Vaccinia virus inducing cell fusion and its use

Abstract

The purpose of the present invention is to provide: a vaccinia virus that induces cell fusion of infected cells; and a method for producing the same.　The present invention is a vaccinia virus which lacks the function of the K2L gene or the HA gene, or both the functions of the K2L gene and the Ha gene and is mutated to cause specific cell fusion of infected cells, in which cell fusion of the infected cells occurs to thereby induce cell death.

Description

[Technical Field]

[0001] The present invention relates to a vaccinia virus that induces cell fusion in infected cells. [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 engineering 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 that induces cell fusion in infected cells and a method for producing the same. [Means for solving the problem]

[0007] While studying the antitumor effect of vaccinia virus, the present inventors found that deleting a gene such as the K2L gene or HA gene of vaccinia virus induces cell fusion in infected cells, resulting in cell death. That is, infection of cancer cells with vaccinia virus lacking the K2L gene or HA gene exerted an anticancer effect. Furthermore, the present inventors found that deleting the K2L gene or HA gene of oncolytic vaccinia virus exerted a synergistic anticancer effect, leading to the completion of the present invention.

[0008] That is, the present invention is as follows. [1] A vaccinia virus that has been mutated to cause cell fusion in infected cells. [2] The vaccinia virus of [1], 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. [3] The vaccinia virus of [1] or [2], which is an oncolytic vaccinia virus. [4] Vaccinia virus [3] does not grow in normal cells, but grows specifically in cancer cells and has oncolytic properties that specifically damage cancer cells. [5] The vaccinia virus of any of [1] to [4], wherein the vaccinia virus is the LC16 strain, the LC16mO strain, or the LC16m8 strain modified to express the B5R gene. [6] A pharmaceutical composition for cancer treatment, comprising any one of the vaccinia viruses [1] to [5]. [7] A vaccinia virus vector in which foreign DNA has been introduced into any of the vaccinia viruses [1] to [5]. [8] The vaccinia virus vector of [7], wherein the foreign DNA is a marker DNA, a therapeutic gene having a cytotoxic or immunostimulatory effect, or DNA encoding an antigen of a cancer, virus, bacterium, or protozoan. [9] A pharmaceutical composition for cancer treatment or use as a vaccine against cancer, viruses, bacteria or protozoa, comprising the vaccinia virus vector of [7] or [8].

[10] A method for producing a vaccinia virus that causes cell fusion in infected cells and induces cell death, comprising deleting the function of the K2L gene or HA gene, or the K2L gene and HA gene, of the vaccinia virus.

[11] A method for producing an oncolytic vaccinia virus,

[10] .

[12] A method for producing

[10] or

[11] , further comprising deleting the function of the vaccinia virus growth factor (VGF) gene or the O1L gene, or the vaccinia virus growth factor (VGF) gene and the O1L gene.

[13] The production method according to any one of

[10] to

[12] , wherein the vaccinia virus is the LC16 strain, the LC16mO strain, or the LC16m8 strain modified to express the B5R gene.

[14] A combination pharmaceutical kit for cancer treatment, comprising any one of the vaccinia viruses [1] to [5] in combination with an immune checkpoint inhibitor.

[15] The combination pharmaceutical kit of

[14] , wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[16] A vaccinia virus of any of [1] to [5] for use in combination with an immune checkpoint inhibitor for cancer treatment.

[17] Vaccinia virus in

[16] , where the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. This specification includes the disclosure of Japanese Patent Application No. 2019-91609, from which this application claims priority. [Effects of the Invention]

[0009] Deletion of genes such as the K2L or HA gene of vaccinia virus induces cell fusion in infected cells, improving its anticancer effect. First, increased viral proliferation and spread improves tumor lysis, and apoptosis and necrosis occur more frequently. Second, cell fusion induces more efficient ICD, resulting in increased CD8 T cell infiltration into both the treated and untreated tumors. Finally, improved tumor immune environments in both treated and untreated tumors facilitate the functioning of cancer immunity. In other words, oncolytic viruses that induce cell fusion exhibit greater anticancer effects than oncolytic viruses that do not induce cell fusion by more efficiently converting immune-resistant cold tumors into immune-resistant hot tumors. [Brief explanation of the drawings]

