Novel recombinant vaccinia virus and use thereof

Abstract

To provide a vaccinia virus having a therapeutic gene introduced therein as a foreign gene, and a therapeutic composition including the vaccinia virus.SOLUTION: A vaccinia virus contains, as a foreign gene, at least one immunoregulatory gene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to vaccinia viruses carrying therapeutic genes. [Background technology]

[0002] Currently, preclinical research and clinical trials of cancer treatments using live viruses are being actively conducted around the world. This cancer virotherapy utilizes the inherent property of viruses, which is to kill infected cells and tissues while multiplying and propagating within them. Compared to conventional radiation and chemotherapy, this anticancer effect is exerted through multiple mechanisms: first, tumor lysis due to viral proliferation, and second, the accompanying induction of antitumor immunity.

[0003] There is a vaccinia virus vaccine strain that was previously established in Japan and used in humans as a smallpox vaccine, and has been proven to be highly safe (see Non-Patent Document 1). However, because it still maintains weak proliferation in normal tissues, it was essential to improve it so that it would proliferate only in cancer cells in order to establish it as a safer cancer virotherapy. Therefore, by using genetic recombination technology to improve this vaccine strain, we succeeded in developing a genetically modified vaccinia virus that proliferates and destroys cancer cells specifically, using dysregulation of the MAPK / ERK pathway in a wide range of cancers as an indicator (see Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2011 / 125469 [Patent Document 2] International Publication No. WO2015 / 076422 [Non-patent literature]

[0005] [Non-Patent Document 1] Protein Nucleic Acid Enzyme Vol.48 No.12(2003), p.1693-1700 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a vaccinia virus into which a therapeutic gene has been introduced as a foreign gene, and a therapeutic composition containing the same. [Means for solving the problem]

[0007] While investigating the development of a vaccinia virus with greater antitumor effects, the present inventors discovered that introducing a gene encoding a protein capable of regulating immunity into an oncolytic vaccinia virus as a therapeutic gene can exert a synergistic anticancer effect, leading to the completion of the present invention.

[0008] That is, the present invention is as follows. [1] Vaccinia virus containing at least one immunoregulatory gene as a foreign gene. [2] The vaccinia virus of [1] containing two or three immunoregulatory genes as foreign genes. [3] The vaccinia virus of [1] or [2], wherein the vaccinia virus is the LC16 strain, the LC16mO strain, or the LC16m8 strain modified to express the B5R gene. [4] A vaccinia virus of any of [1] to [3], which is deficient in the function of the K2L gene or the HA gene, or the K2L gene and the HA gene, and which causes cell fusion in infected cells and induces cell death. [5] An oncolytic vaccinia virus, any of the vaccinia viruses [1] to [4]. [6] Vaccinia virus [4] or [5] that does not grow in normal cells but grows specifically in cancer cells and has oncolytic properties that specifically damage cancer cells. [7] A vaccinia virus according to any one of [1] to [6], wherein the immunoregulatory gene is selected from the group consisting of a gene encoding IL-12, a gene encoding CCL21, a gene encoding IL-7, a gene encoding PD1scFv (scFv of an anti-PD-1 antibody), and a gene encoding CD40L (CD154). [8] A vaccinia virus according to any one of [1] to [7], which contains a combination of a gene encoding IL-12 and a gene encoding CCL21. [9] A vaccinia virus according to any one of [1] to [7], which contains a combination of a gene encoding IL-12, a gene encoding CCL21, and a gene encoding IL-7.

[10] A vaccinia virus of any one of [1] to [7] containing any combination of the following genes (i) to (v): (i) the combination of the gene encoding IL-12 and the gene encoding IL-7; (ii) a combination of a gene encoding IL-12 and a gene encoding PD1scFv; (iii) the combination of the gene encoding IL-7 and the gene encoding CCL21; (iv) the combination of a gene encoding CD40L and a gene encoding IL-7; and (v) A combination of a gene encoding CD40L and a gene encoding CCL21.

[11] A pharmaceutical composition for cancer treatment, comprising any one of the vaccinia viruses [1] to

[10] .

[12] A composition comprising a combination of vaccinia viruses, including two or more types of vaccinia viruses of any of [1] to

[10] that contain one or two immune regulatory genes as foreign genes, wherein each vaccinia virus contains different immune regulatory genes.

[13] The composition of

[12] , comprising a combination of a vaccinia virus containing a combination of a gene encoding IL-12 and a gene encoding IL-7, and a vaccinia virus containing a gene encoding CCL21.

[14] The composition of

[12] or

[13] , which is a pharmaceutical composition for treating cancer.

[15] A kit containing a combination of vaccinia viruses including two or more types of vaccinia viruses of any of [1] to

[10] that contain one or two immune regulatory genes as foreign genes, wherein each vaccinia virus contains different immune regulatory genes.

[16] The kit according to

[15] , comprising a combination of a vaccinia virus containing a combination of a gene encoding IL-12 and a gene encoding IL-7, and a vaccinia virus containing a gene encoding CCL21.

[17] The kit according to

[15] or

[16] , which is a kit for cancer treatment. This specification includes the disclosure of Japanese Patent Application No. 2020-191128, from which this application claims priority. [Effects of the Invention]

[0009] By introducing a gene encoding a protein that can regulate immunity into an oncolytic vaccinia virus as a therapeutic gene, a greater anti-cancer effect is exerted due to the synergistic effect of the anti-tumor effect of the vaccinia virus and the effect of the therapeutic gene. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the partial genome structure of a recombinant vaccinia virus that expresses one type of immunoregulatory gene. [Figure 2-1] FIG. 1 shows cell images showing the cytopathic effect of vaccinia virus carrying and expressing an immunoregulatory gene on A549 cells. [Figure 2-2] FIG. 1 is a graph showing the cytopathic effect of vaccinia viruses carrying and expressing immunoregulatory genes on A549 cells, as measured by cell viability. [Figure 3-1] FIG. 1 shows cell images showing the cytopathic effect of vaccinia virus carrying and expressing an immunoregulatory gene on CT26 cells. [Figure 3-2]FIG. 1 is a graph showing the cytopathic effect of vaccinia viruses carrying and expressing immunoregulatory genes on CT26 cells, as measured by cell viability. [Figure 4] FIG. 1 shows a therapeutic experiment protocol using cancer-bearing model mice. [Figure 5-1] FIG. 1 shows luminescence detection images showing viral proliferation in tumors administered with vaccinia virus carrying and expressing an immune regulatory gene, and the distribution of viral proliferation. [Figure 5-2] FIG. 1 is a graph showing numerical values ​​of viral proliferation in tumors administered with vaccinia viruses carrying and expressing immune regulatory genes. [Figure 6-1] FIG. 1 shows luminescence detection images and the distribution of viral proliferation in tumors not administered with vaccinia virus carrying and expressing an immune regulatory gene. [Figure 6-2] FIG. 10 is a graph showing numerical values ​​of viral proliferation in tumors not administered with vaccinia virus carrying and expressing an immunoregulatory gene. [Figure 7-1] 1 shows tumor growth curves after administration of vaccinia virus carrying and expressing one type of immunoregulatory gene, where A shows the tumor growth curve on the administered side and B shows the tumor growth curve on the unadministered side. [Figure 7-2] FIG. 1 shows a survival curve after administration of a vaccinia virus carrying and expressing one type of immunoregulatory gene. [Figure 8-1] 1 shows tumor growth curves after administration of vaccinia viruses carrying and expressing two types of immune regulatory genes. A shows the tumor growth curve on the administered side, and B shows the tumor growth curve on the unadministered side. [Figure 8-2] FIG. 1 shows survival curves after administration of vaccinia viruses carrying and expressing two types of immunoregulatory genes. [Figure 9] FIG. 1 shows the partial genome structure of a recombinant vaccinia virus that expresses one type of immunoregulatory gene. [Figure 10] 1 shows tumor growth curves after administration of vaccinia virus carrying and expressing one type of immunoregulatory gene, where A shows the tumor growth curve on the administered side and B shows the tumor growth curve on the unadministered side. [Figure 11]1 shows tumor growth curves after administration of vaccinia viruses carrying and expressing two types of immune regulatory genes. A shows the tumor growth curve on the administered side, and B shows the tumor growth curve on the unadministered side. [Figure 12] FIG. 1 shows the partial genome structure of a recombinant vaccinia virus that expresses two types of immunoregulatory genes. [Figure 13] 1 shows the expression levels of immunoregulatory genes in A549 cells infected with vaccinia viruses carrying and expressing immunoregulatory genes, where A shows the expression level of mIL12 and B shows the expression level of mCCL21. [Figure 14] 1 shows tumor growth curves after administration of recombinant vaccinia viruses expressing two types of immunoregulatory genes, where A shows the tumor growth curve on the administered side and B shows the tumor growth curve on the unadministered side. [Figure 15] 1 shows tumor growth curves after administration of a recombinant vaccinia virus that expresses two types of immunoregulatory genes and has cell-fusion ability, or a recombinant vaccinia virus that expresses two types of immunoregulatory genes and does not have cell-fusion ability. A shows the tumor growth curve on the administered side, and B shows the tumor growth curve on the unadministered side. [Figure 16] FIG. 1 shows survival curves after administration of a recombinant vaccinia virus that expresses two types of immunoregulatory genes and has cell fusion ability, or a recombinant vaccinia virus that expresses two types of immunoregulatory genes and does not have cell fusion ability. [Figure 17] 1 shows tumor growth curves after administration of recombinant vaccinia viruses containing a combination of three types of immunoregulatory genes, where A shows the tumor growth curve on the administered side, and B shows the tumor growth curve on the unadministered side. [Figure 18] FIG. 1 shows survival curves after administration of recombinant vaccinia viruses containing combinations of three types of immunoregulatory genes. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below.

