Antitumor immunity induction method

Abstract

The present invention addresses the problem of providing a novel means for treating cancer by activating antitumor immunity in cancer cells. The present invention provides an antitumor immunity induction method comprising introducing a virus-derived gene or a gene whose expression is induced by the virus-derived gene into a cancer cell.

Description

Method for inducing anti-tumor immunity

[0001] The present invention relates to a method for inducing anti-tumor immunity by expressing a virus-derived gene or a gene whose expression is induced by the virus-derived gene.

[0002] In some intractable cancers, antitumor immunity is known to be dysfunctional due to factors such as the expression of immune checkpoint molecules. In response to this, immune checkpoint inhibitors (also known as "checkpoint blockade") have recently been proposed as a means of activating antitumor immunity. However, checkpoint inhibitors are only effective in cases where the immunosuppressive pathway mediated by immune checkpoint molecules such as PD-1 and CTLA4 is the primary cause of carcinogenesis. Many cancers do not exhibit antitumor effects with checkpoint inhibitors alone, resulting in poor prognosis, presenting a major problem. These intractable cancers are known to be poorly immunogenic, and the development of new methods for activating antitumor immunity and cancer treatment is highly desirable.

[0003] In response to this, for example, Non-Patent Document 1 shows that expressing LMP1, an endogenous gene of EB virus, in B-cell lymphoma cells or B-cell leukemia cells promotes the presentation of tumor-associated antigens, activates T cells that recognize cancer antigens in B-cell tumors, and can enhance immunity against B-cell tumors.

[0004] However, the effects of LMP1 expression in non-B cell cancer cells have not been reported.

[0005] Choi IK et al. Mechanism of EBV inducing anti-tumour immunity and its therapeutic use. Nature, 590, 157-162 (2021)

[0006] In view of the above circumstances, an objective of the present invention is to provide a new means for treating cancer by activating anti-tumor immunity in cancer cells.

[0007] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that expressing a gene encoding the LMP1 protein in tumor-causing cancer cells can confer antigen-presenting cell-like functions to the phenotype of non-B cell cancer cells. They have also found that this leads to the generation of new T cells with anti-tumor immune activity that act specifically on the cancer cells, thereby eliminating the cancer cells. They have also found that a similar effect can be achieved by expressing Spib and / or Irf1, genes whose expression is induced by virus-derived genes. Based on these findings, the present invention has been completed. Thus, the present invention provides a novel method for activating anti-tumor immunity and a novel method for treating cancer, based on a novel mechanism of action that is completely different from that of conventional techniques. Specifically, the present invention relates to the following:

[0008] [1] A method for inducing anti-tumor immunity, comprising introducing a virus-derived gene or a gene whose expression is induced by the virus-derived gene into cancer cells. [2] The method, wherein the cancer cells are non-B-cell cancer cells. [3] The method, wherein the virus-derived gene is an EB virus gene. [4] The method, wherein the EB virus gene is LMP1. [5] The method, wherein the virus-derived gene is selected from the group consisting of: (A) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 1; (B) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (C) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein the amino acid sequence comprises substitution, deletion, insertion, and / or addition of one or more amino acid residues, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (D) a gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 2, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2. [6] The method as mentioned above, wherein the gene whose expression is induced is Spib and / or Irf1.[7] The method as described above, wherein the gene whose expression is induced is selected from the group consisting of: (E) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 5; (F) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (G) a gene encoding a protein comprising an amino acid sequence set forth in SEQ ID NO: 4 or 6, wherein the amino acid sequence comprises substitution, deletion, insertion, and / or addition of one or several amino acid residues, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (H) a gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 4 or 6, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (I) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 7 or 9; (J) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; (K) a gene encoding a protein comprising an amino acid sequence comprised of substitution, deletion, insertion, and / or addition of one or several amino acid residues, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; (L) A gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 8 or 10, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10. [8] A composition for inducing anti-tumor immunity by introducing into cancer cells the virus-derived gene or the gene whose expression is induced by the virus-derived gene, the composition comprising a nucleic acid comprising the sequence of a virus-derived gene or a gene whose expression is induced by the virus-derived gene. [9] The composition, wherein the cancer cells are non-B cell cancer cells.

[10] The composition, wherein the virus-derived gene is an EB virus gene.

[11] The composition, wherein the EB virus gene is LMP1.

[12] The composition, wherein the virus-derived gene is selected from the group consisting of: (A) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 1; (B) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (C) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein the amino acid sequence comprises substitution, deletion, insertion, and / or addition of one or several amino acid residues, and the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (D) a gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 2, and the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.

[13] The composition, wherein the gene whose expression is induced is Spib and / or Irf1.

[14] The composition, wherein the gene whose expression is induced is selected from the group consisting of: (E) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 5; (F) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (G) a gene encoding a protein comprising an amino acid sequence set forth in SEQ ID NO: 4 or 6, wherein the amino acid sequence comprises substitution, deletion, insertion, and / or addition of one or several amino acid residues, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (H) a gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 4 or 6, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (I) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 7 or 9; (J) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; (K) a gene encoding a protein comprising an amino acid sequence comprised of substitution, deletion, insertion, and / or addition of one or several amino acid residues, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; (L) A gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 8 or 10, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10.

[15] The composition, wherein the nucleic acid comprising the sequence of the virus-derived gene or the gene whose expression is induced by the virus-derived gene is mRNA.

[16] The composition, wherein the composition further comprises lipid nanoparticles.

[17] The composition, wherein the composition comprises lipid nanoparticles and mRNA comprising the sequence of the virus-derived gene or the gene whose expression is induced by the virus-derived gene, in an amount effective to confer antigen-presenting cell-like function to the trait of cancer cells.

[18] The composition, wherein the lipid nanoparticles comprise, in terms of molar ratios of total lipids in the lipid nanoparticles, 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[19] A method for treating or preventing cancer, comprising administering an effective amount of the composition to a subject.

[20] The method, wherein the cancer is a solid cancer or a liquid cancer.

[21] The method, wherein the solid cancer is one or more selected from the group consisting of nasopharyngeal cancer, gastric cancer, lung cancer, cervical cancer, ovarian cancer, glioblastoma, colorectal cancer, pancreatic cancer, and malignant melanoma.

[22] The method, wherein the liquid cancer is one or more selected from the group consisting of Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-associated B-cell lymphoma, multiple myeloma, central nervous system B-cell lymphoma, post-transplant lymphoproliferative disorder (PTLD), T-cell lymphoma, and B-cell-derived chronic lymphocytic leukemia.

[23] The method, wherein the administration form of the composition is one or more selected from the group consisting of intratumoral administration, intravenous administration, intraarterial administration, intramuscular administration, intraperitoneal administration, intrathecal administration, epidural administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, and oral administration.

[24] The method, wherein the effective amount is 0.1 μg / kg (body weight) to 1000 mg / kg (body weight) of a nucleic acid comprising the sequence of a virus-derived gene or a gene whose expression is induced by the virus-derived gene.

[25] The method, wherein the composition is administered to a subject once daily, twice daily, three times daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, once monthly, twice monthly, three times monthly, four times monthly, five times monthly, six times monthly, seven times monthly, eight times monthly, nine times monthly, or ten times monthly for one day to several years.

[26] The method, wherein the composition is administered to a subject for one day to three years.

[27] The method, wherein the composition is administered in combination with one or more additional therapeutic agents or therapies selected from the group consisting of surgery, radiation, chemotherapy, molecular targeted drugs, immunotherapy, hormone therapy, CAR-T cell therapy, and cancer vaccine.

[0009] The present invention can also be embodied in the following aspects: Use of a nucleic acid comprising a sequence of a virus-derived gene or a gene whose expression is induced by the virus-derived gene, for inducing anti-tumor immunity in cancer cells; A nucleic acid comprising a sequence of a virus-derived gene or a gene whose expression is induced by the virus-derived gene, for use in inducing anti-tumor immunity in cancer cells; Use of a nucleic acid comprising a sequence of a virus-derived gene or a gene whose expression is induced by the virus-derived gene, in the production of a composition for inducing anti-tumor immunity in cancer cells; A method for producing a composition for inducing anti-tumor immunity in cancer cells, comprising formulating a nucleic acid comprising a sequence of a virus-derived gene or a gene whose expression is induced by the virus-derived gene.