[0010] [Figure 1] 1A to 1C show the structures of cell fusion-inducing gene recombinant vaccinia viruses, and FIG. 1D and E show the structures of VGF-Luc / O1L-DsRed and cell fusion-inducing gene recombinant vaccinia viruses derived therefrom. FIG. 1D and E show the structures of VGF-Luc / O1L-LacZ and cell fusion-inducing gene recombinant vaccinia viruses derived therefrom. [Figure 2] FIG. 1 shows images of cells infected with cell fusion-inducing recombinant vaccinia viruses. [Figure 3-1] FIG. 1 shows images of cells infected with cell fusion-inducing recombinant vaccinia viruses. [Figure 3-2] FIG. 1 shows the cytotoxicity of cell fusion-inducing recombinant vaccinia viruses. [Figure 3-3] FIG. 1 shows the production amount of cell fusion-inducing recombinant vaccinia virus. [Figure 4-1] FIG. 1 shows images of cells infected with cell fusion-inducing recombinant vaccinia viruses in 10 types of tumor cells. [Figure 4-2]FIG. 1 shows the cell survival rates of 10 types of tumor cells after infection with cell fusion-inducing recombinant vaccinia virus. [Figure 5-1] 5A and 5B show cell death induced by cell fusion-inducing recombinant vaccinia virus, in which Fig. 5-1A shows apoptosis in A549, and Fig. 5-1B shows necrosis. [Figure 5-2] 5A and 5B show cell death induced by cell fusion-inducing recombinant vaccinia virus, in which Fig. 5-2A shows apoptosis in CT26, and Fig. 5-2B shows necrosis. [Figure 6] 6A and 6B show immunogenic cell death (ICD) induced by fusion-inducing recombinant vaccinia viruses, with A549 showing ICD and CT26 showing ICD, respectively. [Figure 7] 7A and 7B show the experimental system of an allograft model mouse, in which Fig. 7A shows direct administration of the virus to one tumor, and Fig. 7B shows the administration and detection schedule. [Figure 8-1] FIG. 1 shows the growth distribution of cell fusion-inducing recombinant vaccinia viruses in allograft model mice. [Figure 8-2] 8-2A and 8-2B show the quantitative results of cell fusion-inducing recombinant vaccinia virus proliferation in allograft model mice. Figure 8-2A shows the proliferation of vaccinia virus in the administered side, and Figure 8-2B shows the proliferation of vaccinia virus in the unadministered side. [Figure 9] 9A and 9B show tumor volumes after treatment with cell fusion-inducing recombinant vaccinia virus in allograft model mice, with Fig. 9A showing the tumor volume on the administered side and Fig. 9B showing the tumor volume on the unadministered side. [Figure 10-1] FIG. 1 shows the results of immunological analysis of allograft model mice after treatment with cell fusion-inducing recombinant vaccinia virus (part 1). [Figure 10-2] FIG. 1 shows the results of immunological analysis of allograft model mice after treatment with cell fusion-inducing recombinant vaccinia virus (part 2). [Figure 11]These figures show tumor volume after treatment with a cell fusion-inducing recombinant vaccinia virus in an allograft model mouse in which CD8 activity was suppressed. Figure 11A shows the results for a control group administered with an isotype control, and Figure 11B shows the results for a group administered with an anti-CD8 antibody. The left graphs in Figures 11A and 11B show the results for the treated group, and the right graphs show the results for the untreated group. [Figure 12] 12A and 12B show experimental systems for advanced allograft model mice, in which Fig. 12A shows direct administration of the virus to one tumor, and Fig. 12B shows the administration and detection schedule. [Figure 13-1] FIG. 1 shows the distribution of proliferation of cell fusion-inducing recombinant vaccinia viruses in advanced allograft model mice. [Figure 13-2] FIG. 1 is a graph quantifying the proliferation of cell fusion-inducing recombinant vaccinia virus in an advanced allograft model mouse. [Figure 14] FIG. 1 shows tumor volume after treatment with cell fusion-inducing recombinant vaccinia virus in advanced allograft model mice. [Figure 15-1] FIG. 1 shows the structure of a vaccinia virus that has both cell-fusion ability and tumor-targeting ability. [Figure 15-2] FIG. 1 shows the cell survival rate after infection with vaccinia virus that has both cell fusion ability and tumor targeting ability (part 1). [Figure 15-3] FIG. 2 shows the cell survival rate after infection with vaccinia virus that has both cell fusion ability and tumor targeting ability (part 2). [Figure 15-4] FIG. 1 shows immunogenic cell death after infection with vaccinia virus, which has both cell-fusion and tumor-targeting capabilities (part 1). [Figure 15-5] FIG. 2 shows immunogenic cell death after infection with vaccinia virus, which has both cell-fusion and tumor-targeting capabilities (part 2). [Figure 16] FIG. 1 is a diagram summarizing the mechanism by which cell fusion-inducing oncolytic viruses improve the anticancer effect. [Figure 17]Figure 17 shows an experimental system for an allograft model mouse in combination with an anti-PD-1 antibody. Figure 17A shows direct administration of the virus to one tumor, and Figure 17B shows the administration and detection schedule. [Figure 18] Figures 18A and 18B show tumor volume after cell fusion-inducing recombinant vaccinia virus treatment in allograft model mice in combination with an anti-PD-1 antibody. Figure 18A shows the results without the anti-PD-1 antibody, and Figure 18B shows the results with the anti-PD-1 antibody. The left graphs in Figures 18A and 18B show the results for the treated side, and the right graphs show the results for the untreated side. [Figure 19] FIG. 1 shows the survival rate after treatment with cell fusion-inducing recombinant vaccinia virus in allograft model mice in combination with an anti-PD-1 antibody. [Figure 20] Fig. 1 shows the tumor volume after tumor re-implantation into mice in which tumors were in remission after treatment with a cell fusion-inducing gene recombinant vaccinia virus and an anti-PD-1 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below. The vaccinia virus of the present invention is a vaccinia virus that has been mutated to have cell-fusion ability. Here, cell-fusion ability means that when the vaccinia virus infects cells, it can cause cell fusion between infected cells. The vaccinia virus that has been mutated to have cell-fusion ability has either a defect in the function of a gene that suppresses the cell-fusion ability that the vaccinia virus originally has, or has an inserted and expressed gene that promotes cell fusion.

[0012] 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.

[0013] 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).

[0014] 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).

[0015] 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.

[0016] 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: 20, and the nucleotide sequence of the wild-type HA gene is shown in SEQ ID NO: 21.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] Genome editing is a method that utilizes site-specific nucleases to modify target 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.

[0021] 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).

[0022] 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.

[0023] 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.

[0024] 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 enhanced. A decrease in immunosuppressive cells such as Tregs, TAMs, and MDSCs is also observed.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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) Vaccinia viruses that lack the functions of these genes and have oncolytic properties are called oncolytic vaccinia viruses.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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).

[0035] The present invention also encompasses a method for treating cancer, which comprises administering the above-described vaccinia virus to a cancer patient.

[0036] Furthermore, the vaccinia virus of the present invention may contain a foreign gene (foreign DNA or foreign polynucleotide). Examples of foreign genes (foreign DNA or foreign polynucleotide) include marker genes and therapeutic genes encoding products with cytotoxic or immunostimulatory effects, as well as DNA encoding protein antigens of cancer, viruses, bacteria, protozoa, and the like. Marker genes are also called reporter genes, and examples include the luciferase (LUC) gene, fluorescent protein genes such as green fluorescent protein (GFP), fluorescent protein genes such as red fluorescent protein (DsRed), β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, and β-galactosidase (LacZ) gene. Oncolytic vaccinia viruses containing these foreign genes can be called vaccinia virus vectors.

[0037] Therapeutic genes are genes that can be used to treat specific diseases such as cancer and infectious diseases, and include tumor suppressor genes such as p53 and Rb, 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-24, chemokine 2 (CCL2), CCL5, CCL19, CCL21, CXCL9, CXCL10, CXCL11, CD40L, and CD40L. Examples of genes encoding physiologically active substances such as 70, CD80, CD137L, OX-40L, GITRL, LIGHT, α-interferon, β-interferon, γ-interferon, angiostatin, thrombospondin, endostatin, METH-1, METH-2, GM-CSF, G-CSF, M-CSF, MIP1a, FLT3L, HPGD, TRIF, DAI, and tumor necrosis factor, as well as genes encoding antibodies with inhibitory activity against CTLA4, PD1, and PD-L1. Vaccinia viruses expressing luciferase or GFP enable simple and rapid detection of infected cancer cells. When the vaccinia virus of the present invention is used for cancer therapy, the therapeutic gene for cancer can exert a cancer therapeutic effect in addition to the oncolytic properties of the vaccinia virus.

[0038] 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.

[0039] These foreign genes can be introduced, for example, by using the technique of homologous recombination. Homologous recombination can be performed by the method described above. For example, a plasmid (transfer vector) is prepared in which the foreign gene to be introduced is ligated into the DNA sequence at the desired site of introduction, and this can be introduced into cells infected with vaccinia virus. The introduction region for the foreign gene is preferably within a gene that is not essential for the life cycle of vaccinia virus.

[0040] 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, and 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, TaKaRa, EMBO Journal (1987, Vol. 6, pp. 3379-3384).

[0041] The present invention also encompasses a combination therapy of a vaccinia virus capable of cell fusion with an immune checkpoint inhibitor.