[0012] The present invention relates to a method for producing a vaccinia virus that expresses a therapeutic foreign gene, and the resulting vaccinia virus that expresses a specific foreign gene. The vaccinia virus that expresses a therapeutic foreign gene can be suitably used for cancer treatment.

[0013] Vaccinia virus strains for producing the vaccinia virus of the present invention are not limited, but include the Lister strain, the LC16 strain, LC16mO strain, and LC16m8 strain established from the Lister strain (Hashizume Takeshi, Clinical Viruses, Vol. 3, No. 3, 269, 1975, etc.), the New York City Board of Health (NYBH) strain, the Wyeth strain, the Copenhagen strain, the Western Reserve (WR) strain, the Modified Vaccinia Ankara (MVA) strain, the EM63 strain, the Ikeda strain, the Dalian strain, and the Tian Tan strain. The LC16mO strain was developed from the Lister strain via the LC16 strain, and the LC16m8 strain was further developed from the LC16mO strain. A frameshift mutation was found in the B5R gene, which encodes a viral membrane protein, and this protein was no longer expressed or functional, resulting in an attenuated strain (Protein, Nucleic Acid, Enzyme, Vol. 48, No. 12 (2003), p. 1693-1700).

[0014] In order to ensure safety when administered to humans, the vaccinia virus used in the present invention is preferably attenuated and non-pathogenic. Examples of such attenuated strains include strains in which the B5R gene is partially or completely deleted. The B5R gene encodes a protein present in the vaccinia virus envelope, and the B5R gene product is involved in viral infection and proliferation. The B5R gene product is present on the surface of infected cells and in the viral envelope and functions to increase the infection efficiency when the virus infects and spreads to neighboring cells or other sites in the host body. It also contributes to the plaque size and host range of the virus. Deletion of the B5R gene results in smaller plaque and pock size when infected with animal cells. It also reduces the skin proliferation ability and skin pathogenicity. Vaccinia viruses in which the B5R gene is partially or completely deleted lack the normal function of the B5R gene product, exhibit reduced skin proliferation ability, and do not cause side effects when administered to humans. An example of an attenuated strain lacking the B5R gene is the m8Δ strain (also referred to as the LC16m8Δ strain), which was established by completely deleting the B5R gene from the LC16m8 strain. Alternatively, the mOΔ strain (also referred to as the LCmOΔ strain), which was established by completely deleting the B5R gene from the LC16mO strain, can also be used. These attenuated vaccinia virus strains lacking the B5R gene partially or completely are described in International Publication No. WO 2005 / 054451 and can be obtained based on the description therein. Whether a vaccinia virus lacks the B5R gene partially or completely and thus lacks the function of the B5R protein can be determined by, for example, the plaque size or pock size formed when infected with RK13 cells, viral growth in Vero cells, or skin pathogenicity in rabbits. Alternatively, the gene sequence of the vaccinia virus may be examined.

[0015] Vaccinia viruses carrying the B5R gene express the B5R gene in cancer cells, damaging them through the action of the B5R protein. Therefore, it is desirable that the vaccinia virus used in the present invention express the complete B5R gene. When using a vaccinia virus that does not carry the B5R gene and has been attenuated and established for safety as described above, the complete B5R gene is introduced into the B5R gene-deleted vaccinia virus. When using a vaccinia virus with a partial or complete deletion of the B5R gene, the B5R gene can be inserted into the vaccinia virus genome. The B5R gene can be inserted into vaccinia viruses by any method, including known homologous recombination techniques. In this case, the B5R gene can be inserted between the B4R and B6R genes, where the B5R gene was originally located, or at any site in the vaccinia virus genome. Furthermore, the B5R gene can be constructed as a DNA construct and then introduced into vaccinia viruses.

[0016] Examples of vaccinia viruses carrying an exogenous therapeutic gene of the present invention include the vaccinia viruses described in International Publication No. WO2011 / 125469, International Publication No. WO2015 / 076422, or International Publication No. WO2017 / 014296.

[0017] International Publication No. WO2011 / 125469 describes a vaccinia virus in which a marker gene has been inserted into an endogenous gene such as the TK gene or HA gene, thereby deleting the functions of TK and HA.

[0018] International Publication No. WO2015 / 076422 describes a vaccinia virus in which a foreign gene has been inserted into the vaccinia virus growth factor (VGF) gene and the O1L gene, thereby deleting the functions of the vaccinia virus growth factor (VGF) and O1L. This vaccinia virus is called a mitogen-activated protein kinase (MAPK)-dependent recombinant vaccinia virus (MD-RVV (MDRVV)).

[0019] International Publication No. WO2017 / 014296 describes a vaccinia virus into which the UCA1 gene has been introduced in an expressible manner.

[0020] Furthermore, the vaccinia virus of the present invention may be a vaccinia virus mutated to have cell fusion ability. Here, cell fusion ability means that when a vaccinia virus infects a cell, it can cause cell fusion between infected cells. A vaccinia virus mutated to have cell fusion ability either lacks the function of a gene that suppresses the cell fusion ability that the vaccinia virus originally has, or has inserted and expressed a gene that promotes cell fusion. A vaccinia virus mutated to have cell fusion ability is called a fusogenic oncolytic vaccinia virus (FUVAC).

[0021] Examples of genes inherent in vaccinia viruses that are involved in cell fusion and inhibit cell fusion ability include the K2L gene and the HA (A56R) gene. The vaccinia viruses of the present invention are functionally deficient in either the K2L gene or the HA gene, or both the K2L and HA genes, resulting in an altered phenotype that allows for cell fusion. As shown in Figure 8 on page 5159 of Wagenaar et al., Journal of Virology, Vol. 82, No. 11, June 2008, pp. 5153-5160, in cells infected with vaccinia virus, a complex of the HA (A56R) protein and the K2L protein is anchored to the cell membrane via the transmembrane domain of HA. The entry / fusion complex (EFC), which is composed of multiple viral proteins (A21L, A28L, G3L, H2R, J5L, and L5R), is anchored to the membrane of the mature virus in cooperation with the viral proteins G9R and A16L. It is thought that G9R and A16L inhibit fusion between the virus and infected cells by acting on HA and K2L on the cell membrane. Therefore, dysfunction of HA and K2L eliminates the inhibitory function, allowing cell fusion to be induced. Therefore, in addition to K2L and HA, which encode viral proteins, genes inherent in vaccinia viruses that are involved in cell fusion and inhibit cell fusion include A16L, A21L, A25L, A26L, A28L, G3L, G9R, H2R, J5L, and L5R genes. For example, in G9R, mutation of H at position 44 to Y induces fusion even when the fusion-inhibiting molecule is normal. Therefore, cell fusion can be induced or enhanced by deleting or mutating one or more of these functions. An example of a combination is deleting K2L and mutating H at position 44 of G9R to Y.

