for both ensuring dramatic safety (removing lethal side effects) and inducing a strong therapeutic effect." Not only has this concept not been clearly expressed in terms of "safety," but there has also been no suggestion of this concept in terms of "dramatic enhancement of therapeutic effect," and it can be said that this overturns the vague and unfounded dogma (the therapeutic effect itself increases when the expression of a therapeutic gene is enhanced). In addition, in the present invention, both safety and therapeutic effect have been specifically clarified, as well as a promoter that induces the "optimal (moderate)" expression of the immune-inducing gene in oncolytic viruses, creating an innovative cancer treatment drug with performance that surpasses conventional oncolytic immunotherapeutic agents in both safety and therapeutic effect. Even Surv.m-CRA, which does not carry a therapeutic gene, has been shown to be not only extremely safe in investigator-initiated clinical trials conducted by the inventors so far, but has also been shown to be effective in treating all cases of intractable cancer for which standard treatments (previous cancer drugs and cancer treatment methods) have been ineffective (this is actually being proven in human patients). Therefore, the new tumor-lytic immunotherapy agent and method created in this invention are expected to become an even more promising and innovative cancer treatment drug. JPEG0007675451000002.jpg223158JPEG0007675451000003.jpg223158

Claims

1. An oncolytic adenovirus characterized in that it has an immune-inducing gene operably linked downstream of an RSV promoter and that viral proliferation is controlled by the survivin promoter, and that the E1A and E1B genes are located downstream of the survivin promoter.

2. Immune induction genes are Activin A, ANGPTL5, BAFF, BD-2 (β-Defensin-2), BD-3 (β-Defensin-3), BDNF, BMP-2, BMP-4, BMP-6, BMP-7, BMP-10, CCL1, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL6, CCL7 (MCP-3), CCL8 (MCP-2), CCL9 (MIP-1γ), CCL11 (Eotaxin-1), CCL12 (MCP-5), CCL13 (MCP-4), CCL14, CCL15 (MIP-1δ), CCL16, CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3β), CCL20 (MIP-3α), CCL21 (Exodus-2), CCL22, CCL23, CCL24 (Eotaxin-2), CCL25 (TECK), CCL26 (MIP-4α), CCL27, CCL28, CD40-Ligand (TRAP), CD137 (4-1BB)-Ligand, CNTF, CT-1, CX3CL1 (Fractalkine), CXCL1 (GRO1), CXCL2 (MIP-2α, GRO2), CXCL3 (MIP-2β, GRO3), CXCL4 (PF4), CXCL5, CXCL6, CXCL7, CXCL9, CXCL10, CXCL11, CXCL12 (SDF-1α), CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, DKK-1, DLL1, EGFs, EG-VEGF (Prokineticin 1), FasL, FGF-1 (acidic FGF), FGF-2 (basic FGF), FGF-3, FGF-4 (HBGF-4), FGF-5, FGF-6, FGF-7 (KGF, HBGF-7), FGF-8, FGF-9 (HBGF-9), FGF-10((). us--_d��                   -altyârρ!alty_eoly!’ _e-altyoly” . The our- ﵳfit oughtând!altyoly!'r-alty!'r." pHâoficationoly!'sK !nα��-αfit ™ / iveolinusârfitoly!'sl β? . _、_______、、!!Q、!!!!、、 ______、______、Ifi!4、I+!!ILQ!!BILB ___、______、I__、!!!!、!BBB [|_______、________、 ______、|____、I!!!、I3fo!+、I33B+、I our-olousically、olinusfitiveolylolylolyl!'rârfit!l !altyolyolyloly picolateolyoly -olyolousolousolousol The . *、!PPBQPBBBPB 、............ | . The al -βourβoly!‐ finalN butd-α、duousal but-β、on        ivealous udeousalousalousolateolme 、!!!!!!!!!!!!!!!!!!!!!!!!!!!B!BBHBBHBHBHHHHHHH! target!!!!B! In the newspaper, please subscribe to our Facebook page.

3. The oncolytic adenovirus according to claim 1, wherein the immune-inducing gene is GM-CSF.

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