[0042] Immune checkpoint inhibitors include anti-PD-1 (Programmed cell death 1) antibodies, anti-PD-L1 (Programmed cell-death ligand 1) antibodies, anti-CTLA-4 antibodies, anti-PD-L2 antibodies, anti-LAG-3 antibodies, PD-1 antagonists, PD-L1 antagonists, etc. Among these, anti-PD-1 antibodies and anti-PD-L1 antibodies are preferred. Examples of anti-PD-1 antibodies include Nivolumab, Pembrolizumab, Spartalizumab, Cemiplimab, Tislelizumab, and Camrelizumab, and examples of anti-PD-L1 antibodies include Avelumab, Durvalumab, and Atezolizumab.

[0043] The immune checkpoint inhibitor may be administered by a known method. The dosage of the immune checkpoint inhibitor varies depending on symptoms, age, body weight, and other conditions, but may be administered, for example, at a dose of 0.001 mg to 100 mg by subcutaneous injection, intramuscular injection, intravenous injection, or the like, at intervals of several days, weeks, or months.

[0044] Immune checkpoint inhibitors may contain carriers, diluents, excipients, etc. commonly used in the field of pharmaceuticals. For example, lactose, magnesium stearate, etc. are used as carriers and excipients for tablets. Physiological saline, isotonic solutions containing glucose and other adjuvants, etc. are used as aqueous solutions for injection, and solubilizing agents such as alcohol, polyalcohols such as propylene glycol, and nonionic surfactants may be used in combination. Sesame oil, soybean oil, etc. are used as oily solutions, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination.

[0045] The cell-fusogenic vaccinia virus and immune checkpoint inhibitor of the present invention can exert a synergistic effect in cancer treatment. The cancer therapeutic effect of the combined use of the cell-fusogenic vaccinia virus and immune checkpoint inhibitor is significantly higher than the cancer therapeutic effect of the use of the cell-fusogenic vaccinia virus alone or the use of the immune checkpoint inhibitor alone.

[0046] The cell-fusogenic vaccinia virus of the present invention can be administered simultaneously with, separately from, or consecutively with the administration of an immune checkpoint inhibitor. The cell-fusogenic vaccinia virus can also be administered before or after the administration of an immune checkpoint inhibitor. Preferably, the cell-fusogenic vaccinia virus is administered before the administration of an immune checkpoint inhibitor.

[0047] The present invention also encompasses a combination, a combined preparation, or a combined pharmaceutical kit comprising a vaccinia virus capable of cell fusion and an immune checkpoint inhibitor.

[0048] The present invention also encompasses a method of using a cell-fusion capable vaccinia virus in combination with an immune checkpoint inhibitor in the manufacture of a medicament for treating cancer.

[0049] The present invention also includes a pharmaceutical comprising a vaccinia virus capable of cell fusion and an immune checkpoint inhibitor.

[0050] The present invention further encompasses a vaccinia virus having cell-fusion ability for use in combination with an immune checkpoint inhibitor. [Example]

[0051] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0052] Example 1: Structure of vaccinia virus capable of cell fusion 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. WO 2015 / 076422, we constructed pUC19-O1L-P-DsRed to express DsRed in the same orientation as O1L under the control of the p7.5K promoter rather than a synthetic vaccinia virus promoter. CV1 cells cultured to 80% confluence in a 24-well plate were infected with VGF-LucGFP / O1L-DsRed at an MOI of 0.1–0.5 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 the cells were cultured at 37°C for 2–3 days.Cells were harvested, freeze-thawed, and sonicated. Appropriate dilutions were then inoculated onto nearly confluent BSC1 cells. The cells were then cultured at 37°C for 2–4 days in Eagle MEM containing 0.5% methylcellulose and 5% FBS medium. 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. For O1L, PCR was performed using two primers (SEQ ID NO: 3 and SEQ ID NO: 4). For clones in which PCR products of the desired size were detected, the nucleotide sequences of the PCR products were confirmed by direct sequencing. A virus clone with a clean nucleotide sequence, VGF-LucGFP / O1L-BFP, was selected and amplified in A549 cells. The virus titer was measured in RK13 cells and used for experiments. Using this vaccinia virus (VGF-LucGFP / O1L-BFP) and transfer vector plasmid DNA (pUC19-O1L-P-DsRed), a recombinant virus was recovered using the same method as above, using DsRed expression as an indicator. This virus was named VGF-LucGFP / O1L-DsRed.

[0053] During the process of generating this recombinant vaccinia virus, viruses with unusual cell-fusion ability emerged at an extremely high frequency. Therefore, the medium was removed, and the fusogenic plaques were scraped with the tip of a tip and suspended in opti-MEM medium (Invitrogen). This process was repeated three more times in BSC1 cells, and two cell-fusion-competent virus clones were isolated by plaque purification. These two clones were subjected to direct sequencing using a PacBio RSII (Pacific Bioscience) next-generation sequencer or PCR. The resulting sequence information revealed mutations in the K2L or HA gene, which had the nucleotide sequences shown in SEQ ID NO:5 or SEQ ID NO:6. K2L is a 1110-bp gene, and a guanine-to-adenine mutation at bp 762 was detected. This mutation resulted in a tryptophan-to-termination codon at 254, indicating a nonsense mutation. HA is a 933-bp gene, and an adenine deletion at bp 70 was detected. Based on these findings, the two cell-fusion-competent viral clones obtained were designated VGF-LucGFP / K2Lmut / O1L-DsRed and VGF-LucGFP / O1L-BFP / HAmut (Fig. 1B and C), respectively, based on the parent virus VGF-LucGFP / O1L-DsRed (International Publication No. WO2015 / 076422) (Fig. 1A). Each recombinant virus was cultured in A549 cells, purified, and then titered in RK13 cells for subsequent experiments.

[0054] Next, the Luc2 gene region was amplified using pGL4.20 (F6751, Promega) DNA as a template with two primers (SEQ ID NO: 7 and SEQ ID NO: 8). Each PCR product was digested with restriction enzymes BspEI and NheI and cloned into the AgeI and NheI restriction sites of the pTNshuttle / VGF-SP-BFP vector to replace the BFP gene, constructing pTNshuttle / VGF-SP-Luc. Meanwhile, the E. coli LacZ gene (SEQ ID NO: 9), which had been synthesized to be codon-optimized for H. sapiens, was digested with restriction enzymes AgeI and NheI and cloned into the same restriction sites of the pTNshuttle / O1L-SP-BFP vector to replace the BFP gene, constructing pTNshuttle / O1L-SP-LacZ.

[0055] To recover recombinant vaccinia viruses containing the viral genome shown in Figure 1B, we used vaccinia virus (VGF-LucGFP / O1L-DsRed or VGF-LucGF P / K2Lmut / O1L-DsRed) and transfer vector plasmid DNA (pTNshuttle / VGF-SP-Luc) to recover recombinant viruses designated VGF-Luc / O1L-DsRed or VGF-Luc / K2Lmut / O1L-DsRed, as described above, using the loss of GFP expression as an indicator. These vaccinia viruses (VGF-Luc / O1L-DsRed or VGF-Luc / K2Lmut / O1L-DsRed) and transfer vector plasmid DNA (pTNshuttle / O1L-SP-LacZ) were then recovered using the same method as described above to recover recombinant viruses using the loss of DsRed expression as an indicator, and then subjected to PCR screening. 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 K2L. For clones in which PCR products of the desired size were detected, the nucleotide sequences of the PCR products were confirmed by direct sequencing. The virus clones with intact nucleotide sequences were designated VGF-Luc / O1L-LacZ and VGF-Luc / K2Lmut / O1L-LacZ (Fig. 1D and E). Each recombinant virus was cultured in A549 cells and purified. The virus titer was then measured in RK13 cells and used for the experiments.