[0010] The present invention provides a new means for treating cancer by activating anti-tumor immunity in cancer cells. The method of the present invention converts non-B cell cancer cells, in particular, into antigen-presenting cell-like cells and newly generates T cells with anti-tumor immune activity. This method has not existed until now, and the present invention may provide an entirely new cancer treatment method. Furthermore, the technology of the present invention may further improve survival rates when used in combination with conventional anti-cancer agents or cancer therapeutic agents.

[0011] Figure 1 shows the results of a study on the induction of antigen-presenting cell-associated T cell stimulatory molecules in LMP1-expressing melanoma cells. Expression of antigen-presenting cell-associated T cell stimulatory molecules (MHC class I, CD80, ICOSL, CD112, CD155, and ICAM-1) was induced in melanoma cells overexpressing LMP1. (A) Using a mouse melanoma cell line (B16F10 melanoma cell line), we established cells expressing both LMP1 and the reporter molecule human CD2 in a doxycycline-inducible manner (LMP1 B16) and, as a control, cells expressing human CD2 alone (Ctrl B16). (B) Flow cytometry analysis confirmed that LMP1 protein was normally expressed after doxycycline induction. (C) Induction of LMP1 expression resulted in increased expression of the MHC class I molecule (H-2Kb), which is involved in T cell recognition. (D) Induction of LMP1 expression confirmed increased expression of costimulatory molecules involved in T cell activation: ICAM-1, CD80, ICOSL, CD112, and CD155. Figure 2 shows the results of a test to determine the induction of expression of antigen-presenting cell-associated T cell stimulatory molecules in LMP1-expressing colon cancer cells and pancreatic cancer cells. (A) Using a mouse colon cancer cell line (MC38), induction of LMP1 expression confirmed increased expression of MHC class I, costimulatory molecules ICAM-1, CD80, ICOSL, CD112, and CD155, which are important for T cell activation. (B) Using a mouse pancreatic cancer cell line (Panc02), induction of LMP1 expression confirmed increased expression of MHC class I, ICAM-1, CD112, and CD155, which are important for T cell activation. Figure 3 shows the results of a test to determine the induction of CD8 by LMP1-expressing cancer cells. ＋ Figure 1 shows the results of a test of T cell activation. LMP1-expressing cancer cells co-cultured with IL-2 stimulated CD8 + Activates T cells. (A) LMP1-expressing cancer cells and syngenic mouse CD8 +A co-culture system for T cells was established to verify whether LMP1 expression induces T cell activation. (B) T cell activation was assessed using the cell proliferation detection reagent CellTrace, and it was confirmed that co-culture with LMP1 B16 cells induced cell division associated with T cell activation. (C) A similar experiment was performed with LMP1 MC38 cells, confirming that T cell activation was also induced in colon cancer cells. Figure 4 shows the results of a test on the antigen recognition and killing activity of tumor cells by activated cytotoxic T cells. Cytotoxic CD8 activated by LMP1 +T cells recognize and kill tumor cell antigens in an MHC class I-dependent manner. (A) LMP1-activated T cells were cocultured with wild-type B16F10 cells or MHC class I-deficient B16F10 cells (B16F10 β2m - / -). T cells exhibited cytotoxic activity against wild-type B16F10 cells, which depended on MHC class I expression. (B) The results in (A) suggest that tumor antigens are presented on MHC molecules induced by LMP1, which T cells recognize and kill cancer cells. Figure 5 shows the results of an analysis of the intracellular signaling required for the enhanced immunogenicity of LMP1-expressing cancer cells. The immunogenicity-enhancing effect of LMP1 is dependent on the NF-κB pathway, and it can induce MHC expression even in IFN-γ-refractory cancer cells. (A) The LMP1-induced enhanced MHC class I expression was abolished by an NF-κB inhibitor, demonstrating that it is regulated by NF-κB signaling. (B) IFN-γ signaling is known to be important for cancer immunogenicity, and IFN-γ-refractory cancers with genetic mutations present a therapeutic challenge. In contrast, LMP1 induced increased expression of MHC molecules even in IFN-γ-refractory cancers lacking JAK1. Figure 6 shows the results of an analysis of transcription factors required for the increased immunogenicity of LMP1-expressing cancer cells. LMP1 induces the expression of the transcription factor Spi-B and regulates T cell activation. (A) RNA sequencing quantified the expression of transcription factors that regulate gene expression upon LMP1 introduction. The Spib gene, which is characteristically expressed in immune cells with antigen-presenting function, was particularly upregulated by LMP1 expression. (B) A B16 cell line with Spib gene knockout using CRISPR-Cas9 was created, and co-cultured with T cells suppressed T cell proliferation. These results demonstrate that Spi-B-mediated gene expression regulation is involved in the LMP1-induced immunogenicity enhancement. Figure 7 shows the results of a test to induce MHC class I expression by introducing the LMP1 gene into B16F10 cells using mRNA. MHC class I expression is induced by introducing the LMP1 gene into B16F10 cells using mRNA. (A) B16F10 cells were transfected with mRNA that simultaneously expresses LMP1 and the fluorescent protein GFP, and protein expression was confirmed.The LMP1 gene and GFP gene were linked by a 2A self-cleaving peptide. (B) Transfected B16F10 cells expressed GFP, confirming increased expression of MHC class I molecules. Figure 8 shows the results of an experiment to examine in vivo LMP1 protein expression following intratumoral administration of LMP1 mRNA-LNP. Intratumoral administration of LMP1 mRNA-LNP resulted in LMP1 protein expression in mouse B16F10 tumor cells. (A) LMP1 mRNA-LNP, which contained LMP1 mRNA in lipid nanoparticles (LNPs), was directly administered into the engrafted B16F10 melanoma tumor in mice. (B) Tumor cells were isolated 12 hours after LMP1 mRNA-LNP administration, and LMP1 protein expression was confirmed by flow cytometry. (C) B16 cells were implanted into C57BL / 6 wild-type mice, and LMP1-mRNA-LNP was intratumorally administered on days 7, 10, and 13 after implantation. Tumor growth was monitored by measuring the long and short diameters with a digital caliper and calculating the volume up to day 15 (photograph substitutes for drawing). Figure 9 shows the results of a study to identify transcription factors that regulate MHC expression. IRF1, a downstream transcription factor of LMP1, was identified as the main MHC expression regulator. (A) Schematic diagram of the study method. (B) Test results. *: p<0.05 Figure 10 shows H-2K induced by Spi-B / IRF1 co-transfection. b This figure shows the results of verifying changes in CD80 molecule expression and T cell activation. It was shown that co-transfection of Spi-B and CD80 is necessary for efficient T cell activation. (A) MHC class I (H-2K b (B) The left panel shows a flow cytometry histogram of CellTrace. The right panel shows the mean CellTrace signal value for each group. *: p<0.05, ***: p<0.001, ****: p<0.0001. Figure 11 shows the results of a test on the induction of T cell immune surveillance by Spib / Irf1 gene transfer. Co-transfection of Spi-B / IRF1 suppressed the expression of tumor-infiltrating cytotoxic CD8 +This increased T cells and induced anti-tumor immunity. (A) A schematic diagram of the test method. (B) Tumor weight 10 days after subcutaneous transplantation into mice (photograph substitute for drawing). (C) The characteristics of tumor-infiltrating immune cells with or without Spi-B / IRF1 transfection are shown. *: p<0.05, ***: p<0.001

[0012] The present invention will be described below. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention.

[0013] <Method for Inducing Anti-Tumor Immunity> One aspect of the present invention relates to a method for inducing anti-tumor immunity (hereinafter, sometimes referred to as the "method for inducing anti-tumor immunity of the present invention"), which comprises introducing a virus-derived gene or a gene whose expression is induced by the virus-derived gene into cancer cells. In the present invention, the term "cancer cells" is not particularly limited, as long as the method of the present invention exerts an anti-tumor immunity induction effect. The method for inducing anti-tumor immunity of the present invention involves introducing a virus-derived gene (e.g., LMP1, etc.) or a gene whose expression is induced by the virus-derived gene (e.g., Spib, Irf1, etc.) into cancer cells and expressing the corresponding protein, thereby activating the antigen-presenting function of B-cell cancer cells and / or conferring antigen-presenting cell-like functions to the phenotype of non-B-cell cancer cells, thereby inducing anti-tumor immunity. Hereinafter, "virus-derived genes or genes whose expression is induced by the virus-derived genes" may be collectively referred to as "virus-derived genes or their derived genes."