[0022] Several types of viral proteins with fusion ability are known, and by inserting and expressing these genes in viruses, including different oncolytic viruses, they can be mutated to have cell fusion ability. Examples of such genes include the genes encoding the H (hemagglutinin) protein and F (fusion) protein derived from measles virus. Furthermore, as shown in U.S. Patent No. 7,635,752, it is possible to induce fusion in specific cells by modifying the H gene, and it has been demonstrated that expressing this gene in adenovirus or vesicular stomatitis virus (VSV) can be used in cancer therapy (Nakamura et al., Nature Biotechnology, Volume 22, Number 3, March 2004, pp. 331-336). Another example is the gene encoding the GaLV envelope derived from the Gibbon ape leukemia virus (GaLV), which has been demonstrated to be applicable to cancer therapy when expressed by herpesviruses, adenoviruses, and lentiviruses (Krabee et al., Cancers 2018, 10, 216; doi: 10.3390 / cancers10070216).

[0023] Other reported viruses include VSV expressing the FAST protein derived from Reovirus, adenovirus expressing the HIV envelope derived from HIV, VSV expressing the F protein derived from Newcastle disease virus (NDV), and adenovirus expressing the F protein derived from SV5 (Krabee et al., Cancers 2018, 10, 216; doi: 10.3390 / cancers10070216).

[0024] That is, examples of genes that promote cell fusion include a gene encoding a FAST protein derived from reovirus, a gene encoding an HIV envelope derived from HIV, a gene encoding an F protein derived from NDV, and a gene encoding an F protein derived from SV5.

[0025] The K2L gene is known as a serine protease inhibitor, but its function remains largely unknown. The HA gene is a glycoprotein induced on the surface of infected cells and is known as a hemagglutinin. The nucleotide sequence of the wild-type K2L gene is shown in SEQ ID NO: 15, and the nucleotide sequence of the wild-type HA gene is shown in SEQ ID NO: 16.

[0026] Deficiency of the function of the vaccinia virus K2L gene or HA gene refers to the absence of expression of the K2L gene or HA gene, or the expression of the expressed protein, but the normal function of the K2L protein or HA protein. Deficiency of the function of the vaccinia virus K2L gene or HA gene can be achieved by deleting all or part of the K2L gene or HA gene. Alternatively, the gene may be mutated by substituting, deleting, or adding bases, thereby preventing expression of the normal K2L protein or HA protein. Alternatively, a foreign gene may be inserted into the K2L gene or HA gene. While cell fusion was induced by the deficiency of K2L and HA in the present invention, the induction of cell fusion is important for the anticancer effect, and deficiency of other viral genes is also acceptable.

[0027] The function of a gene can be impaired by, for example, known techniques such as genome editing, homologous recombination, RNA interference, antisense, gene insertion, artificial mutation, and PTGS using a viral vector. In the present invention, a gene is said to be defective when a normal gene product is not expressed due to deletion or mutation of the gene.

[0028] Homologous recombination is a phenomenon in which two DNA molecules recombine with each other via the same base sequence within a cell. This method is often used to recombine viruses with large genomic DNA, such as vaccinia virus. First, a plasmid (called a transfer vector) is constructed in which the sequence of the target vaccinia virus K2L or HA gene is split in the middle and other DNA is ligated. This plasmid is then introduced into cells infected with vaccinia virus. During viral replication, the naked viral DNA is swapped with the same sequence on the transfer vector, resulting in the integration of the inserted DNA into the target gene in the viral genome, rendering the gene functionally incompetent. Cells that can be infected with vaccinia virus, such as BSC-1 cells, HTK-143 cells, Hep2 cells, MDCK cells, Vero cells, HeLa cells, CV1 cells, COS cells, RK13 cells, BHK-21 cells, and primary rabbit kidney cells, can be used. Furthermore, vectors can be introduced into cells by known methods such as the calcium phosphate method, the cationic liposome method, and the electroporation method.

[0029] Genome editing is a method that utilizes site-specific nucleases to modify targeted genes. Genome editing methods, depending on the nuclease used, include the ZFN (zinc finger nuclease) method (Urnov, Fyodor D. et al., Natur, Vol. 435, 2 June 2005, pp. 642-651), the TALEN (Tale nuclease) method (Mahfouz, Magdy M et al., PNAS February 8, 2011, 108(6), pp. 2623-2628), CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 (Crispr Associated protein 9) (Jinek, Martin, et al., Science, Vol. 337, 17 August 2012, pp. 816-821), and methods using CRISPR / Cas systems such as CRISPR / Cas3. These methods also include methods using modified nucleases, such as those using nickase-modified Cas. Among these, methods using the CRISPR / Cas9 system are preferred. In the CRISPR / Cas9 system, a desired sequence is cleaved using guide RNA (crRNA, tracrRNA) containing a sequence complementary to the target sequence of a gene whose function is to be disrupted by cleavage, and the nuclease Cas9. After genome cleavage, repair occurs via non-homologous end joining (NHEJ), inducing base deletions and knocking out the gene. Alternatively, after genome cleavage, mutations can be induced in the target gene by homologous recombination repair (HDR). To disrupt the GBSS gene and / or SBE gene by genome editing, a target sequence within the gene is selected and a guide RNA sequence containing a sequence complementary to that sequence is designed. The guide RNA preferably has a length of 20 or more bases. When genome editing is performed using the CRISPR / Cas9 system, the Cas9 protein and guide RNA can be co-expressed, for example, by introducing a vector that co-expresses both.Genome editing using CRISPR / Cas9 can be performed using commercially available CRISPR / Cas9 tools.

[0030] Deletion of the K2L or HA gene of vaccinia virus results in impaired K2L or HA function, which increases the virus's proliferation and propagation ability, improving its oncolytic ability and inducing cell death in infected cancer cells. Cell death includes apoptosis and necrosis. Cell fusion is also induced in infected cancer cells. This cell fusion enhances the ability to induce immunogenic cell death (ICD). This results in active infiltration of CD8 T cells into cancer cells, which attack them. Furthermore, systemic anti-cancer immune activity is improved. A decrease in immunosuppressive cells such as Tregs, TAMs, and MDSCs is also observed.

[0031] As a result, the K2L gene or HA gene of the vaccinia virus becomes deleted, and the function of K2L or HA is no longer functioning, thereby improving the anti-cancer effect of the vaccinia virus.

[0032] As described above, cell fusion occurs in cells infected with vaccinia virus, improving the anticancer effect, and the anticancer effect and ICD induction ability are improved regardless of whether the virus has tumor specificity or not. However, if the vaccinia virus also has tumor specificity, the anticancer effect is synergistically improved. Therefore, the vaccinia virus used in the present invention is preferably an oncolytic virus that has tumor cell-specific cytolytic activity and can infect cancer cells and cause cell death. This can be achieved by genetic modification, such as by deleting the function of a specific protein or suppressing the expression of a specific gene or protein.

[0033] Such genes include the hemagglutinin (HA) gene; the thymidine kinase (TK) gene; the F fragment; the F3 gene; the vaccinia virus growth factor (VGF) gene (U.S. Patent Application Publication No. 2003 / 0031681); O1L; the hemorrhagic region or type A inclusion body region (U.S. Patent No. 6,596,279); the Hind III F, F13L, or Hind III M region (U.S. Patent No. 6,548,068); the A33R, A34R, or A36R gene (Katz et al., J. Virology 77:12266-12275 (2003)); the SalF7L gene (Moore et al., EMBO J. 1992 11:1973-1980); the N1L gene (Kotwal et al., Virology 1989 171:579-58); the M1 gene (Child et al. al., Virology, 1990 174:625-629); HR, HindIII-MK, HindIII-MKF, HindIII-CNM, RR, or BamF region (Lee et al., J. Virol. 1992 66:2617-2630); C21L gene (Isaacs et al., Proc. Natl. Acad. Sci. USA. 1992 89:628-632). Among these genes, the VGF gene, O1L gene, TK gene, HA gene, and F fragment are preferred.