[0056] Example 2 Characterization of cell-fusion-competent vaccinia viruses To compare the cell-fusion-incompetent VGF-LucGFP / O1L-DsRed with the cell-fusion-competent VGF-LucGFP / K2Lmut / O1L-DsRed and VGF-LucGFP / O1L-BFP / HAmut viruses, various cancer cells (human ovarian cancer RMG1 cells, human colon cancer CaCO2 cells, human lung cancer A549 cells, and mouse colon cancer CT26 cells) were infected with each virus. First, various cancer cells (human ovarian cancer RMG1 cells, human colon cancer CaCO2 cells, human lung cancer A549 cells, and mouse colon cancer CT26 cells) were infected with each virus in a 96-well plate. 4 / well, A549 1.0×10 4 / well, CT26 6.0 × 103 Cells were seeded at 1 / well and cultured for 24 hours. After reaching 80% confluence, cells were infected with VGF-LucGFP / O1L-DsRed, VGF-LucGFP / K2Lmut / O1L-DsRed, or VGF-LucGFP / O1L-BFP / HAmut at an MOI of 0.1 for RMG1, CaCO2, and A549, and at an MOI of 10 for CT26. Seventy-two hours after infection, infection patterns were observed using a BZ-X700 (Keyence). Compared to VGF-LucGFP / O1L-DsRed, VGF-LucGFP / K2Lmut / O1L-DsRed and VGF-LucGFP / O1L-BFP / HAmut were found to infect various cancer cell lines, resulting in cell fusion (Figure 2).

[0057] Next, we examined whether the cell fusion alteration affected the anti-cancer effect using VGF-LucGFP / O1L-DsRed and VGF-LucGFP / K2Lmut / O1L-DsRed. First, we placed 1.0 × 10 A549 cells in a 96-well plate. 4Cells were seeded per well and cultured for 24 hours. When the cells reached 80-90% confluence, they were infected with each virus solution at an MOI of 0.01, 0.1, or 1. Seventy-two hours after infection, infection images were observed using a BZ-X700 (Keyence). Results indicated that VGF-LucGFP / K2Lmut / O1L-DsRed, compared with VGF-LucGFP / O1L-DsRed, infected cells and fused them in an MOI-dependent manner. Images of infected cells are shown in Figure 3-1. Seventy-two hours after infection, cell viability was measured using the CellTiter96® Aqueous Non-radioactive Cell Proliferation Assay (Promega). At MOIs of 0.1 and 1, VGF-LucGFP / K2Lmut / O1L-DsRed exhibited reduced cell viability compared with VGF-LucGFP / O1L-DsRed, demonstrating its improved anticancer effect. Figure 3-2 shows the cytotoxicity as measured by cell viability. To investigate whether changes in cell fusion altered the amount of virus produced, the amounts of virus produced by VGF-LucGFP / O1L-DsRed and VGF-LucGFP / K2Lmut / O1L-DsRed were compared by virus titration. First, 5.0 × 10 cells were placed in a 24-well plate. 4 Human lung cancer cell line A549 cells were seeded as hosts at 1 / well and, after 24 hours of culture, when they reached 60-80% confluence, were infected with each virus solution at an MOI of 0.1. 24, 48, and 72 hours after infection, the cells and supernatant were collected with a scraper, disrupted by freeze-thawing and sonication, and centrifuged at 2000 rpm at 4°C for 10 minutes before collecting the supernatant. RK13 cells were seeded to measure the virus titer, and the dilution rate was varied depending on the expected virus titer. Titer measurements were performed by serially diluting (10 -1 ~10 -5Cells were infected with each virus (2x diluted) and cultured for three days in Eagle MEM containing 0.8% methylcellulose and 5% FBS medium, after which the virus titer was calculated by counting the number of virus plaques formed. The results confirmed that there was no difference in the amount of virus produced between VGF-LucGFP / O1L-DsRed and VGF-LucGFP / K2Lmut / O1L-DsRed. The amount produced is shown in terms of titer in Figure 3-3.

[0058] Example 3 Anti-cancer effect of vaccinia virus with cell fusion ability Next, we investigated cell viability using various human and mouse cancer cells other than A549. Human ovarian cancer cells (SKOV3: 2.0 × 10 4 / well), human pancreatic cancer cells (Panc1: 2.0 × 10 4 / well), human colon cancer cells (CaCO2:2.0×10 4 / well), human breast cancer cells (MDA-MB-231: 2.0 × 10 4 / well), human lung cancer cells (A549: 1.0 × 10 4 / well), human prostate cancer cells (PC3: 2.5 × 10 4 / well), human skin cancer cells (A431:2.0×10 4 / well), mouse melanoma cells (B16-F10: 1.5 × 10 4 / well), mouse colon cancer cells (CT26:1.0×10 4 / well) and mouse lung cancer cells (TC1: 4.0 × 10 3The cells were seeded into a 96-well plate with either VGF-LucGFP / O1L-DsRed or VGF-LucGFP / K2Lmut / O1L-DsRed virus solution (MOI: 0.1 for Panc1, CaCO2, MDA-MB-231, A549, and A431; MOI: 1 for SKOV3 and PC3; and MOI: 5 for B16-F10, CT26, and TC1) (n=3). Cell viability was measured 48 hours after infection for TC1 and 72 hours after infection for the other cells using the CellTiter 96® Aqueous Nonradioactive Cell Proliferation Assay (Promega). When the infection pattern was observed, cell fusion infection was confirmed with VGF-LucGFP / K2Lmut / O1L-DsRed, indicating that the infection pattern differed from that of VGF-LucGFP / O1L-DsRed in a wide range of cancer types (Figure 4-1). Furthermore, cell viability was significantly reduced in VGF-LucGFP / K2Lmut / O1L-DsRed compared to VGF-LucGFP / O1L-DsRed by t-test (SKOV3: **P = 0.0021, Panc1: ***P = 0.0003, CaCO2: ***P < 0.0001, MDA-MB-231: ***P = 0.0004, A549: ***P < 0.0001, PC3: *P = 0.0284, A431: *P = 0.0480, B16-F10: ***P = 0.0004, CT26: **P = 0.0030, TC1: ***P = 0.0006). This indicates that VGF-LucGFP / K2Lmut / O1L-DsRed has improved antitumor effects across a wide range of cancer types (Figure 4-2).