[0014] <Cancer Cells> The method for inducing anti-tumor immunity of the present invention involves introducing a virus-derived gene (e.g., LMP1) or a gene whose expression is induced by the virus-derived gene (e.g., Spib, Irf1) into non-B cell cancer cells, in particular, to express the corresponding protein, thereby conferring antigen-presenting cell-like functions to the non-B cell cancer cells, thereby generating T cells that act specifically on the cancer cells and exhibit anti-tumor activity, thereby inducing anti-tumor immunity. A preferred aspect of the present invention relates to a method for inducing anti-tumor immunity, comprising introducing a virus-derived gene or a derived gene thereof into non-B cell cancer cells. In this aspect, "introducing a virus-derived gene or a derived gene thereof into non-B cell cancer cells" may mean "introducing a virus-derived gene or a derived gene thereof into a cell population containing only non-B cell cancer cells" or "introducing a virus-derived gene or a derived gene thereof into a cell population containing non-B cell cancer cells and other cancer cells." Hereinafter, the present invention may be described using as an example a "method for inducing anti-tumor immunity, which comprises introducing a virus-derived gene or its derivative into non-B cell cancer cells," but the present invention is not limited to this embodiment.

[0015] In the present invention, "non-B cell cells" can be rephrased as "cells that do not have antigen-presenting cell-like functions." Non-B cell cells refer to cells that do not have antigen-presenting cell-like functions, and do not refer to a cell type. In other words, any cell that does not have antigen-presenting cell-like functions is encompassed by the non-B cell cells of the present invention. Furthermore, as long as the non-B cell cells do not have antigen-presenting cell-like functions, it also includes cells that do not originally have antigen-presenting cell-like functions, and cells that have lost antigen-presenting cell-like functions or cells with reduced antigen-presenting cell-like functions. "Does not have antigen-presenting cell-like functions" includes cells that do not have any antigen-presenting cell-like functions, and cells that do not have sufficient antigen-presenting cell-like functions for T cell activation.

[0016] Furthermore, in the present invention, "antigen-presenting cell-like function" can be rephrased as "antigen-presenting cell-associated T cell-stimulatory molecule-presenting function." The "antigen-presenting cell-associated T cell-stimulatory molecule" is, for example, one or more selected from MHC molecules and costimulatory molecules. Although not limited thereto, the MHC molecule is preferably MHC class I, and the costimulatory molecule is preferably one or more selected from CD80, ICOSL, CD112, CD155, and ICAM-1. Note that, in the present invention, "one or more selected from MHC molecules and costimulatory molecules" encompasses "embodiments comprising one or more MHC molecules and one or more costimulatory molecules." That is, one preferred embodiment of the present invention is a method for inducing anti-tumor immunity by introducing a virus-derived gene or its derivative into non-B cell cancer cells and expressing the corresponding protein, thereby endowing the non-B cell cancer cells with the ability to express and present on the cell surface one or more MHC molecules and costimulatory molecules, thereby generating T cells that act specifically on the cancer cells and exhibit anti-tumor activity.

[0017] <Virus-Derived Genes> In the method for inducing anti-tumor immunity of the present invention, the "virus-derived gene" to be introduced into cancer cells and used to induce anti-tumor immunity is not particularly limited as long as it exerts the anti-tumor immunity-inducing effect of the present invention, and may be, for example, a cancer-associated gene. In the present invention, the "cancer-associated gene" includes not only oncogenes but also genes that ultimately promote carcinogenesis or cancer growth through the activation of oncogenes or the inactivation of tumor suppressor genes.

[0018] The virus-derived gene used in the method of inducing anti-tumor immunity of the present invention is preferably an Epstein-Barr virus (EBV) gene. EBV was isolated in 1964 as the first human cancer virus from Burkitt's lymphoma (BL). Since then, its involvement in nasopharyngeal carcinoma, Hodgkin's lymphoma, NK / T lymphoma, opportunistic lymphoma associated with AIDS or organ transplantation, gastric cancer, and other cancers has been revealed, which are referred to as EBV-associated cancers. EBV maintains a latent infection state in these cancer cells, and the function of the expressed viral genes is involved in carcinogenesis.

[0019] Specific examples of EBV genes include LMP1, LMP2A, LMP2B, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP, and EBER, more preferably LMP1, LMP2A, and LMP2B, and even more preferably latent membrane protein 1 (LMP1). LMP1 is an important viral oncogene involved in the oncogenesis of many EBV-associated cancers.

[0020] The LMP1 gene specific to EB virus (scientific name: human gammaherpesvirus 4) has the nucleotide sequence shown in SEQ ID NO: 1, and the amino acid sequence of the LMP1 protein encoded by this gene is shown in SEQ ID NO: 2.

[0021] In one embodiment, the LMP1 gene used in the present invention may be a gene having the nucleotide sequence shown in SEQ ID NO: 1, and the LMP1 protein may be a protein having the amino acid sequence shown in SEQ ID NO: 2. Unless otherwise specified, the expression "a gene or protein has a nucleotide sequence or an amino acid sequence" may mean that a gene or protein contains the nucleotide sequence or the amino acid sequence in a longer sequence, or may mean that a gene or protein has only the nucleotide sequence or the amino acid sequence.

[0022] Although there may be differences in DNA sequence between EB virus strains, etc., any gene may be used in the present invention as long as it achieves the effects of the present invention. Therefore, the LMP1 gene is not limited to a gene having the nucleotide sequence shown in SEQ ID NO: 1, but may also include genes having a mutant nucleotide sequence of SEQ ID NO: 1 and encoding an LMP1 protein (including a protein having the amino acid sequence shown in SEQ ID NO: 2 and mutant proteins thereof). Similarly, the LMP1 protein is not limited to a protein having the amino acid sequence shown in SEQ ID NO: 2, but may also include proteins having mutant amino acid sequences of SEQ ID NO: 2.

[0023] The term "variant nucleotide sequence" may refer to a nucleotide sequence that encodes an LMP1 protein (e.g., a protein having the amino acid sequence set forth in SEQ ID NO: 2) using any synonymous amino acid codon according to the standard genetic code (see, e.g., Lewin B., "Genes VIII", 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458). Thus, a DNA encoding an LMP1 protein having the amino acid sequence set forth in SEQ ID NO: 2 may be a gene having a variant nucleotide sequence of SEQ ID NO: 1 due to the degeneracy of the genetic code.

[0024] The term "mutant nucleotide sequence" may also refer to a nucleotide sequence that can hybridize under stringent conditions with a nucleotide sequence complementary to the sequence set forth in SEQ ID NO: 1 or a probe that can be prepared from said nucleotide sequence, as long as the nucleotide sequence encodes a protein that maintains the activity or function of a protein having the amino acid sequence set forth in SEQ ID NO: 2 or whose three-dimensional structure is not significantly altered compared to the unmodified protein having the amino acid sequence set forth in SEQ ID NO: 2. The term "stringent conditions" may refer to conditions under which specific hybrids are formed, for example, hybrids with a homology of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, as defined by the parameter "identity" when using the computer program BLASTn, but not nonspecific hybrids, such as hybrids with lower homology than the above. The method for obtaining a "mutant nucleotide sequence" is not limited and can be performed by known techniques.

[0025] The term "mutant nucleotide sequence" may also refer to a nucleotide sequence that encodes a mutant protein.

[0026] The term "variant protein" may refer to a protein having a variant amino acid sequence of SEQ ID NO:2.