[0034] In addition, multiple genetic modifications may be combined. Examples of multiple genetic modifications include the following modifications. · Deficiency of TK, HA, and F14.5L functions (Cancer Research, 2007, Vol. 67, pp. 10038-10046) · Deficiency of TK and B18R functions (PLoS Medicine, 2007, Vol. 4, p. e353) - Deficiency of TK and ribonucleotide reductase functions (PLoS Pathogens, 2010, Vol.6, p.e1000984) Loss of SPI-1 and SPI-2 function (Cancer Research, 2005, Vol. 65, p. 9991-9998) -Deficiency of SPI-1, SPI-2 and TK functions (Gene Therapy, 2007, Vol.14, p.638-647) Introduction of mutations into the E3L and K3L regions (WO 2005 / 007824)

[0035] More than one of these genes may be deleted. For example, two genes, the VGF gene and the O1L gene, may be deleted. A vaccinia virus in which the functions of the VGF gene and the O1L gene are deleted is described in International Publication No. WO2015 / 076422.

[0036] For example, loss of TK gene function reduces the proliferation ability of vaccinia virus in normal cells. However, because cancer cells contain abundant enzymes that complement the function of this gene, the proliferation ability is not reduced in cancer cells. Reduced proliferation ability in normal cells means reduced pathogenicity to normal cells, which improves safety when applied to living organisms. Furthermore, when vaccinia virus lacking the functions of the VGF and O1L genes infects normal cells, ERK is not activated in the normal cells, preventing cell proliferation. As a result, vaccinia virus proliferation is significantly reduced. On the other hand, because the Ras / Raf / MEK / ERK metabolic pathway is abnormally active in cancer cells, this pathway complements the ERK activation function of vaccinia virus VGF and O1L, allowing the vaccinia virus to proliferate. As a result, vaccinia virus proliferates specifically in cancer cells, destroying and damaging the cancer cells.

[0037] The use of oncolytic vaccinia virus, coupled with the increased cell fusion ability due to deletion of the K2L gene or HA gene, synergistically improves the ability to induce cell death in cancer cells.

[0038] These gene deletions can be achieved by the above-mentioned genome editing, homologous recombination, RNA interference, antisense, gene insertion, artificial mutation, PTGS using viral vectors, and the like.

[0039] Vaccinia viruses that have oncolytic properties are called oncolytic vaccinia viruses.

[0040] The vaccinia virus of the present invention is an oncolytic vaccinia virus that contains a foreign therapeutic gene.

[0041] Therapeutic genes include interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL- 15, IL-17, IL-18, IL-21, IL-24, chemokine 2 (CCL2), CCL5, CCL19, CCL21, CXCL9, CXCL10, CXCL11, CD40L, CD70, CD80, CD13 These genes include those encoding physiologically active substances such as cytokines and chemokines, including 7L, OX40L, GITRL, LIGHT, α-interferon, β-interferon, γ-interferon, GM-CSF, G-CSF, M-CSF, MIP1a, FLT3L, HPGD, TRIF, DAI, and tumor necrosis factor, as well as those encoding immune checkpoint inhibitors, such as antibodies that inhibit CTLA4, PD1, and PD-L1. These genes encode proteins that can regulate the immune system and are called immunoregulatory genes. They are also called immunostimulatory genes because they have immunostimulatory effects.

[0042] The gene encoding the antibody may be a gene encoding a full-length antibody, or a gene encoding a functional fragment of the antibody. A functional antibody fragment is a fragment containing a portion of the antigen-binding region or antibody variable region of a full-length antibody and retains antigen-binding activity. Examples of functional antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, and single-chain antibody molecules (scFv). Multimers of these antibody fragments are also included in the functional antibody fragments of the present invention. Fab is a fragment obtained by treating an antibody with the protease papain. It is an antibody fragment with a molecular weight of approximately 50,000, in which approximately the amino-terminal half of the H chain and the entire L chain are linked via disulfide bonds and have antigen-binding activity. F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000, in which Fab is linked via disulfide bonds in the hinge region, among fragments obtained by treating IgG with the protease pepsin. Fab' is an antibody fragment with a molecular weight of approximately 50,000, obtained by cleaving the disulfide bonds in the hinge region of the above-mentioned F(ab')2. A single-chain antibody molecule (scFv), which is one type of Fv fragment, is an antibody fragment in which one heavy chain variable region (VH) and one light chain variable region (VL) are linked via a peptide linker. Diabodies are antibody fragments formed by dimerizing scFvs and have bivalent antigen-binding activity.

[0043] Furthermore, therapeutic genes include tumor suppressor genes such as p53 and Rb, and angiogenesis suppressor genes such as angiostatin, thrombospondin, endostatin, METH-1, and METH-2.

[0044] When the vaccinia virus of the present invention is used for cancer therapy, the therapeutic gene for cancer can exert a cancer therapeutic effect together with the oncolytic properties of the vaccinia virus.

[0045] Furthermore, by introducing DNA encoding antigens of viruses, bacteria, protozoa, cancers, etc. as a foreign gene (foreign DNA), the vaccinia virus vector into which the foreign gene has been introduced can be used as a vaccine against various viruses, bacteria, protozoa, and cancers. For example, protective antigens (neutralizing antigens) of human immunodeficiency virus, hepatitis virus, herpes virus, mycobacteria, malaria parasites, severe acute respiratory syndrome (SARS) virus, etc., or WT1, MART-1, NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, Glypican-3, KIF20A, Survivin, AFP-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, SART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, MESOTHEL Genes encoding cancer antigens such as proteins such as IN, NKG2D, P1A, 5T4, B7-H6, BCMA, CD123, CD133, CD138, CD171, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CEA, cMet, CS1, EGFR, EGFRvIII, EphA2, ErbB2, FAP, FR-α, HER2, IL13Ra2, MUC1, MUC16, NKG2D, PSCA, PSMA, ROR1, TARP, DLL3, PRSS21, Claudin18.2, Claudin18, CAIX, L1-CAM, FAP-α, CTAG1B, and FR-α, or glycolipids such as GD2 and GM2 may be introduced.

[0046] These foreign genes can be introduced, for example, by homologous recombination. Homologous recombination can be performed using the methods described above. For example, a plasmid (transfer vector) containing the foreign gene to be introduced is constructed by ligating the foreign gene into the DNA sequence at the desired site, and this vector is then introduced into cells infected with vaccinia virus. During viral replication, the naked viral DNA is swapped with the same sequence on the transfer vector, resulting in the integration of the inserted foreign gene into the viral genome. Cells that can be infected with vaccinia virus, such as CV1 cells, RK13 cells, BSC-1 cells, HTK-143 cells, Hep2 cells, MDCK cells, Vero cells, HeLa cells, COS cells, BHK-21 cells, and primary rabbit kidney cells, can be used. Furthermore, vectors can be introduced into cells using known methods, such as the calcium phosphate method, cationic liposome method, and electroporation.

[0047] The region for introducing a foreign gene is preferably inserted into a gene that is not essential for the life cycle of vaccinia virus, for example, the vaccinia virus growth factor (VGF) gene or the O1L gene.

[0048] Furthermore, when introducing a foreign gene, it is desirable to functionally link an appropriate promoter upstream of the foreign gene. The promoter is not limited, but examples that can be used include the aforementioned PSFJ1-10, PSFJ2-16, p7.5K promoter, p11K promoter, T7.10 promoter, CPX promoter, HF promoter, H6 promoter, and T7 hybrid promoter. Introducing a foreign gene into the vaccinia virus vector of the present invention can be carried out by known methods for constructing recombinant vaccinia virus vectors, such as those described in "Experimental Medicine: The Protocol Series, Gene Transfer & Expression Analysis Experimental Methods," edited by Saito Izumi et al., Yodosha (published September 1, 1997), or "DNA Cloning 4: Mammalian Systems," edited by D.M. Glover et al., translated and supervised by Kato Ikunoshin, EMBO Journal (1987), Vol. 6, pp. 3379-3384.