[0059] Example 4 Analysis of the mechanism of anti-cancer effect of vaccinia virus with cell fusion ability in vitro We investigated whether apoptosis and necrosis were significantly occurring in human lung cancer cell line (A549) and mouse colon cancer cell line (CT26) using the Apoptotic / Necrotic / Healthy Cells Detection Kit (Takara Bio). First, A549 or CT26 cells were cultured in a 96-well plate at 1.0 × 10 4 Cells were seeded at 1 / well and cultured at 37°C for 24 hours. Then, cells were infected with either VGF-Luc / O1L-lacZ or VGF-Luc / K2Lmut / O1L-lacZ virus at an MOI of 1 for A549 and 10 for CT26 (n=3). Thirty or 22 hours after infection, the cells were assayed using the kit described above, photographed with a BZ-X700 (Keyence), and quantified. The results showed that the number of apoptotic cells was significantly higher in both A549 and CT26 cells treated with VGF-Luc / K2Lmut / O1L-lacZ than in those treated with VGF-Luc / O1L-lacZ (A549: *P=0.0355, CT26: *P=0.0264). Furthermore, the number of cells detected as not only apoptotic but also necrotic was significantly increased by t-test (Figure 5) (A549: *P=0.0181, CT26: *P=0.0264). Furthermore, we also examined immunogenic cell death (ICD), which is said to be important in the induction of antitumor immunity. The mechanisms of immune induction by ICD have been reported to include extracellular release of HMGB1 and cell surface exposure of calreticulin. In this study, extracellular HMGB1 was quantified using the HMGB1 ELISA Kit II (Shinotest). A549 or CT26 were plated in a 24-well plate, with 5.25 x 10 4 / well, CT26 is 3.15 × 10 4Cells were seeded per well and cultured at 37°C for 24 hours. Then, they were infected with either VGF-Luc / O1L-lacZ or VGF-Luc / K2Lmut / O1L-lacZ at an MOI of 1 for A549 and 5 for CT26 (n=3). Cell supernatants were collected 60 hours postinfection and analyzed by ELISA. A t-test confirmed that VGF-Luc / K2Lmut / O1L-lacZ significantly increased extracellular HMGB1 release compared with VGF-Luc / O1L-lacZ in both A549 and CT26 cells (Figure 6) (***P=0.0006 for A549, ***P=0.0004 for CT26). These results suggest that enhanced apoptosis and necrosis induction, as well as enhanced ICD-mediated antitumor immunity, contribute to the enhanced anticancer effect of VGF-Luc / K2Lmut / O1L-lacZ.

[0060] Example 5 Therapeutic effect of vaccinia virus with cell fusion ability in vivo Next, we used an allograft model to examine the proliferation and spread of the virus in vivo and the therapeutic effects of the virus. Mouse colon cancer cell line (CT26) was added to 5.0 × 10 5 The cells were subcutaneously transplanted into both sides of the abdomen of BALB / cAjcl mice, and tumors were grown to 42-94 mm 3 (Average 60mm 3 After tumor growth, VGF-Luc / O1L-lacZ and VGF-Luc / K2Lmut / O1L-lacZ were injected every other day for three doses at 5.0 × 10 7Viral FLuc expression was directly administered into one tumor at a dose of 1000 PFU (Days 0, 2, and 4). Viral FLuc expression (i.e., viral proliferation and spread) was detected noninvasively using an in vivo imaging system (Berthold, NightSHADE LB985) after administration of Vivo Glo Luciferin (Promega) (Days 1, 3, 5, and 7) (Figure 7B). Figure 8-1 shows the viral FLuc detection results 3 days after virus administration. At 3 days after virus administration, VGF-Luc / K2Lmut / O1L-lacZ expressed a higher viral FLuc signal than VGF-Luc / O1L-lacZ in the virus-injected side. In contrast, no viral signal was detected in the non-virus-injected side, even after virus administration. Furthermore, when viral FLuc expression was quantified, a significant difference was observed in the injected side by two-way ANOVA (*P<0.05) (Figure 8-2). These results confirmed that VGF-Luc / K2Lmut / O1L-lacZ virus had more active viral replication and spread in vivo than VGF-Luc / O1L-lacZ virus. We next examined the therapeutic efficacy of the virus by measuring tumor size. The results showed that tumor growth in both the virus-treated and non-treated sides of VGF-Luc / K2Lmut / O1L-lacZ virus-injected mice was suppressed compared with uninjected (PBS) and VGF-Luc / O1L-lacZ virus-injected mice. Furthermore, two-way ANOVA statistical analysis confirmed that tumor volumes in the virus-injected and non-injected sides of VGF-Luc / K2Lmut / O1L-lacZ virus-injected mice were significantly different from those in PBS and VGF-Luc / O1L-lacZ virus-injected mice after 22 days (**P<0.01, ***P<0.001) (Figure 9).

[0061] Example 6 Analysis of the mechanism of therapeutic effect of vaccinia virus with cell fusion ability in vivo Next, to analyze the mechanism of the improved therapeutic effect in vivo, immunological analysis was performed using flow cytometry. 5The cells were subcutaneously transplanted into both sides of the abdomen of BALB / cAjcl mice, and tumors were grown to 50-111 mm 3 (Average 60mm 3 After tumor growth, VGF-Luc / O1L-lacZ and VGF-Luc / K2Lmut / O1L-lacZ were injected three times every other day at a dose of 5.0 × 10 7 The virus was directly administered into one tumor at 100 PFU (Day 0, 2, 4). Five days after virus administration (Day 5), tumors on both sides of the abdomen were collected in serum-free RPMI, and tumor tissue was dispersed using gentleMACS (Miltenyi Biotec). The cells were filtered through a 100 μm strainer and subjected to hemolysis treatment, after which the cells were counted and collected at 5.0 × 10 5The cells were blocked with Fc block (BD Bioscience), stained for cell surface antigens and intracellular antigens, and analyzed for CD8, CD4, Treg, TAM, and MDSC using CytoFLEX (Beckman Coulter). Dead cell staining was performed with 7AAD (Beckman Coulter). Cell surface antigen staining was performed with antibodies against CD45 (30-F11; BioLegend), CD3 (145-2C11; BioLegend), CD8 (53-6.7; BioLegend), CD4 (GK1.5; Thermo), CD25 (PC61.5; Thermo), F4 / 80 (BM8; BioLegend), CD11b (M1 / 70; BioLegend), Ly6G (1A8; BioLegend), and Ly6C (AL21; BD Bioscience). Intracellular antigen staining was performed with antibodies against FoxP3 (FJK-16s; Thermo). The results of immunological analysis of immune cell infiltration are shown in Figures 10-1 and 10-2. CD8 T cell infiltration only tended to increase with VGF-Luc / K2Lmut / O1L-lacZ administration in the virus-injected side, but was significantly elevated compared to VGF-Luc / O1L-lacZ in the uninjected side by t-test (*P=0.0413). CD4 T cell infiltration was significantly decreased with VGF-Luc / K2Lmut / O1L-lacZ compared to PBS in the virus-injected side, but remained unchanged in the uninjected side (**P=0.0025). Treg infiltration was also decreased with VGF-Luc / K2Lmut / O1L-lacZ compared to PBS in the virus-injected side, but not in the uninjected side (*P=0.0362, **P=0.0094). Furthermore, TAM was significantly reduced in both the virus-treated and non-treated sides by VGF-luc / K2Lmut / O1L-lacZ administration compared with PBS (*P=0.0383, ***P=0.0002). Finally, G-MDSCs were significantly increased in the treated side by virus administration (*P=0.0179, **P=0.0051). On the other hand, M-MDSCs were reduced in the treated side only by VGF-Luc / K2Lmut / O1L-lacZ administration compared with PBS (*P=0.0417).This suggests that the reduction of immunosuppressive cells such as Tregs, TAMs, and MDSCs in the virus-treated side, and the increase in infiltration of CD8 T cells in the non-treated side, are important for the therapeutic effect.