[0027] The term "mutant protein" specifically refers to a protein having one or more mutations in its sequence, whether substitution, deletion, insertion, and / or addition of one or several amino acid residues, compared to the amino acid sequence set forth in SEQ ID NO:2, which maintains the activity or function of the protein having the amino acid sequence set forth in SEQ ID NO:2, or whose three-dimensional structure is not significantly altered compared to the unmodified protein having the amino acid sequence set forth in SEQ ID NO:2. The number of mutations in a mutant protein depends on the position of the amino acid residue in the three-dimensional structure of the protein or the type of amino acid residue. The number of mutations in a mutant protein is not strictly limited, and may be 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1, 2, 3, 4, or 5, based on SEQ ID NO:2. This is possible because amino acids can be highly homologous to each other, and the activity or function of the protein is not affected or the three-dimensional structure of the protein is not significantly changed compared to the unmodified protein having the amino acid sequence set forth in SEQ ID NO: 2. Thus, a mutant protein may be a protein having an amino acid sequence that is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the entire amino acid sequence of SEQ ID NO: 2, as defined by the parameter "identity" when using the computer program BLASTp, as long as the activity or function of the protein having the amino acid sequence set forth in SEQ ID NO: 2 is maintained or the three-dimensional structure of the protein is not significantly altered compared to the unmodified protein having the amino acid sequence set forth in SEQ ID NO: 2. As used herein, the term "homology" may refer to "identity" (which is the identity between amino acid residues). Sequence identity between two sequences is calculated as the percentage of matching residues between the two sequences when the two sequences are aligned for maximum matching.

[0028] Examples of substitution, deletion, insertion, and / or addition of one or several amino acid residues include conservative mutations. A representative example of conservative mutations can be conservative substitutions. Conservative substitutions include, but are not limited to, substitutions between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid; between Ala, Leu, Ile, and Val when the substitution site is a hydrophobic amino acid; between Glu, Asp, Gln, Asn, Ser, His, and Thr when the substitution site is a hydrophilic amino acid; between Gln and Asn when the substitution site is a polar amino acid; between Lys, Arg, and His when the substitution site is a basic amino acid; between Asp and Glu when the substitution site is an acidic amino acid; and between Ser and Thr when the substitution site is an amino acid with a hydroxyl group. Examples of conservative substitutions include substitutions of Ala with Ser or Thr, Arg with Gln, His, or Lys, Asn with Glu, Gln, Lys, His, or Asp, Asp with Asn, Glu, or Gln, Cys with Ser or Ala, Gln with Asn, Glu, Lys, His, Asp, or Arg, Glu with Asn, Gln, Lys, or Asp, Gly with Pro, His with Asn, Lys, Gln, Arg, or Tyr, Ile with Leu, Me Examples of such substitutions include substitutions of Lys with Ile, Met, Val, or Phe, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. The above-mentioned substitutions, deletions, insertions, additions, etc. of amino acid residues also encompass naturally occurring mutations due to individual differences in the organism from which the amino acid sequence is derived.

[0029] Examples of substitutions, deletions, insertions, and / or additions of one or several amino acid residues also include non-conservative mutations, provided that the mutations are compensated for by one or more second mutations at different positions in the amino acid sequence such that the activity or function of the mutant protein is maintained or the three-dimensional structure of the protein is not significantly altered relative to the unmodified protein having the amino acid sequence shown in SEQ ID NO:2.

[0030] The percentage of polypeptide identity can be calculated using the BLASTp algorithm. More specifically, the percentage of polypeptide identity can be calculated using the BLASTp algorithm provided by the National Center for Biotechnology Information (NCBI) with default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The percentage of polynucleotide identity can be calculated using the BLASTn algorithm. More specifically, the percentage of polynucleotide identity can be calculated using the BLASTn algorithm provided by NCBI with default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0031] In the present invention, one virus-derived gene may be used alone, or two or more virus-derived genes may be used in any combination.

[0032] <<Gene whose expression is induced by a virus-derived gene>> One embodiment of the present invention relates to a method for inducing anti-tumor immunity, comprising introducing into cancer cells a gene whose expression is induced by a virus-derived gene (e.g., Spib, Irf1, etc.). In this method, the gene whose expression is induced by the virus-derived gene in the cancer cells is activated (e.g., expression is enhanced), thereby inducing anti-tumor immunity in the cancer cells. Here, "introducing a gene whose expression is induced by a virus-derived gene" may mean actually introducing the gene whose expression is induced by the virus-derived gene into the cancer cells so that the gene is activated, or may mean activating the gene whose expression is induced by the virus-derived gene in the cancer cells using known genome editing techniques, nucleic acids, low-molecular-weight compounds, or the like.

[0033] In the method for inducing anti-tumor immunity of the present invention, the "gene whose expression is induced by a virus-derived gene" that is introduced into cancer cells and used to induce anti-tumor immunity is a gene whose expression is induced by the virus-derived gene. It is not particularly limited as long as it exerts the anti-tumor immunity-inducing effect of the present invention, and may be, for example, Spib, Irf1, Irf9, STAT1, STAT2, etc., whose expression is induced by the LMP1 gene. The gene whose expression is induced by a virus-derived gene may be used alone or in any combination of two or more. Among these, preferred are, but not limited to, Spib or Irf1, and combinations thereof. A particularly preferred embodiment is to use a combination of Spib and Irf1.

[0034] The human Spib gene has the nucleotide sequence shown in SEQ ID NO: 3, and the amino acid sequence of the Spi-B protein encoded by this gene is shown in SEQ ID NO: 4. The mouse Spib gene has the nucleotide sequence shown in SEQ ID NO: 5, and the amino acid sequence of the Spi-B protein encoded by this gene is shown in SEQ ID NO: 6. The human Irf1 gene has the nucleotide sequence shown in SEQ ID NO: 7, and the amino acid sequence of the IRF1 protein encoded by this gene is shown in SEQ ID NO: 8. The mouse Irf1 gene has the nucleotide sequence shown in SEQ ID NO: 9, and the amino acid sequence of the IRF1 protein encoded by this gene is shown in SEQ ID NO: 10.

[0035] In one embodiment, the Spib gene used in the present invention may be a gene having the nucleotide sequence shown in SEQ ID NO: 3 or 5, and the Spi-B protein may be a protein having the amino acid sequence shown in SEQ ID NO: 4 or 6. In another embodiment, the Irf1 gene used in the present invention may be a gene having the nucleotide sequence shown in SEQ ID NO: 7 or 9, and the IRF1 protein may be a protein having the amino acid sequence shown in SEQ ID NO: 8 or 10. Unless otherwise specified, the expression "a gene or protein has a nucleotide sequence or an amino acid sequence" may mean that a gene or protein contains the nucleotide sequence or the amino acid sequence in a longer sequence, and may also mean that a gene or protein has only the nucleotide sequence or the amino acid sequence.

[0036] Although the DNA sequences of these genes may differ between species, etc., they may be used in the present invention as long as they achieve the effects of the present invention. Therefore, the Spib gene is not limited to a gene having the nucleotide sequence set forth in SEQ ID NO: 3 or 5, but may also include a gene having a variant nucleotide sequence of SEQ ID NO: 3 or 5 and encoding an Spi-B protein (including a protein having the amino acid sequence set forth in SEQ ID NO: 4 or 6 or a variant thereof). Similarly, the Spi-B protein is not limited to a protein having the amino acid sequence set forth in SEQ ID NO: 4 or 6, but may also include a protein having a variant amino acid sequence of SEQ ID NO: 4 or 6. Similarly, the Irf1 gene is not limited to a gene having the nucleotide sequence set forth in SEQ ID NO: 7 or 9, but may also include a gene having a variant nucleotide sequence of SEQ ID NO: 7 or 9 and encoding an IRF1 protein (including a protein having the amino acid sequence set forth in SEQ ID NO: 8 or 10 or a variant thereof). Similarly, the IRF1 protein is not limited to a protein having the amino acid sequence set forth in SEQ ID NO: 8 or 10, but may also include a protein having a variant amino acid sequence of SEQ ID NO: 8 or 10.

[0037] The following description will be given using the Spib gene having the nucleotide sequence of SEQ ID NO: 3 as an example, but this description also applies to Spib genes having other nucleotide sequences and to the Irf1 gene, substituting the respective sequences. The term "variant nucleotide sequence" may refer to a nucleotide sequence encoding an Spi-B protein (e.g., a protein having the amino acid sequence set forth in SEQ ID NO: 4) using any synonymous amino acid codon according to the standard genetic code (see, e.g., Lewin B., "Genes VIII," 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458). Therefore, a DNA encoding an Spi-B protein having the amino acid sequence set forth in SEQ ID NO: 4 may be a gene having a variant nucleotide sequence of SEQ ID NO: 3 due to the degeneracy of the genetic code.