[0049] At least one foreign gene is introduced. Alternatively, two or more foreign genes may be introduced. For example, two, three, four, five, six, seven, eight, nine, ten, or more foreign genes may be introduced. The foreign gene to be introduced may be a full-length gene or a fragment of a functional site. Here, a functional site refers to a site that, when expressed as a protein, can exert the same function as the full-length protein. Furthermore, the foreign gene to be introduced is a DNA that has at least 85% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more sequence identity with the gene sequence of a wild-type foreign gene when calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., default, i.e., initial setting parameters). Genes encoding proteins with activity equivalent to that of the protein encoded by the wild-type gene are also encompassed within the foreign gene of the present invention. The amino acid sequence of the protein encoded by these genes may be such that at least one, preferably one or several (e.g., 1 to 10, more preferably 1 to 5, and particularly preferably 1 or 2) amino acids are deleted from the amino acid sequence of the protein encoded by the wild-type gene, or at least one, preferably one or several (e.g., 1 to 9, more preferably 1 to 5, and particularly preferably 1 or 2) amino acids are added to the amino acid sequence of the protein encoded by the wild-type gene, or at least one, preferably one or several (e.g., 1 to 9, more preferably 1 to 5, and particularly preferably 1 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 6 may be substituted with other amino acids. Proteins having such amino acid sequences have activity equivalent to that of the protein encoded by the wild-type gene.Furthermore, an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence of a protein encoded by such a wild-type gene has at least 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more sequence identity with the amino acid sequence of the protein encoded by the wild-type gene when calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, default, i.e., initial setting parameters), and a protein having such an amino acid sequence is a protein having activity equivalent to that of the protein encoded by the wild-type gene.

[0050] Among the above exogenous therapeutic genes, genes encoding IL-12, CCL21, IL-7, PD1scFv (scFv of anti-PD-1 antibody), and CD40L (CD154) are preferred. Furthermore, two, three, four, or five of these genes can be used in combination. Examples of combinations of two of these genes include the combination of genes encoding IL-12 and CCL21, the combination of genes encoding IL-12 and IL-7, the combination of genes encoding IL-12 and PD1scFv, the combination of genes encoding IL-12 and CD40L (CD154), the combination of genes encoding CCL21 and IL-7, the combination of genes encoding CCL21 and PD1scFv, the combination of genes encoding CCL21 and CD40L (CD154), the combination of genes encoding IL-7 and PD1scFv, the combination of genes encoding IL-7 and CD40L (CD154), and the combination of genes encoding PD1scFv and CD40L (CD154). The three types of combinations include a combination of genes encoding IL-12, CCL21, and IL-7, a combination of genes encoding IL-12, CCL21, and PD1scFv, a combination of genes encoding IL-12, CCL21, and CD40L (CD154), a combination of genes encoding IL-12, IL-7, and PD1scFv, a combination of genes encoding IL-12, IL-7, and CD40L (CD154), a combination of genes encoding IL-12, PD1scFv, and CD40L (CD154), a combination of genes encoding CCL21, IL-7, and PD1scFv, a combination of genes encoding CCL21, IL-7, and CD40L (CD154), a combination of genes encoding CCL21, PD1scFv, and CD40L (CD154), and a combination of genes encoding IL-7, PD1scFv, and CD40L (CD154).Four types of combinations include a combination of genes encoding IL-12, CCL21, IL-7, and PD1scFv, a combination of genes encoding IL-12, CCL21, IL-7, and CD40L (CD154), a combination of genes encoding IL-12, CCL21, PD1scFv, and CD40L (CD154), a combination of genes encoding IL-12, IL-7, PD1scFv, and CD40L (CD154), and a combination of genes encoding CCL21, IL-7, PD1scFv, and CD40L (CD154). Five types of combinations include a combination of IL-12, CCL21, IL-7, PD1scFv, and CD40L (CD154).

[0051] Viruses into which foreign genes have been introduced are designated as MDRVV-IL12 (MDRVV into which the gene encoding IL-12 has been introduced), MDRVV-IL7 / CCL21 (MDRVV into which two genes encoding IL-7 and CCL21 have been introduced), FUVAC-IL12 (FUVAC into which the gene encoding IL-12 has been introduced), and FUVAC-IL7 / CCL21 (FUVAC into which two genes encoding IL-7 and CCL21 have been introduced).

[0052] Multiple exogenous therapeutic genes may be introduced into a single vaccinia virus and that vaccinia virus may be used for therapy, or one exogenous gene may be introduced into a single vaccinia virus, and multiple vaccinia viruses carrying different exogenous genes may be used for therapy. For example, a vaccinia virus carrying any one of the genes encoding IL-7, IL-12, CD40L (CD154), CCL21, or PD1scFv (scFv of an anti-PD-1 antibody) may be prepared, and a vaccinia virus carrying an IL-7 gene, a vaccinia virus carrying an IL-12 gene, a vaccinia virus carrying a CD40L (CD154) gene, a vaccinia virus carrying a CCL21 gene, or a vaccinia virus carrying a PD1scFv (scFv of an anti-PD-1 antibody) gene may be prepared, and two, three, four, or five of these vaccinia viruses may be combined for use in therapy.

[0053] Furthermore, a vaccinia virus into which one or more, e.g., one or two, foreign genes have been introduced may be used in combination with a vaccinia virus into which a different one or more, e.g., one or two, foreign genes have been introduced. In this case, multiple vaccinia viruses can be used in combination. For example, a vaccinia virus into which any one of the genes encoding IL-7, IL-12, CD40L (CD154), CCL21, or PD1scFv (scFv of an anti-PD-1 antibody) has been introduced may be used in combination with a vaccinia virus into which any two or more genes encoding IL-7, IL-12, CD40L (CD154), CCL21, or PD1scFv (scFv of an anti-PD-1 antibody) different from the gene introduced by the vaccinia virus have been introduced. Examples include combinations of three types of viruses, such as the combination of FUVAC-IL12 / IL7 and FUVAC-CCL21, the combination of FUVAC-CCL21 / IL7 and FUVAC-IL12, the combination of FUVAC-IL12 / CCL21 and FUVAC-IL7, the combination of MDRVV-IL12 / IL7 and MDRVV-CCL21, the combination of MDRVV-CCL21 / IL7 and MDRVV-IL12, and the combination of MDRVV-IL12 / CCL21 and MDRVV-IL7.

[0054] The present invention also includes a composition containing multiple vaccinia viruses with different introduced foreign genes in the same composition, and a therapeutic kit that combines multiple vaccinia viruses with different introduced foreign genes as separate compositions.

[0055] By introducing the above-mentioned therapeutic gene into an oncolytic vaccinia virus and using it, the ability to induce cell death in cancer cells is synergistically improved.

[0056] Cancers that can be targeted by cancer virus therapy using vaccinia virus are not limited, and include any type of cancer, such as ovarian cancer, lung cancer, pancreatic cancer, skin cancer, stomach cancer, liver cancer, hepatocellular carcinoma, colon cancer, anal / rectal cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, prostate cancer, testicular cancer, head and neck cancer, brain / nerve tumors, thymus cancer, lymphoma / leukemia, bone / osteosarcoma, leiomyoma, rhabdomyoma, and melanoma.

[0057] The pharmaceutical composition for cancer treatment containing the vaccinia virus of the present invention contains a pharmaceutically effective amount of the vaccinia virus of the present invention as an active ingredient, and may be in the form of a sterile aqueous or non-aqueous solution, suspension, or emulsion. It may further contain pharmaceutically acceptable diluents, auxiliary agents, carriers, etc., such as salts, buffers, and adjuvants. Administration may be via various parenteral routes, such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, and transdermal routes. It may also be administered locally to the cancer site. The effective dose can be determined appropriately depending on the age, sex, health, weight, etc., of the subject. For example, but not limited to, a dose of about 10 mg / kg / day for an adult human can be administered. 2 ~10 10 Plaque-forming units (PFU).