[0062] Immunological analysis suggested that CD8 T cell infiltration was important for the therapeutic effect in the non-vaccinated side, so we suppressed the activity of CD8 T cells with an inhibitor. 5 The cells were subcutaneously transplanted into both sides of the abdomen of BALB / cAjcl mice, and tumors were grown to 42-128 mm 3 (Average 60mm 3 After tumor growth, VGF-Luc / O1L-lacZ and VGF-Luc / K2Lmut / O1L-lacZ were injected three times every other day. 7PFU was administered directly into one tumor (Days 0, 2, and 4). In addition, InVivoPlus rat IgG2b isotype control, anti-keyhole limpet hemocyanin clone LTF-2 (isotype control), and InVivoPlus anti-mouse CD8α Clone 2.43 (BioXCell) were administered intraperitoneally at 200 μg / mouse on Days -4, -2, 1, 3, 5, and 7. The therapeutic effect was assessed by measuring tumor volume, and the results are shown in Figure 11. Figure 11A shows the results after administration of the isotype control, and Figure 11B shows the results after administration of the anti-CD8 antibody. In the control group administered with the isotype control, tumor volume was suppressed in both the virus-injected and non-injected sides of VGF-Luc / O1L-lacZ and VGF-Luc / K2Lmut / O1L-lacZ mice compared to PBS mice, as shown in Figure 9. Two-way ANOVA statistical analysis revealed a significant difference (*P<0.05, ***P<0.001). On the other hand, in the case of administration of anti-CD8 antibody, tumor volume was slightly reduced in the virus-injected group compared to PBS in the virus-injected side, but no difference was observed in any of the non-injected groups. This suggests that CD8 T cell infiltration is important not only in the non-injected side but also in the injected side.

[0063] Example 7 Therapeutic effect of cell-fusion-competent vaccinia virus in advanced cancer-bearing mouse model We investigated the proliferation and spread of the virus in vivo and the therapeutic effect of the virus when the tumor volume in the mouse body increased further using an allograft model. Mouse colon cancer cell line (CT26) was added to 5.0 × 10 5 The cells were subcutaneously transplanted into BALB / cAjcl mice on one side of the abdomen, and tumors were grown to 87-253 mm 3 (Average 150mm 3 After tumor growth, VGF-Luc / O1L-lacZ and VGF-Luc / K2Lmut / O1L-lacZ were injected three times every other day at a total cell density of 5.0 × 10 7The virus was directly administered into the tumor at 1000 PFU (Days 0, 2, and 4). VivoGlo Luciferin (Promega) was administered, and viral FLuc expression (i.e., viral proliferation and spread) was noninvasively detected using an in vivo imaging system (Berthold, NightSHADE LB985) (Days 1, 3, 5, and 7) (Figure 12B). Figure 13-1 shows the results of viral FLuc expression detection one day after virus administration. The VGF-Luc / K2Lmut / O1L-lacZ virus expressed a higher signal than the VGF-Luc / O1L-lacZ virus one day after virus administration (Figure 13-1). Furthermore, when FLuc expression was quantified, a significant difference was observed in two-way ANOVA statistical analysis one day after virus administration (Figure 13-2) (***P<0.001). These results confirmed that VGF-Luc / K2Lmut / O1L-lacZ virus replicates and spreads actively in vivo. We then examined the anticancer effect of the virus by measuring tumor diameter. Two-way ANOVA statistical analysis confirmed that tumor volume was significantly reduced in VGF-Luc / K2Lmut / O1L-lacZ virus-injected mice 19 days after virus injection compared with uninjected (PBS) mice and VGF-Luc / O1L-lacZ virus-injected mice (*<0.05, ***<0.001) (Figure 14).

[0064] Example 8 Anticancer effect of vaccinia virus with both cell fusion and tumor targeting abilities To demonstrate that the improvement of anticancer effects through cell fusion can be achieved by the above-mentioned K2L mutations not only through cell fusion but also through any mutation, deletion, or foreign gene insertion, and that tumor specificity can be achieved not only through the above-mentioned deletion of VGF and O1 but also through any other method, we constructed vaccinia viruses with various cell fusion and tumor targeting abilities as described below and compared their anticancer effects in vitro.

[0065] The following clones were used as vaccinia viruses having both cell fusion ability and tumor targeting ability. VGF-LucGFP / ΔK2L-BFP / O1L-DsRed (Figure 15-1A) Clones lacking K2L, capable of cell fusion, and lacking VGF and O1L to ensure tumor specificity VGF-LucGFP (Figure 15-1B) Clones that ensure tumor specificity by lacking VGF TK-GFP (Figure 15-1C) Clones that ensure tumor specificity by lacking TK Unmodified virus (Figure 15-1D) Clones that are not deficient in any of K2L, VGF, O1L, and TK VGF-LucGFP / K2L-BFP (Figure 15-1E) K2L-deficient clones with cell fusion ability and VGF-deficient clones that ensure tumor specificity K2L-BFP / TK-GFP (Figure 15-1F) A clone lacking K2L that retains cell fusion ability and TK that ensures tumor specificity K2L-BFP (Figure 15-1G) K2L-deficient clones with cell fusion ability The method for producing these clones is described below.