[0038] The term "mutant nucleotide sequence" may also refer to a nucleotide sequence that can hybridize under stringent conditions with a nucleotide sequence complementary to the sequence set forth in SEQ ID NO: 3 or a probe that can be prepared from said nucleotide sequence, as long as the nucleotide sequence encodes a protein that maintains the activity or function of a protein having the amino acid sequence set forth in SEQ ID NO: 4 or whose three-dimensional structure is not significantly altered compared to the unmodified protein having the amino acid sequence set forth in SEQ ID NO: 4. The term "stringent conditions" may refer to conditions under which specific hybrids are formed, for example, hybrids with a homology of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, as defined by the parameter "identity" when using the computer program BLASTn, but not nonspecific hybrids, such as hybrids with lower homology than the above. The method for obtaining a "mutant nucleotide sequence" is not limited and can be performed by known techniques.

[0039] The above transcription factors may be used in gene families, provided that they achieve the effects of the present invention. Specific examples of such families include, but are not limited to, the Spib (also referred to as SpiB) gene, which may be substituted for the Spib gene by family members such as Spi1 (also referred to as PU.1) and Spic (also referred to as SpiC) (Nat Rev Mol Cell Biol. 2001 Nov;2(11):827-37. doi: 10.1038 / 35099076). Furthermore, for example, in the case of the Irf (also referred to as IRF) 1 gene, examples of the family include Irf2, Irf3, Irf4 / PIP / LSIRF / ICSAT, Irf5, Irf6, Irf7, Irf8 / ICSBP, and Irf9 / ISGF3γ (Cold Spring Harb Perspect Biol. 2018 Nov 1;10(11):a028423. doi: 10.1101 / cshperspect.a028423.), and these can be used in place of the Irf1 gene. That is, one embodiment of the present invention relates to a method for inducing anti-tumor immunity, comprising introducing into cancer cells at least one gene (preferably Spib) selected from the Spib gene encoding the transcription factor Spi-B or its family genes (e.g., Spi1, Spic, etc.). One embodiment of the present invention relates to a method for inducing anti-tumor immunity, which comprises introducing into cancer cells at least one gene (preferably Irf1) selected from the Irf1 gene encoding the transcription factor IRF1 or its family genes (e.g., Irf2, Irf3, Irf4 / PIP / LSIRF / ICSAT, Irf5, Irf6, Irf7, Irf8 / ICSBP, Irf9 / ISGF3γ, etc.). Another embodiment of the present invention relates to a method for inducing anti-tumor immunity, which comprises introducing into cancer cells at least one gene selected from the Spib gene or its family genes, and at least one gene selected from the Irf1 gene or its family genes.

[0040] The term "mutant nucleotide sequence" may also refer to a nucleotide sequence that encodes a mutant protein.

[0041] The term "variant protein" may refer to a protein having a variant amino acid sequence of SEQ ID NO:4.

[0042] The term "mutant protein" specifically refers to a protein having one or more mutations in its sequence, whether substitution, deletion, insertion, and / or addition of one or several amino acid residues, compared to the amino acid sequence set forth in SEQ ID NO:4, which maintains the activity or function of the protein having the amino acid sequence set forth in SEQ ID NO:4 or whose three-dimensional structure is not significantly altered compared to the unmodified protein having the amino acid sequence set forth in SEQ ID NO:4. The number of mutations in a mutant protein depends on the position of the amino acid residue in the three-dimensional structure of the protein or the type of amino acid residue. The number of mutations in a mutant protein is not strictly limited, and may be 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1, 2, 3, 4, or 5, based on SEQ ID NO:4. This is possible because amino acids can be highly homologous to each other, and the activity or function of the protein is not affected or the three-dimensional structure of the protein is not significantly changed compared to the unmodified protein having the amino acid sequence set forth in SEQ ID NO: 4. Thus, a mutant protein may be a protein having an amino acid sequence that is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the entire amino acid sequence of SEQ ID NO: 4, as defined by the parameter "identity" when using the computer program BLASTp, as long as the activity or function of the protein having the amino acid sequence set forth in SEQ ID NO: 4 is maintained or the three-dimensional structure of the protein is not significantly altered compared to the unmodified protein having the amino acid sequence set forth in SEQ ID NO: 4. As used herein, the term "homology" may refer to "identity" (which is the identity between amino acid residues). Sequence identity between two sequences is calculated as the percentage of matching residues between the two sequences when the two sequences are aligned for maximum matching.

[0043] Examples of substitution, deletion, insertion, and / or addition of one or several amino acid residues include conservative mutations. A representative example of conservative mutations can be conservative substitutions. Conservative substitutions include, but are not limited to, substitutions between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid; between Ala, Leu, Ile, and Val when the substitution site is a hydrophobic amino acid; between Glu, Asp, Gln, Asn, Ser, His, and Thr when the substitution site is a hydrophilic amino acid; between Gln and Asn when the substitution site is a polar amino acid; between Lys, Arg, and His when the substitution site is a basic amino acid; between Asp and Glu when the substitution site is an acidic amino acid; and between Ser and Thr when the substitution site is an amino acid with a hydroxyl group. Examples of conservative substitutions include substitutions of Ala with Ser or Thr, Arg with Gln, His, or Lys, Asn with Glu, Gln, Lys, His, or Asp, Asp with Asn, Glu, or Gln, Cys with Ser or Ala, Gln with Asn, Glu, Lys, His, Asp, or Arg, Glu with Asn, Gln, Lys, or Asp, Gly with Pro, His with Asn, Lys, Gln, Arg, or Tyr, Ile with Leu, Me Examples of such substitutions include substitutions of Lys with Ile, Met, Val, or Phe, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. The above-mentioned substitutions, deletions, insertions, additions, etc. of amino acid residues also encompass naturally occurring mutations due to individual differences in the organism from which the amino acid sequence is derived.

[0044] Examples of substitutions, deletions, insertions, and / or additions of one or several amino acid residues also include non-conservative mutations, provided that the mutations are compensated for by one or more second mutations at different positions in the amino acid sequence such that the activity or function of the mutant protein is maintained or the three-dimensional structure of the protein is not significantly altered relative to the unmodified protein having the amino acid sequence shown in SEQ ID NO:4.

[0045] The percentage of polypeptide identity can be calculated using the BLASTp algorithm. More specifically, the percentage of polypeptide identity can be calculated using the BLASTp algorithm provided by the National Center for Biotechnology Information (NCBI) with default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The percentage of polynucleotide identity can be calculated using the BLASTn algorithm. More specifically, the percentage of polynucleotide identity can be calculated using the BLASTn algorithm provided by NCBI with default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0046] A virus-derived gene or a gene derived from a virus is specifically composed of nucleic acid. The nucleic acid may be any molecule as long as it is a molecule formed by polymerizing nucleotides and / or substances having the same function as the nucleotides. For example, RNA, which is a polymer of ribonucleotides, DNA, which is a polymer of deoxyribonucleotides, chimeric nucleic acids consisting of RNA and DNA, and nucleic acids in which at least one nucleotide is a substance having the same function as the nucleotide (for example, pseudouridine or N-terminal amino acid). 1Examples of such nucleotide polymers include nucleotide polymers substituted with uridine (nucleotide derivatives such as uridine-1-methyl-2-methyl ...

[0047] In the present invention, nucleic acids preferably include ribonucleic acid (RNA), including messenger RNA (mRNA), which may encode a polypeptide of interest, including naturally occurring or non-naturally occurring or modified polypeptides. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure.

[0048] The nucleic acid may include, but is not limited to, a base sequence encoding an expression control region sequence, a gene, an intergenic sequence, a signal peptide, a protein pro-region, an artificial amino acid sequence, a reporter molecule, etc. Expression control regions include promoters, enhancers, operators, attenuators and termination signals, anti-termination signals, ribosome binding sites (RBS), and other expression control elements (e.g., regions to which repressors or activators bind, and / or binding sites for transcription and translation control proteins in, for example, transcribed mRNA). Such control regions can be found, for example, in known literature (e.g., Sambrook J., Fritsch EF and Maniatis T., "Molecular Cloning: A Laboratory Manual", 2004). nded., Cold Spring Harbor Laboratory Press (1989); Pfleger BF et al., Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes, Nat. Biotechnol., 2006, 24:1027-1032; Mutalik VK et al., Precise and reliable gene expression via standard transcription and translation initiation elements, Nat. Methods, 2013, 10:354-360).