[0058] The present invention also encompasses a method for treating cancer, which comprises administering the above-described vaccinia virus to a cancer patient. [Example]

[0059] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0060] Example 1: Construction of recombinant vaccinia virus carrying and expressing immunoregulatory genes To insert expression units for different foreign genes into the VGF and O1L genes of a mitogen-activated protein kinase-dependent recombinant vaccinia virus (WO 2015 / 076422) in which both VGF and O1L genes are nonfunctional, the BFP gene region was amplified using pTagBFP-N (FP172, Evrogen) DNA as a template with two primers (SEQ ID NO: 1 and SEQ ID NO: 2). Each PCR product was digested with restriction enzymes SfiI and EcoRI and cloned into the same restriction enzyme sites of the pTK-SP-LG vector (WO 2015 / 076422) to construct pTNshuttle / TK-SP-BFP, in which BFP is ligated under the control of a synthetic vaccinia virus promoter (Hammond JM. et al., Journal of Virological Methods. 1997; 66(1):135-138). Next, pTNshuttle / TK-SP-BFP was cleaved with the restriction enzymes SphI and EcoRI and blunt-treated. The SP-BFP fragment was then cloned into the pUC19-VGF vector (International Publication No. WO2015 / 076422) which had been cleaved with the restriction enzyme AccI and blunt-treated, or into the pUC19-O1L vector (International Publication No. WO2015 / 076422) which had been cleaved with the restriction enzyme XbaI and blunt-treated, to construct the shuttle vector pTNshuttle / VGF-ST-BFP for expressing BFP in the opposite orientation to VGF, or pTNshuttle / O1L-SP-BFP for expressing BFP in the opposite orientation to O1L. Separately, using a method similar to that described in International Publication No. WO2015 / 076422, we constructed pUC19-O1L-p7.5-DsRed to express DsRed in the same orientation as O1L under the control of the p7.5K promoter (p7.5) rather than the synthetic vaccinia virus promoter (SP). CV1 cells cultured to 80% confluence in a 24-well plate were infected with MDRVV virus (International Publication No. WO2015 / 076422) at an MOI of 0.1 to 0.5 and allowed to adsorb at room temperature for 1 hour.The transfer vector plasmid pTNshuttle / O1L-SP-BFP mixed with FuGENE HD (Roche) was then added to the cells according to the manufacturer's instructions and allowed to integrate. The cells were then cultured at 37°C for 2–3 days. The cells were harvested, freeze-thawed, and sonicated. The appropriate dilutions were then inoculated onto nearly confluent BSC1 cells. The cells were then added to Eagle MEM containing 0.5% methylcellulose and 5% FBS medium and cultured at 37°C for 2–4 days. The medium was removed, and BFP-expressing plaques were scraped with a tip and suspended in Opti-MEM medium (Invitrogen). This procedure was repeated three more times with BSC1 cells for plaque purification. After plaque purification, the plaque suspension was sonicated, and 200 μL of the resulting solution was used to extract genomic DNA using the High Pure Viral Nucleic Acid Kit (Roche) according to the manufacturer's instructions. Genomic DNA was then extracted from 200 μL of the resulting solution and subjected to PCR screening. For O1L, PCR was performed using two primers (SEQ ID NO: 3 and SEQ ID NO: 4). For clones in which a PCR product of the expected size was detected, the nucleotide sequence of the PCR product was confirmed by direct sequencing. A viral clone with an intact nucleotide sequence, VGF-LucGFP / O1L-BFP, was selected and amplified in A549 cells. The viral titer was then measured in RK13 cells and used for further experiments. Using this vaccinia virus (VGF-LucGFP / O1L-BFP) and transfer vector plasmid DNA (pUC19-O1L-p7.5-DsRed), a recombinant virus was recovered using the same method as above, using DsRed expression as an indicator. This virus was designated VGF-LucGFP / O1L-DsRed.

[0061] During the process of generating this recombinant vaccinia virus, VGF-LucGFP / O1L-DsRed, we observed a high frequency of viruses with unusual cell-fusion capabilities. The medium was then removed, and the fusogenic plaques were scraped with a tip and suspended in Opti-MEM medium (Invitrogen). This process was repeated three more times in BSC1 cells, and plaques were purified to obtain the fusogenic virus clone FUVAC. This clone was subjected to direct sequencing using a next-generation sequencer, PacBio RSII (Pacific Bioscience), and PCR. The resulting sequence revealed a mutation in the K2L gene, which mutated the 254th amino acid from tryptophan to a stop codon, resulting in a nonsense mutation (Patent Application No. 2019-091609, PCT / JP2020 / 018976).

[0062] To recover a recombinant vaccinia virus having the viral genome shown in Figure 1, a gene encoding mouse IL-12, consisting of the IL-12 p40 subunit (SEQ ID NO: 6) upstream of the IRES sequence (SEQ ID NO: 5) derived from encephalomyocarditis virus and the IL-12 p35 subunit (SEQ ID NO: 7) downstream, was synthesized by adding the restriction enzymes AgeI and NheI to both ends of the gene. After cleavage with AgeI and NheI, the gene was cloned into the same restriction enzyme sites of the pTNshuttle / VGF-SP-BFP vector so as to replace the BFP gene, constructing pTNshuttle / VGF-SP-mIL12. Similarly, genes encoding human IL-7 (SEQ ID NO: 8), mouse CCL21 (SEQ ID NO: 9), or mouse PD1 single-chain fragments (scFv) (Oncoimmunology. 2016;5(10):e1220467) were synthesized by adding restriction enzymes AgeI and NheI to both ends. After digestion with AgeI and NheI, the resulting genes were cloned into the same restriction enzyme sites of the pTNshuttle / O1L-SP-BFP vector, replacing the BFP gene. pTNshuttle / O1L-SP-hIL7, pTNshuttle / O1L-SP-mCCL21, or pTNshuttle / O1L-SP-mPD1scFv were constructed. Next, using the vaccinia virus (FUVAC) and transfer vector plasmid DNA (pTNshuttle / VGF-SP-mIL12), recombinant viruses were recovered using the same method as above, using the loss of GFP expression as an indicator, and designated FUVAC-IL12. Similarly, recombinant viruses were recovered using vaccinia virus (FUVAC) and transfer vector plasmid DNA (pTNshuttle / O1L-SP-hIL7, pTNshuttle / O1L-SP-mCCL21, or pTNshuttle / O1L-SP-mPD1scFv) in the same manner as above, using the disappearance of DsRed expression as an indicator, and named FUVAC-IL7, FUVAC-CCL21, or FUVAC-PD1scFv.PCR was performed using two primers (SEQ ID NO: 10 and SEQ ID NO: 11) for VGF and two primers (SEQ ID NO: 12 and SEQ ID NO: 13) for O1L. For clones in which PCR products of the specified size were detected, the nucleotide sequences of the PCR products were confirmed by direct sequencing. Recombinant viruses with intact nucleotide sequences were cultured in large quantities in A549 cells and purified, and then the viral titers were measured in RK13 cells and used for experiments.

[0063] To recover the recombinant vaccinia virus containing the viral genome shown in Figure 9, a sequence containing the EspEI and AvrII restriction enzyme sites under the p7.5K promoter (p7.5) was inserted into pTNshuttle / O1L-SP-BFP in the opposite direction to BFP expression to construct pTNshuttle / O1L-SP-BFP+p7.5. A gene encoding mouse CD40L (SEQ ID NO: 14) was synthesized with the AgeI and NheI restriction enzymes added to both ends, digested with AgeI and NheI, and cloned into the EspEI and AvrII restriction enzyme sites of the pTNshuttle / O1L-SP-BFP+p7.5 vector to construct pTNshuttle / O1L-SP-BFP+p7.5-mCD40L. As described above, recombinant viruses were isolated using vaccinia virus (FUVAC) and transfer vector plasmid DNA (pTNshuttle / O1L-SP-BFP+p7.5-mCD40L) using the same method as above, using the loss of DsRed expression and the expression of BFP as indicators. These viruses were designated FUVAC-CD40L. Recombinant viruses with intact nucleotide sequences were cultured in large quantities in A549 cells and purified. The viral titers were then measured in RK13 cells and used for the experiments.

[0064] To recover recombinant vaccinia viruses containing the viral genome shown in Figure 12, the above-mentioned vaccinia virus (FUVAC) and transfer vector plasmid DNA (pTNshuttle / VGF-SP-mIL12) were used to recover recombinant viruses, designated FUVAC-IL12, using the same method as above, based on the loss of GFP expression. Next, recombinant viruses were recovered using the same method as above, based on the loss of DsRed expression, based on the vaccinia virus (FUVAC-IL12) and transfer vector plasmid DNA (pTNshuttle / O1L-SP-mCCL21 or pTNshuttle / O1L-SP-hIL7), using the same method as above, based on the loss of DsRed expression. These viruses were designated FUVAC-IL12 / CCL21 or FUVAC-IL12 / IL7. Recombinant viruses with intact nucleotide sequences were cultured in large quantities in A549 cells and purified. The virus titers were then measured in RK13 cells and used for experiments.