[0066] To generate a recombinant vaccinia virus lacking the K2L gene, we constructed the plasmid pTNshuttle / ΔK2L-BFP, which contains the K2L gene and its 5'- and 3'-flanking regions (SEQ ID NO: 14) replaced with the BFP gene (SEQ ID NO: 15). Using vaccinia virus (VGF-LucGFP / O1L-DsRed) and transfer vector plasmid DNA (pTNshuttle / ΔK2L-BFP), recombinant viruses were isolated using the same method as above, using BFP expression as an indicator, and then subjected to PCR screening. PCR for K2L was performed using the same primers (SEQ ID NO: 12 and SEQ ID NO: 13) as above. For clones in which a PCR product of the desired size was detected, the nucleotide sequence of the PCR product was confirmed by direct sequencing, and the resulting clone was designated VGF-LucGFP / ΔK2L-BFP / O1L-DsRed (Figure 15-1A). To insert an expression unit for a different foreign gene into the K2L gene, the K2L gene region was amplified using two primers (SEQ ID NO: 16 and SEQ ID NO: 17) with genomic DNA from the LC16mO strain as a template. The PCR product was digested with restriction enzymes XbaI and MfeI and cloned into the XbaI and EcoRI sites of the pUC19 vector to construct pUC19-K2L. Next, pTNshuttle / TK-SP-BFP was digested with restriction enzymes SphI and EcoRI and blunt-treated. The SP-BFP fragment was then cloned into the ClaI-digested and blunt-treated pUC19-K2L vector to construct pTNshuttle / K2L-SP-BFP. Using vaccinia virus (LC16mO strain, an unmodified virus that has not undergone genetic modification) and transfer vector plasmid DNA (pTNshuttle / K2L-SP-BFP), recombinant viruses were recovered using the same method as above, using BFP expression as an indicator, and subjected to PCR screening. For K2L, PCR was performed using the same primers as above (SEQ ID NO: 12 and SEQ ID NO: 13). For clones in which a PCR product of the specified size was detected, the nucleotide sequence of the PCR product was confirmed by direct sequencing and designated K2L-BFP (Figure 15-1G).To insert an expression unit for a different foreign gene into the TK gene, pEGFP-N1 (Clontech) was digested with the restriction enzymes AgeI and NotI and cloned into the same restriction enzyme sites of the pTNshuttle / TK-SP-BFP vector, replacing the BFP gene, to construct pTNshuttle / TK-SP-GFP. Using vaccinia virus (LC16mO strain, an unmodified virus) and transfer vector plasmid DNA (pTNshuttle / TK-SP-GFP), recombinant viruses were isolated using the same method as above and screened by PCR using GFP expression as an indicator. For TK, PCR was performed using two primers (SEQ ID NO: 18 and SEQ ID NO: 19). For clones in which a PCR product of the desired size was detected, the nucleotide sequence of the PCR product was confirmed by direct sequencing and designated TK-GFP (Figure 15-1C). To generate vaccinia viruses with both cell fusion and tumor targeting abilities, recombinant vaccinia viruses (VGF-LucGFP, a VGF-null vaccinia virus (WO 2015 / 076422) or TK-null vaccinia viruses (TK-GFP, a TK-null vaccinia virus) were used in combination with the transfer vector plasmid DNA (pTNshuttle / K2L-SP-BFP). Recombinant viruses were isolated using the same method as described above, and then subjected to PCR screening for BFP expression. PCR was performed using the same primers (SEQ ID NOs: 10 and 11) for VGF, the same primers (SEQ ID NOs: 12 and 13) for K2L, and the same primers (SEQ ID NOs: 18 and 19) for TK. For clones with PCR products of the desired size, the nucleotide sequences of the PCR products were confirmed by direct sequencing. These clones were designated VGF-LucGFP / K2L-BFP (Figure 15-1E) or K2L-BFP / TK-GFP (Figure 15-1F). Each recombinant virus was cultured in large quantities in A549 cells, and then the viral titer was measured in RK13 cells and used in the experiment.

[0067] Next, we investigated cell viability and ICD in vitro. Figures 15-2 and 15-3 show the results of cell viability when infected with 10 types of viruses: VGF-LucGFP / O1L-DsRed (Figure 1A), VGF-LucGFP / K2Lmut / O1L-DsRed (Figure 1B), VGF-LucGFP / ΔK2L-BFP / O1L-DsRed (Figure 15-1A), VGF-LucGFP / O1L-BFP / HAmut (Figure 1C), VGF-LucGFP (Figure 15-1B), VGF-LucGFP / K2L-BFP (Figure 15-1E), TK-GFP (Figure 15-1C), K2L-BFP / TK-GFP (Figure 15-1F), unmodified virus (Figure 15-1D), and K2L-BFP (Figure 15-1G). 4Human lung cancer cells (A549) were seeded in 96-well plates at 1 / well and incubated at 37°C for 24 hours. Then, they were infected with the viruses shown in Figures 1 and 15-1 at an MOI of 0.1 (n=3). Seventy-two hours after infection, cell viability was measured using the CellTiter 96® Aqueous Nonradioactive Cell Proliferation Assay (Promega). Compared with the non-deleted virus (VGF-LucGFP / O1L-DsRed), the spontaneous K2L mutations, the total K2L deletion, and the spontaneous HA mutation all significantly reduced cell viability (VGF-LucGFP / K2Lmut / O1L-DsRed, VGF-LucGFP / ΔK2L-BFP / O1L-DsRed, VGF-LucGFP / O1L-BFP / HAmut: ***P<0.0001). These results demonstrate that regardless of the method of mutation or deletion, cell fusion due to the loss of K2L or HA function leads to improved anticancer effects. Furthermore, K2L insertion / deletion significantly reduced cell viability in all of the following viruses: VGF-LucGFP, TK-GFP, and unmodified viruses (unmodified virus). (VGF-LucGFP / K2L-BFP, TK-GFP / K2L-BFP, K2L-BFP: ***P≦0.0001). Therefore, cell fusion enhances anticancer effects regardless of the type or presence of tumor specificity. Figures 15-4 and 15-5 show the results of ICD induction when the same 10 viruses were infected. A549 cells were cultured in a 24-well plate with 5.25 × 10 4Cells were seeded per well and incubated at 37°C for 24 hours. Then, they were infected with the viruses shown in Figure 1 and Figure 15-1 at an MOI of 1 (n=3). Sixty hours after infection, cell supernatants were collected and quantified using the HMGB1 ELISA Kit II (Shinotest). The results showed that the spontaneous K2L mutations, the complete K2L deletion, and the spontaneous HA mutations all significantly increased extracellular HMGB1 release compared with the virus without mutations or deletions (VGF-LucGFP / O1L-DsRed: ***P<0.0001, VGF-LucGFP / ΔK2L-BFP / O1L-DsRed: *P=0.0102, VGF-LucGFP / O1L-BFP / HAmut: **P=0.0011). These results indicate that cell fusion, mediated by loss of K2L or HA function, regardless of the mutation or deletion, enhances ICD induction. Furthermore, K2L insertion / deletion significantly enhanced extracellular HMGB1 release in all of the following recombinant viruses: VGF-LucGFP, TK-GFP, and unmodified virus (VGF-LucGFP / K2L-BFP: **P=0.0010, TK-GFP / K2L-BFP: ***P=0.0002, K2L-BFP: **P=0.0042). These results demonstrate that cell fusion enhances ICD induction regardless of the type or presence of tumor specificity.

[0068] These results demonstrate that vaccinia virus induces cell fusion, resulting in improved anticancer effects. Analysis of the underlying mechanism revealed the following three factors. First, increased viral proliferation and spread enhances tumor lysis, leading to increased apoptosis and necrosis. Second, fusion induces more efficient ICD, resulting in increased CD8 T cell infiltration into both the treated and untreated tumors. Finally, improved tumor immune environments in both treated and untreated tumors facilitate the activation of cancer immunity. Thus, oncolytic viruses that induce cell fusion exhibit greater anticancer effects than oncolytic viruses that do not induce cell fusion by more efficiently converting immune-resistant cold tumors into immune-resistant hot tumors (Figure 16).