[0049] The nucleic acids used in the present invention may be produced using known RNA or DNA synthesis methods and RNA or DNA modification methods.

[0050] The nucleic acid used in the present invention may be incorporated into an expression vector in a state in which the gene of interest can be expressed. As the expression vector, for example, one that can be expressed in mammalian cells and can be transformed can be used, and examples of the expression vector that can be used include plasmids derived from Escherichia coli, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai viruses, and vaccinia viruses.

[0051] Known methods for introducing genes into cells include the lipofection method using lipids, the lipid nanoparticle (LNP) method, the physical electroporation method and microinjection method, and the viral vector method that utilizes the cell entry mechanism of viruses. Any of these may be used, but the LNP method is preferred from the viewpoint that it can effectively deliver an introduced compound such as a nucleic acid to a target cell or tissue in the body and that the expression of the foreign gene does not persist for a long period of time. Introduction of a gene into cells can be carried out by genetic engineering techniques well known to those skilled in the art.

[0052] For example, but not limited to, the method for inducing anti-tumor immunity of the present invention involves administering the composition of the present invention intratumorally or intravenously to a mammal, including a human, so that the composition is delivered to, for example, an organ or site where cancer has developed, and a virus-derived gene or its derived gene can be introduced into cells in the delivered organ or site. By expressing the corresponding protein within the cells, antigen-presenting cell-like functions are imparted to the traits of non-B cell cancer cells, thereby generating T cells that act specifically on the cancer cells and exhibit anti-tumor activity, which can be used to induce anti-tumor immunity.

[0053] The anti-tumor immunity-inducing effect of the methods of the present invention can be evaluated, for example, by an increase (preferably a statistically significant increase) in the expression level of one or more antigen-presenting cell-associated T cell stimulatory molecules (e.g., MHC class I, costimulatory molecules CD80, ICOSL, CD112, CD155, ICAM-1, etc.) in cancer cells transfected with a virus-derived gene or its derivative, compared to when the virus-derived gene or its derivative is not transfected (e.g., when a control gene is transfected, no transfection is performed, or before transfection). More specifically, the anti-tumor immunity-inducing effect of the methods of the present invention can be evaluated by transfecting cancer cells with a virus-derived gene or its derivative, measuring the expression level of the antigen-presenting cell-associated T cell stimulatory molecule in the cells after transfection, and comparing it with a control. If the expression level of the antigen-presenting cell-associated T cell stimulatory molecule is increased (preferably a statistically significant increase) compared to the control, it can be determined that the transfection of the virus-derived gene or its derivative has an anti-tumor immunity-inducing effect.

[0054] The cancer for which anti-tumor immunity is induced by the method for inducing anti-tumor immunity of the present invention may be any type of cancer, and may be a solid cancer or a liquid cancer.

[0055] Examples of solid cancers include cancers of the brain and nervous system (e.g., brain tumors, spinal cord tumors, glioblastomas, etc.), head and neck cancers (e.g., nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, salivary gland cancer, paranasal sinus cancer, thyroid cancer, etc.), digestive system cancers (e.g., stomach cancer, esophageal cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, etc.), urinary or reproductive system cancers (e.g., kidney cancer, renal cell carcinoma, bladder cancer, prostate cancer, renal pelvis and ureter cancer, gallbladder cancer, bile duct cancer, testicular cancer, penile cancer, uterine cancer, endometrial cancer, uterine sarcoma, cervical cancer, vaginal cancer, vulvar cancer, ovarian cancer, fallopian tube cancer, etc.), cancers of the respiratory system (for example, lung cancer (including small cell lung cancer, non-small cell lung cancer, and metastatic lung cancer), bronchial cancer, etc.), breast cancer, skin cancer (for example, malignant melanoma, etc.), bone cancer (for example, osteosarcoma, etc.), muscle cancer (for example, rhabdomyosarcoma, etc.), etc., and preferably one or more selected from the group consisting of nasopharyngeal cancer, gastric cancer, lung cancer, cervical cancer, ovarian cancer, glioblastoma, colorectal cancer, pancreatic cancer, and malignant melanoma.

[0056] Liquid cancers include leukemia, malignant lymphoma, multiple myeloma, myelodysplastic syndrome, etc. Leukemias include acute myeloid leukemia, acute lymphocytic leukemia (B-cell precursor type, mature B-cell type, and T-cell type), chronic myeloid leukemia, and chronic lymphocytic leukemia. Malignant lymphomas are classified into Hodgkin's lymphoma and non-Hodgkin's lymphoma. Non-Hodgkin's lymphomas include B-cell lymphomas (diffuse large cell lymphoma, Burkitt's lymphoma, AIDS-associated B-cell lymphoma, central nervous system B-cell lymphoma, post-transplant lymphoproliferative disorder (PTLD), follicular lymphoma, MALT lymphoma, mantle cell lymphoma, etc.), and T-cell lymphomas and NK-cell lymphomas (adult T-cell lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, etc.). Preferably, the cancer is one or more selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, and chronic lymphocytic leukemia, and more preferably, one or more selected from the group consisting of Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-associated B-cell lymphoma, central nervous system B-cell lymphoma, post-transplant lymphoproliferative disorder (PTLD), multiple myeloma, T-cell lymphoma, and chronic lymphocytic leukemia of B-cell origin.

[0057] In one embodiment, the cancer that is the target of the method of inducing anti-tumor immunity of the present invention is a cancer associated with non-B cell cancer cells, i.e., cancer cells that do not have antigen-presenting cell-like functions.

[0058] Furthermore, the method for inducing anti-tumor immunity of the present invention can also be used as a method for imparting antigen-presenting cell-like functions to the phenotype of non-B cell cancer cells, which involves introducing a virus-derived gene or its derivative into non-B cell cancer cells.

[0059] Furthermore, the method for inducing anti-tumor immunity of the present invention can also be used as a method for imparting antigen-presenting cell-like functions to the characteristics of non-B cell cancer cells, including introducing a virus-derived gene or its derived gene into non-B cell cancer cells, and generating T cells that act specifically on the cancer cells and exhibit anti-tumor activity.

[0060] <Composition for inducing anti-tumor immunity> Another aspect of the present invention relates to a composition for inducing anti-tumor immunity by introducing a virus-derived gene or its derived gene into cancer cells, the composition comprising a nucleic acid containing the sequence of the virus-derived gene or its derived gene (hereinafter, sometimes referred to as the "composition of the present invention"). Note that all of the matters explained in the section <Method for inducing anti-tumor immunity> above apply to the explanation in this section.

[0061] The composition of the present invention contains, as an active ingredient, a nucleic acid comprising the sequence of a virus-derived gene or a gene derived therefrom. By allowing the composition of the present invention to enter a cell, the virus-derived gene or a gene derived therefrom is introduced into the cell, and the composition is effective, for example, for inducing anti-tumor immunity as described above in the section <Method for inducing anti-tumor immunity>, and for preventing, ameliorating, or treating cancer as described above in the section <Method for treating or preventing cancer>.

[0062] The compositions of the present invention contain an active ingredient in an amount (i.e., an effective amount) necessary to achieve the expected anti-tumor immunity induction, and preventive, ameliorative, and therapeutic effects. The amount of nucleic acid comprising the sequence of a virus-derived gene or its derived gene, which is the active ingredient of the compositions of the present invention, generally varies depending on the dosage form, but the amount of the active ingredient can be set within the range of, for example, 0.01 to 100% by mass, 0.1 to 99.9% by mass, or 1 to 80% by mass so as to achieve the desired dosage.

[0063] Although not particularly limited, as a preferred embodiment, the LNP method can be used as a method for introducing the gene according to the present invention into cells.

[0064] The lipid nanoparticles used in the LNP method are those described in known literature (e.g., Ugur Sahin et al., COVID-19 vaccine BNT 162b1 elicits human antibody and T H1T cell responses, Nature, 2020 Oct, 586(7830), 594-599.; KS Corbett et al., Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates, N. Engl. J. Med., 2020, 383. 1544-55.; Yan Zong et al., Lipid Nanoparticle (LNP) Enables mRNA Delivery for Cancer Therapy, Adv. Mater., 2023, 35:2303261-2303261; Xuexiang Han et al., An ionizable lipid toolbox for RNA delivery, Nat. Commun., 2021, 12, 7233; Japanese Patent No. 6946384; JP 2024-071384; Japanese Patent No. 6666391; JP 2024-052664; JP 2023-526178; JP 2024-027144, etc.) can be used.