[0065] Example 2: Characterization of recombinant vaccinia viruses carrying and expressing immune regulatory genes To compare recombinant vaccinia viruses carrying and expressing immune regulatory genes, cancer cells were infected with each virus. First, 1.0 × 10 human lung cancer A549 cells or 1.0 × 10 mouse colon cancer CT26 cells were placed in a 96-well plate. 4Cells were seeded at 1 / well and cultured for 24 hours. A549 cells were infected with each virus at an MOI of 0.1 or 1, and CT26 cells at an MOI of 1 or 10. Seventy-two hours after infection, infection patterns were observed using a BZ-X700 (Keyence). Results indicated that, compared with FUVAC without immunoregulatory gene expression, FUVAC carrying and expressing immunoregulatory genes infects, proliferates, and spreads in a viral dose-dependent manner, fusing cells with each other in both A549 (Figure 2-1) and CT26 (Figure 3-1) cells, regardless of the gene expression. Cell viability was also measured 72 hours after infection using the CellTiter96® Aqueous Non-radioactive Cell Proliferation Assay (Promega). Compared to FUVAC, FUVAC carrying and expressing immunoregulatory genes exhibited cytotoxic effects in both A549 cells (MOI = 1) and CT26 cells (MOI = 10), regardless of the gene expression. Figures 2-2 and 3-2 show the cell viability of cells infected with each virus, with the cell viability of mock cells treated in the same way but without virus infection set at 100% (n=3).

[0066] Example 3 Therapeutic effect of recombinant vaccinia virus carrying and expressing immunoregulatory genes Using the allograft model shown in Figure 4, we investigated the proliferation and spread of the virus in vivo and the therapeutic effect of the virus. CT26 cells were injected at 5.0 × 10 5 The cells were transplanted subcutaneously into both sides of the abdomen of each mouse. The tumor size was an average of 100 mm. 3 The tumors were grown for 6 to 7 days until they exceeded 2.5 × 10 cells / cm. Tumor volume was calculated using the formula: minor axis × major axis × major axis × 0.5. After tumor growth, PBS or each virus was administered three times every other day at a dose of 2.5 × 10 cells / cm. 7PFU of each virus was directly administered into one tumor (Days 0, 2, and 4). Furthermore, the luciferase-containing virus expressed in tumor cells infected with each virus was detected noninvasively by administering the luciferase substrate (luciferin) VivoGlo Luciferin (Promega) and observing the presence or absence of luminescence. Viral proliferation and spread were then noninvasively monitored using an in vivo imaging system (Berthold, NightSHADE LB985) (Days 1, 3, 5, and 7). Figure 5-1 shows the detected images of viral FLUC after virus administration, and Figure 5-2 shows the quantified results. Viral FLUC in the virus-administered side showed equally high signals on Days 1, 3, and 5 after administration, and had uniformly disappeared by Day 7 after administration. Figure 6-1 shows the detected images of viral FLUC after non-administration, and Figure 6-2 shows the quantified results. No viral signal was observed in the non-administered side. From the above, it was found that compared to FUVAC that does not carry and express immune regulatory genes, FUVAC that carries and expresses immune regulatory genes showed no difference in proliferation within the administered tumor, regardless of the type of gene carried and expressed, and that the virus did not spread to tumors on the non-administered side.

[0067] Next, we examined the therapeutic efficacy of the virus by measuring tumor diameter and survival curves. Two-way ANOVA statistical analysis confirmed that tumor diameters in both the virus-injected and non-injected sides were significantly smaller in mice treated with FUVAC without immunoregulatory genes and with each immunoregulatory gene compared with mice treated with PBS (Figure 7-1, ****: P<0.0001, ***: P<0.001, **: P<0.01, *: P<0.05). There was no significant difference in tumor diameter between the virus-injected and non-injected sides in mice treated with viruses expressing each immunoregulatory gene compared with mice treated with FUVAC (Figure 7-1). Log-rank statistical analysis demonstrated that mice treated with FUVAC without immunoregulatory genes and with viruses expressing each immunoregulatory gene had significantly longer survival times than mice treated with PBS (*: P<0.05). On the other hand, there was no significant difference in survival time between mice administered with viruses carrying and expressing each immune regulatory gene and mice administered with FUVAC (Figure 7-2).

[0068] Example 4 Therapeutic effect of a combination of two types of recombinant vaccinia viruses carrying and expressing immune regulatory genes As in Example 3, the allograft model shown in Figure 4 was used. PBS or each virus was administered directly into one tumor, three times every other day (Days 0, 2, and 4). The therapeutic effect of the virus combinations was assessed by measuring tumor diameter and analyzing survival curves. Two-way ANOVA statistical analysis confirmed that the combinations of FUVAC-IL12 and FUVAC-CCL21, FUVAC-IL12 and FUVAC-PD1, FUVAC-IL7 and FUVAC-CCL21, and FUVAC-IL7 and FUVAC-PD1scFv significantly reduced tumor diameters in both the virus-treated and non-treated sides compared with FUVAC, which does not express immune regulatory genes (Figure 8-1, ****: P<0.0001, ***: P<0.001, **: P<0.01). Log-rank statistical analysis showed that mice treated with the combination of FUVAC-IL12 and FUVAC-CCL21, FUVAC-IL12 and FUVAC-PD1, FUVAC-IL7 and FUVAC-CCL21, and FUVAC-IL7 and FUVAC-PD1scFv had significantly longer survival times than mice treated with FUVAC (Figure 8-2). Notably, 5 / 5 mice treated with FUVAC-IL12 and FUVAC-CCL21, 4 / 5 mice treated with FUVAC-IL12 and FUVAC-PD1, and 2 / 5 mice treated with FUVAC-IL7 and FUVAC-CCL21 achieved complete remission of both tumors.

[0069] Based on the above results, compared to monotherapy with recombinant vaccinia virus carrying and expressing immune regulatory genes, the combination therapy exerted an unexpectedly high therapeutic effect and extended survival time not only in the virus-treated but also in the non-treated groups.

[0070] Example 5: Therapeutic effects of a combination of two types of recombinant vaccinia viruses carrying and expressing other immune regulatory genes The therapeutic effect of the recombinant vaccinia virus FUVAC-CD40L (Figure 9) carrying and expressing CD40L was investigated using the allograft model shown in Figure 4, as in Example 3. PBS or each virus was administered directly into the tumor on one side of the body three times every other day (Days 0, 2, and 4). Two-way ANOVA statistical analysis confirmed that tumor diameters in both the virus-administered and non-administered sides were significantly smaller in FUVAC-CD40L compared with PBS (Figure 10, ****: P<0.0001, ***: P<0.001). Furthermore, while there was no significant difference in tumor diameter in the virus-administered side compared with FUVAC, two-way ANOVA statistical analysis confirmed that tumor diameters in the non-administered side were significantly smaller in FUVAC-CD40L compared with FUVAC (Figure 10, **: P<0.01). Furthermore, we examined the therapeutic effect of the combination of FUVAC-CD40L and FUVAC-IL7, or FUVAC-CD40L and FUVAC-CCL21. Two-way ANOVA statistical analysis confirmed that the combination of FUVAC-CD40L and FUVAC-IL7, and FUVAC-CD40L and FUVAC-CCL21, resulted in significantly smaller tumor diameters in both the virus-administered and non-administered sides compared with FUVAC (Figure 11, **: P<0.01, *: P<0.05).

[0071] Example 6: Recombinant vaccinia virus carrying and expressing two types of immune regulatory genes Recombinant vaccinia viruses carrying and expressing two immune regulatory genes, IL12 and CCL21, or IL12 and IL7, were prepared (FUVAC-IL12 / CCL21 or FUVAC-IL12 / IL7) (Figure 12). 1.0 × 10 A549 cells were cultured in a 96-well plate. 4Cells were seeded at 1 / well and cultured for 24 hours, after which they were infected with FUVAC-IL12 / CCL21 at an MOI of 0.1. After 48 hours of culture at 37°C, the supernatants were collected, and IL12 and CCL21 in the supernatants were measured using a Mouse IL12 p40 / p70 ELISA Kit (RayBiotech) and a Mouse CCL21 / 6Ckine Quantikine ELISA Kit (R&D System). As a result, FUVAC-IL12 / CCL21 expressed and produced both IL12 and CCL21 (Figure 13). Figure 13 shows the concentrations of IL12 and CCL21 in the supernatants (n=1). Next, the therapeutic effects of FUVAC-IL12 / CCL21 or FUVAC-IL12 / IL7 were evaluated using the allograft model shown in Figure 4 in the same manner as in Example 3. PBS or virus was administered three times every other day (2.5 × 10 7 The tumor diameter was measured after direct intratumoral administration of FUVAC-IL12 / CCL21 or FUVAC-IL12 / IL7 (PFU) into one of the tumors (Days 0, 2, and 4). Two-way ANOVA statistical analysis confirmed that tumor diameters on both the virus-injected and non-injected sides 19 days after administration were significantly smaller in the FUVAC-IL12 / CCL21 or FUVAC-IL12 / IL7 groups than in the PBS group (Figure 14 ****: P<0.0001).