[0069] Example 8 Therapeutic effect of combined use of cell-fusion-competent vaccinia virus and anti-PD-1 antibody Since it was suggested that the function of CD8 T cells is important for the therapeutic effect of the virus, especially in the untreated group, the function of CD8 T cells was promoted by combining it with an anti-PD-1 antibody, an immune checkpoint inhibitor. 5 The cells were subcutaneously transplanted into both sides of the abdomen of BALB / cAjcl mice, and tumors were grown to 42-135 mm 3 (Average 70mm 3 After tumor growth, VGF-Luc / O1L-lacZ and VGF-Luc / K2Lmut / O1L-lacZ were injected three times every other day. 7 PFU was administered directly into one tumor (Days 0, 2, and 4). In addition, PBS or InVivoPlus anti-mouse PD-1 Clone RMP1-14 (BioXCell) was administered intraperitoneally at 200 μg / mouse on Days 3, 5, 7, 9, and 11 (Figure 17). The antibody was administered the day after vaccinia virus administration. Starting with the second vaccinia virus administration, vaccinia virus and antibody were administered alternately every day, for a total of three virus administrations and five antibody administrations. The therapeutic effect was verified by measuring tumor volume, and the results are shown in Figure 18.

[0070] In the absence of antibody administration, tumor volume was suppressed in both VGF-Luc / O1L-lacZ- and VGF-Luc / K2Lmut / O1L-lacZ-treated mice in the virus-injected group, as shown in Figure 9. Two-way ANOVA statistical analysis revealed a significant difference compared to PBS-injected mice (***P<0.001). Also, as shown in Figure 9, VGF-Luc / K2Lmut / O1L-lacZ administration in the non-injected group resulted in tumor suppression greater than that achieved by PBS or VGF-Luc / O1L-lacZ administration, but tumor remission was not achieved in most mice. On the other hand, when anti-PD-1 antibodies were administered, the tumor suppression effect of virus administration was enhanced in the virus-injected group, with tumor remission achieved in 3 / 5 VGF-Luc / O1L-lacZ-treated mice and in all (6 / 6) VGF-Luc / K2Lmut / O1L-lacZ-treated mice. Furthermore, the concomitant administration of anti-PD-1 antibodies significantly enhanced the therapeutic effect of the virus in the untreated side, but did not result in tumor remission in the untreated side of VGF-Luc / O1L-lacZ-treated mice. On the other hand, two-way ANOVA statistical analysis confirmed that VGF-Luc / K2Lmut / O1L-lacZ-treated mice had significantly smaller tumor volumes than VGF-Luc / O1L-lacZ-treated mice (***<0.001), and 3 / 6 mice achieved tumor remission in both the treated and untreated sides. Figure 19 shows the survival curves of the anti-PD-1 antibody-treated mice after virus administration. Log-rank statistical analysis showed that VGF-Luc / K2Lmut / O1L-lacZ-treated mice had significantly longer survival times than PBS-treated mice without antibody administration (*: P=0.0112), but there was no significant difference from VGF-Luc / O1L-lacZ-treated mice. On the other hand, when combined with anti-PD-1 antibodies, VGF-Luc / K2Lmut / O1L-lacZ-treated mice showed a significant survival benefit compared to both PBS-treated and VGF-Luc / O1L-lacZ-treated mice (***: P = 0.0007, **: P = 0.0066).

[0071] Next, to confirm whether or not the tumors in the cured mice had immune memory, tumor cells were re-implanted into the cured mice. On day 101 after viral administration, 5.0 × 10 of a mouse colon cancer cell line (CT26) was re-implanted into the cured mice. 5 The mice were then subcutaneously transplanted with tumor cells into both sides of the abdomen. For comparison, similar tumor cell transplants were also performed subcutaneously in naive BALB / cAjcl mice of the same age. Figure 20 shows the changes in tumor size after tumor re-implantation. The control mice showed tumor growth similar to that shown in Figure 9, but no tumor growth was observed in the mice that were re-implanted after tumor recovery.

[0072] These findings suggest that the combined use of anti-PD-1 antibodies significantly enhances the therapeutic effect of vaccinia viruses with cell fusion ability, and promotes the induction of antitumor immunity and the establishment of immune memory. [Industrial Applicability]

[0073] Vaccinia virus, which induces cell fusion in infected cells, can be used for cancer therapy. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Sequence List Free Text]

[0074] 1-4, 7, 8, 10-13, 16-19 Primers

Claims

1. An oncolytic vaccinia virus that is deficient in the functions of the K2L gene or the HA gene, or the K2L gene and the HA gene, and further deficient in the functions of the vaccinia virus growth factor (VGF) gene and the O1L gene, and that does not grow in normal cells but grows specifically in cancer cells, has oncolytic properties that specifically damage cancer cells, causes cell fusion in infected cells, improves systemic anti-cancer immune activity, and induces cell death.

2. A vaccinia virus as described in claim 1, which further improves the ability to induce immunogenic cell death, increases the infiltration of CD8 T cells into cancer cells, attacks cancer cells, and reduces immunosuppressive cells such as Tregs, TAMs, or MDSCs.

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

4. A pharmaceutical composition for cancer treatment, comprising the vaccinia virus according to any one of claims 1 to 3.

5. A vaccinia virus vector, in which a foreign DNA is introduced into the vaccinia virus according to any one of claims 1 to 3.

6. 6. The vaccinia virus vector according to claim 5, wherein the foreign DNA is a marker DNA, a therapeutic gene having a cytotoxic effect or an immunostimulatory effect, or a DNA encoding an antigen of a cancer, virus, bacterium, or protozoa.

7. A pharmaceutical composition for cancer treatment or for use as a vaccine against cancer, viruses, bacteria or protozoa, comprising the vaccinia virus vector of claim 5 or 6.

8. A method for producing a vaccinia virus according to any one of claims 1 to 6, which comprises deleting the function of the K2L gene or HA gene, or the K2L gene and the HA gene, of the vaccinia virus, and further deleting the function of the vaccinia virus growth factor (VGF) gene and the O1L gene, and which does not grow in normal cells but grows specifically in cancer cells, has oncolytic properties that specifically damage cancer cells, causes cell fusion in infected cells, improves systemic anti-cancer immune activity, and induces cell death.

9. The production method according to claim 8, wherein the vaccinia virus is the LC16 strain, the LC16mO strain, or the LC16m8 strain modified to express the B5R gene.

10. A combination pharmaceutical kit for cancer treatment, comprising the vaccinia virus according to any one of claims 1 to 3 in combination with an immune checkpoint inhibitor.

11. The pharmaceutical combination kit according to claim 10, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

12. The vaccinia virus according to any one of claims 1 to 3, for use in combination with an immune checkpoint inhibitor for cancer treatment.

13. The vaccinia virus of claim 12, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

Images