[0065] In one embodiment, the composition of the present invention comprises lipid nanoparticles and mRNA containing the sequence of the virus-derived gene or its derivative gene in an amount effective to confer antigen-presenting cell-like function to the phenotype of cancer cells. In another embodiment, the lipid nanoparticles contained in the composition of the present invention comprise, in molar ratios relative to the total lipid content of the lipid nanoparticles, 20-60% ionizable cationic lipids, 5-25% non-cationic lipids, 25-55% sterols, and 0.5-15% polyethylene glycol (PEG)-modified lipids. Without limitation, the molar ratios relative to the total lipid content of the lipid nanoparticles may be 30-50% or 40-45%, the non-cationic lipids may be 7.5-20% or 10-15%, the sterols may be 30-50% or 40-45%, and the PEG-modified lipids may be 1-10%, or 1.5-5%. Here, an ionizable cationic lipid is a lipid that is nearly electrically neutral at physiological pH (for example, around pH 7) and becomes cationic in an acidic environment.

[0066] Ionizable cationic lipids include, but are not limited to, DLin-MC3-DMA (MC3), SM-102, ALC-0315, and 306O. i10 , cKK-E12, C12-200, 5A2-SC8, TT3, FTT5, ZA3-Ep10, OF-Deg-Lin, AA3-DLin, OC2-K3-E10, 4N4T(MIC1 and MIC2), IC8, unsaturated trialkylated lipid 10, YK009, 93-O17S, LNP-A10, 306-N16B, OF-02, A18-Iso5-2DC18, 98N 12 -5, 9AIP9, 7C1, G0-C14, L319, 304O 13, 306-O12B, A9, potentially Lipid 2,2(8,8)4CCH3, CL1, LP01, ATX-100, LP-01, Lipid 5, SGL0806, DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, L319, etc. Ionizable cationic lipids can be used singly or in combination of two or more.

[0067] Among the ionizable cationic lipids, preferred specific examples include, but are not limited to, a lipid represented by the following formula (Ia) (SGL0806).

[0068]

[0069] Examples of non-cationic lipids include neutral phospholipids derived from biological membranes, such as phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and dipalmitoylphosphatidylcholine. One type of non-cationic lipid can be used alone, or two or more types can be used in combination.

[0070] Examples of sterols include cholesterol, dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brassicasterol, ergocastol, fucosterol, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), etc. Sterols can be used singly or in combination of two or more.

[0071] Examples of polyethylene glycol-modified lipids include PEG2000-DMG (PEG2000-dimyristylglycerol), PEG2000-DPG (PEG2000-dipalmitoylglycerol), PEG2000-DSG (PEG2000-distearoylglycerol), PEG5000-DMG (PEG5000-dimyristylglycerol), PEG5000-DPG (PEG5000-dipalmitoylglycerol), PEG5000-DSG (PEG5000-distearoylglycerol), Examples of polyethylene glycol-modified lipids include PEG-cDMA (N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxylpropyl-3-amine), PEG-C-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxylpropyl-3-amine), polyethylene glycol (PEG)-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, and PEG-ceramide (Cer). Polyethylene glycol-modified lipids can be used alone or in combination of two or more.

[0072] The ionizable cationic lipids, non-cationic lipids, sterols, and PEG-modified lipids can be produced by known methods. Some are also commercially available, and any of them can be used.

[0073] In the present invention, lipid nanoparticles can be formed by assembling and organizing the above-mentioned lipids. Here, "organization" refers to the assembly of constituent lipid molecules via non-covalent bonds such as hydrophobic bonds. Organized assemblies include bilayers formed by hydrophobic bonds between the hydrophobic portions of the constituent molecules, liposomes, multivesicles, string-like assemblies, disk-like assemblies, lamellar assemblies, rod-like assemblies, and mixtures thereof. Lipid nanoparticles can be obtained by incorporating nucleic acids containing the sequence of a virus-derived gene or its derivative gene into the interior during the organization process.

[0074] The lipid nanoparticles used in the present invention may further contain molecules other than the essential constituent lipids, such as surfactants (e.g., CHAPS, sodium cholate, octylglucoside, ND-gluco-N-methylalkanamides, etc.), glycolipids, peptide lipids, proteins, etc., as long as the advantages of the present invention, such as the efficiency of intracellular introduction of the compound to be introduced and low cytotoxicity, are not impaired.

[0075] Lipid nanoparticles encapsulating nucleic acids containing the sequence of a virus-derived gene or a gene derived therefrom can be formed by known methods without any particular limitations.

[0076] Lipid nanoparticles can be prepared, without limitation, by preparing an aqueous solution such as a citrate buffer solution containing a nucleic acid containing a sequence of a virus-derived gene or a derived gene thereof, preparing a polar organic solvent-containing solution containing the lipid used in the composition of the present invention, mixing the aqueous solution with the polar organic solvent-containing solution to obtain a mixed solution, and removing the polar organic solvent from the mixed solution.

[0077] In addition to the ionizable cationic lipid, non-cationic lipid, sterol, and PEG-modified lipid, the polar organic solvent-containing solution may further contain surfactants (e.g., CHAPS, sodium cholate, octylglucoside, ND-gluco-N-methylalkanamides, etc.), glycolipids, peptide lipids, proteins, etc.

[0078] Furthermore, the polar organic solvent of the polar organic solvent-containing solution is not particularly limited, but examples thereof include alcohols such as ethanol, isopropanol, and t-butanol, taking into account the polarity of the solvent and the ease of removing the solvent after the formation of lipid nanoparticles.

[0079] The step of obtaining the mixture can be performed using, for example, a vortex mixer or a microchannel. Furthermore, by the step of obtaining the mixture, lipid nanoparticles having the compound to be introduced encapsulated therein can be formed in the mixture.

[0080] In the step of removing the polar organic solvent, the content of the polar organic solvent can be reduced by, for example, diafiltration, ultrafiltration, or evaporation under reduced pressure.

[0081] An incubation step can be carried out between the step of obtaining the mixed solution and the step of reducing the content of the polar organic solvent.

[0082] In addition to the above-mentioned components, the composition of the present invention may contain other additives such as sugars such as sucrose, glucose, sorbitol, and lactose; amino acids such as glutamine, glutamic acid, sodium glutamate, and histidine; and salts of acids such as citric acid, phosphoric acid, acetic acid, lactic acid, carbonic acid, and tartaric acid.

[0083] The composition of the present invention can be used as a reagent, a pharmaceutical, or the like for humans or animals.

[0084] As described above, the compositions of the present invention contain, as an active ingredient, a nucleic acid comprising the sequence of a cancer-related gene or a gene derived therefrom. However, the compositions of the present invention may consist solely of the active ingredient, or may be formulated by appropriately combining other pharmaceutically acceptable ingredients in addition to the active ingredient (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antioxidants, antiseptics, surfactants, lubricants, diluents, coating agents, sugar-coating agents, flavorings, emulsifying / solubilizing / dispersing agents, pH adjusters, isotonic agents, solubilizing agents, fragrances, colorants, solubilizing aids, sterilized water, physiological saline, etc.) as long as the effects of the present invention are not impaired.

[0085] The dosage form when formulated is not particularly limited. Examples of dosage forms include tablets, powders, fine granules, granules, capsules, syrups, liquids, suspensions, emulsions, jellies, injections, topical preparations, inhalants, nasal drops, eye drops, and suppositories. Formulation can be carried out based on known formulation methods.

[0086] The composition of the present invention may be in, for example, a solid state, a powder state from which the solvent has been removed by freeze-drying or the like, or a liquid state.

[0087] A preferred embodiment of the dosage form of the composition of the present invention is an injection.In this case, if the composition is in a powder state, it can be used as an injection by suspending or dissolving it in a pharmaceutically acceptable medium before use.If the composition is in a liquid state, it can be used as an injection as it is or by suspending or dissolving it in a pharmaceutically acceptable medium.