[0072] From the above, it was shown that the therapeutic effect of combining two types of FUVAC carrying and expressing a single different type of immune regulatory gene is equivalent to the therapeutic effect of FUVAC carrying and expressing two types of immune regulatory genes simultaneously, and that in addition to being able to use a combination of FUVAC carrying and expressing a single different type of immune regulatory gene, it can also be used when two types of immune regulatory genes are carried and expressed simultaneously.

[0073] Example 7 Effect of cell fusion ability of recombinant vaccinia virus carrying and expressing two types of immune regulatory genes Using the above-mentioned vaccinia virus (VGF-LucGFP / O1L-DsRed:MDRVV) and transfer vector plasmid DNA (pTNshuttle / VGF-SP-mIL12), a recombinant virus was recovered using the same method as above, using the loss of GFP expression as an indicator, and named MDRVV-IL12. Next, using the above-mentioned vaccinia virus (MDRVV-IL12) and transfer vector plasmid DNA (pTNshuttle / O1L-SP-mCCL21), a recombinant virus was recovered using the same method as above, using the loss of DsRed expression as an indicator, and named MDRVV-IL12 / CCL21. Each recombinant virus with a nucleotide sequence that was intact was cultured in large quantities in A549 cells and purified, and then the virus titer was measured in RK13 cells and used for experiments. The therapeutic effects of FUVAC, MDRVV-IL12 / CCL21, or FUVAC-IL12 / CCL21 were evaluated using the allograft model shown in Figure 4. 7 A single dose of 1000 PFU (1000 ng / mL) was directly injected into one tumor (Day 0) and tumor diameter was measured. Two-way ANOVA statistical analysis confirmed that tumor diameters in both the virus-injected and non-injected sides were significantly smaller in MDRVV-IL12 / CCL21 and FUVAC-IL12 / CCL21 groups compared with FUVAC-injected groups 19 days after injection (Figure 15, *: P<0.05, ****: P<0.0001). Next, in a similar allograft model, PBS, MDRVV-IL12 / CCL21 (5 × 10 7 PFU), or FUVAC-IL12 / CCL21 (5 × 10 7Survival was assessed after a single intratumoral injection of 1000 PFU (PFU) of FUVAC-IL12 / CCL21 directly into one tumor (Day 0). Log-rank statistical analysis revealed that mice treated with MDRVV-IL12 / CCL21 or FUVAC-IL12 / CCL21 had significantly longer survival times than mice treated with PBS. Furthermore, FUVAC-IL12 / CCL21 significantly prolonged survival times compared with MDRVV-IL12 / CCL21 (Figure 16). Notably, complete remission of both tumors was achieved in 13 / 18 mice treated with FUVAC-IL12 / CCL21 and in 5 / 17 mice treated with MDRVV-IL12 / CCL21 (Table 1). [Table 1] These results suggest that even with a vaccinia virus (MDRVV) lacking cell-fusion ability, when two immune-regulating genes (IL12 and CCL21) are simultaneously loaded and expressed, a single administration of the virus exerts an extremely high therapeutic effect and extends survival time in both the virus-treated and non-treated groups. Furthermore, when cell-fusion ability is added to the vaccinia virus in addition to these two immune-regulating genes, i.e., when IL12 and CCL21 are simultaneously loaded and expressed in a vaccinia virus (FUVAC) capable of cell-fusion, the anti-cancer effect is enhanced to an unexpected degree, extending survival time.

[0074] Example 8 Therapeutic effect of a combination of three immunoregulatory genes carried and expressed in a recombinant vaccinia virus As in Example 7, the allograft model shown in Figure 4 was used. PBS or each virus was administered directly into one tumor in a single dose (Day 0). The therapeutic effect of the virus combination was examined by measuring tumor diameter and analyzing survival curves. Compared with FUVAC-IL12 / CCL21, the combination of FUVAC-IL12 / IL7 and FUVAC-CCL21 showed no significant difference in tumor diameter on the virus-injected side 26 days after administration, but the tumor diameter on the non-injected side was significantly smaller. This was confirmed by two-way ANOVA statistical analysis (Figure 17, ***: P<0.001). Log-rank statistical analysis revealed no significant difference in survival time between FUVAC-IL12 / CCL21 and FUVAC-IL12 / IL7 and FUVAC-CCL21, but both treatments significantly extended survival time compared to PBS-injected mice (Figure 18). It is noteworthy that the combination of FUVAC-IL12 / IL7 and FUVAC-CCL21 resulted in complete remission of both tumors in 7 / 7 mice, and in 4 / 7 mice treated with FUVAC-IL12 / CCL21 (Table 2). [Table 2] Based on the above results, treatment with a combination of three types of immune regulatory genes, even with a single administration of the virus, demonstrated an unexpectedly high therapeutic effect and extended survival time not only in the virus-treated but also in the non-treated patients. [Industrial Applicability]

[0075] The vaccinia viruses of the present invention can be used for cancer therapy. [Sequence List Free Text]

[0076] SEQ ID NOs: 1 to 4, 10 to 13 Primers All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A vaccinia virus in which the functions of the K2L gene or the HA gene, or the K2L gene and the HA gene, are defective, and which causes cell fusion in infected cells and induces cell death, and which contains any combination of the following genes (i) to (v) as foreign genes: (i) a combination of a gene encoding IL-12 and a gene encoding IL-7; (ii) a combination of a gene encoding IL-12 and a gene encoding PD1scFv; (iii) a combination of a gene encoding IL-7 and a gene encoding CCL21; (iv) a combination of a gene encoding CD40L and a gene encoding IL-7; and (v) A combination of a gene encoding CD40L and a gene encoding CCL21.

2. The vaccinia virus according to claim 1, wherein the vaccinia virus is an LC16 strain, an LC16mO strain, or an LC16m8 strain modified to express the B5R gene.

3. The vaccinia virus of claim 1 or 2, which is an oncolytic vaccinia virus.

4. 4. The vaccinia virus according to claim 3, which does not grow in normal cells but grows specifically in cancer cells and has oncolytic properties that specifically damage cancer cells.

5. A pharmaceutical composition for cancer treatment, comprising the vaccinia virus according to any one of claims 1 to 4.

6. A composition comprising, in combination, a vaccinia virus containing a gene encoding IL-12 and a vaccinia virus containing a gene encoding CCL21.

7. The composition according to claim 5 or 6, which is a pharmaceutical composition for treating cancer.

8. A kit for cancer treatment comprising a combination of a vaccinia virus containing a gene encoding IL-12 and a vaccinia virus containing a gene encoding CCL21.

9. A vaccinia virus comprising at least one immunoregulatory gene as a foreign gene, the vaccinia virus comprising any combination of the following genes (i) to (iii): (i) a combination of a gene encoding IL-7 and a gene encoding CCL21; (ii) a combination of a gene encoding CD40L and a gene encoding IL-7; and (iii) the combination of the gene encoding CD40L and the gene encoding CCL21.

10. The vaccinia virus according to claim 9, wherein the vaccinia virus is an LC16 strain, an LC16mO strain, or an LC16m8 strain modified to express the B5R gene.

11. The vaccinia virus of claim 9 or 10, which is an oncolytic vaccinia virus.

12. The vaccinia virus according to claim 11, which does not grow in normal cells but grows specifically in cancer cells and has oncolytic properties that specifically damage cancer cells.

13. A pharmaceutical composition for cancer treatment, comprising the vaccinia virus according to any one of claims 9 to 12.

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