[0088] Pharmaceutically acceptable vehicles include sterilized water, physiological saline, and isotonic solutions containing adjuvants such as glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. The composition of the present invention may further contain additives such as solubilizers (e.g., alcohols such as ethanol, propylene glycol, and polyethylene glycol), stabilizers, antioxidants, and preservatives.

[0089] For example, but not limited to, the composition of the present invention can be delivered to, for example, an organ or site where cancer has developed by intratumoral or intravenous administration to a mammal, including a human, and a nucleic acid containing the sequence of a virus-derived gene or its derived gene in the composition of the present invention can be introduced into cells of the delivered organ or site. By expressing the corresponding protein within the cells, antigen-presenting cell-like functions are imparted to the traits of non-B cell cancer cells, thereby generating T cells that act specifically on the cancer cells and exhibit anti-tumor activity, which can be used to induce anti-tumor immunity and for cancer treatment or prevention.

[0090] Although not particularly limited, a preferred embodiment of the present invention includes a composition for inducing antitumor immunity, comprising an mRNA containing the sequence of the virus-derived gene or its derivative in an amount effective to confer antigen-presenting cell-like function to the phenotype of non-B cell cancer cells, and lipid nanoparticles. Such a composition of the present invention can be directed to induce a specific immune response and can therefore be applied to a wide range of therapeutic and preventive mRNA vaccines for low-immunogenic tumors, etc.

[0091] <Method for treating or preventing cancer> One aspect of the present invention relates to a method for treating or preventing cancer (hereinafter, sometimes referred to as the "cancer treatment or prevention method of the present invention"), which comprises administering an effective amount of a composition of the present invention to a subject (patient). The method for treating or preventing cancer may be a method for inhibiting cancer progression or a method for preventing cancer recurrence. Note that the matters explained in the above sections <Method for inducing anti-tumor immunity> and <Composition for inducing anti-tumor immunity> all apply to the explanation in this section.

[0092] In the present invention, the term "effective amount" refers to the amount required to achieve a desired effect, such as induction of anti-tumor immunity, or therapeutic, ameliorative, or preventive effect, when administered to a subject or to cells, tissues, or organs of the subject.

[0093] In the cancer treatment or prevention method of the present invention, the composition of the present invention can be administered by any administration route, such as parenteral or oral administration, but parenteral administration is preferred, and it can be administered, for example, intratumorally, intravenously, intraarterially, intramuscularly, intraperitoneally, intrathecally, epidurally, subcutaneously, intradermally, intranasally, pulmonary, etc. The composition of the present invention may be administered systemically or locally.

[0094] The subject is preferably a mammal, and may be, for example, a primate such as a human, chimpanzee, or gorilla, a rodent such as a mouse, rat, or guinea pig, a cow, a horse, a pig, a sheep, a goat, a llama, a camel, a dog, a cat, or a rabbit, but is preferably a human. The subject may be a subject experiencing or suspected of experiencing anti-tumor immunosuppression. Typically, the subject may be a subject with cancer or suspected of having cancer. The subject may be a subject that is refractory to cancer immunotherapy or has reduced responsiveness to cancer immunotherapy.

[0095] In this specification, "administering a composition to an animal" may be synonymous with "inducing an animal to ingest the composition." Intake may be voluntary (ad libitum intake) or forced (forced intake).

[0096] The cancer that is the target of the cancer treatment or prevention method of the present invention may be any type of cancer, and more specifically, the details explained in the section <Method for inducing anti-tumor immunity> above can be referred to.

[0097] The administration method can be appropriately selected by those skilled in the art depending on the species, age, body weight, sex, symptoms, etc. of the subject (patient). An effective amount of nucleic acid comprising the sequence of a virus-derived gene or its derived gene may be, for example, in the range of 0.0001 mg (0.1 μg) to 1,000 mg, 0.001 mg to 500 mg, 0.01 mg to 100 mg, or 0.1 mg to 10 mg per kg of body weight. An effective amount of nucleic acid comprising the sequence of a virus-derived gene or its derived gene may be administered in divided doses, or in a controlled-release or sustained-release form, two or three or more times a day.

[0098] An effective amount of a nucleic acid comprising the sequence of a virus-derived gene or its derivative gene can be administered to a subject daily (e.g., once or multiple times per day, e.g., twice or three times), weekly (e.g., once or multiple times per week, e.g., twice, three times, four times, or five times), or monthly (e.g., once or multiple times per month, e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times). Determining an appropriate dosing schedule can be performed by one skilled in the art.

[0099] There are no particular limitations on the administration period, and it may be a period necessary to induce anti-tumor immunity or achieve a therapeutic or preventive effect, but it may be, for example, one day or more, one week or more, one month or more, or three months or more, or it may be several years or less, three years or less, one year or less, or six months or less, or any compatible combination of these.

[0100] For example, but not limited to, the cancer treatment or prevention method of the present invention involves intratumoral or intravenous administration of the composition of the present invention to a mammal, including a human, so that the composition is delivered to, for example, an organ or site where cancer has developed, and a virus-derived gene or its derivative gene can be introduced into cells in the delivered organ or site. By expressing the corresponding protein in the cells, antigen-presenting cell-like functions are imparted to the traits of non-B cell cancer cells, thereby generating T cells that act specifically on the cancer cells and exhibit anti-tumor activity, which can be used to induce anti-tumor immunity and for cancer treatment or prevention.

[0101] In the cancer treatment or prevention method of the present invention, for example, tumor size (e.g., tumor volume) is measured (preferably over time) in a subject (cancer patient) or a cancer-bearing animal model to which the composition of the present invention has been administered, and if a decrease in tumor size or suppression of tumor growth (preferably a statistically significant decrease or suppression) is observed compared to a control group (when a control gene is introduced, when not introduced, before introduction, etc.), it can be determined that the introduction of the virus-derived gene or its derived gene has a cancer treatment or prevention effect.

[0102] As described above, administration of an effective amount of the composition of the present invention to a subject results in intracellular expression of a protein corresponding to a virus-derived gene or its derived gene, thereby endowing the phenotype of non-B cell cancer cells with antigen-presenting cell-like functions, thereby generating T cells that act specifically on the cancer cells and exhibit anti-tumor activity, thereby inducing anti-tumor immunity and providing cancer treatment and prevention effects. In one embodiment, the composition and method of the present invention reprogram non-B cell cancer cells by inducing the expression of transcription factors, such as Spi-B and IRF1, that are specifically expressed in B cells, and thereby producing CD8 + Acquires antigen-presenting ability to activate T cells, enhances expression of MHC-I and costimulatory molecules CD80, CD155, CD112, ICOSL, and ICAM-1, and induces CD8 + This method is based on a completely different concept from conventional methods, namely, activating T, and can provide a method for inducing antitumor immunity and a method for treating and preventing cancer. Furthermore, cancer immunogenicity is generally controlled by IFN signaling, and IFNγ-refractory cancers, in which genetic mutations have occurred, present a therapeutic challenge. In contrast, the increased expression of MHC molecules and other molecules by the compositions and methods of the present invention is controlled by NF-κB signaling, which uses a different pathway. Therefore, this method can provide a useful method for inducing antitumor immunity and a method for treating and preventing cancer, in that it can induce increased expression of MHC molecules even in IFNγ-refractory cancers.

[0103] In the cancer treatment or prevention methods of the present invention, the composition of the present invention may be used in combination (co-administered) with an additional therapeutic agent or therapy.

[0104] The cancer treatment or prevention method of the present invention may be carried out in combination with a known additional therapeutic agent or therapy, such as one or more additional therapeutic agents or therapies selected from the group consisting of surgery, radiation, chemotherapeutic agents, molecularly targeted drugs (such as vascular endothelial growth factor (VEGF) inhibitors), immunotherapeutic agents (such as checkpoint inhibitors and recombinant chemokines), immunomodulators, hormone therapy agents, CAR-T cell therapy, and cancer vaccines.

[0105] The additional therapeutic agent or therapy may be administered to the subject prior to, after, or simultaneously with the administration of the compositions of the invention.

[0106] The additional therapeutic agent may be present in the same pharmaceutical composition as the composition of the present invention, or the additional therapeutic agent may be present in a separate pharmaceutical composition from the composition of the present invention.

[0107] Chemotherapeutic agents include, but are not limited to, alkylating agents such as mitomycin C, cyclophosphamide, busulfan, ifosfamide, isosphamide, melphalan, hexamethylmelamine, thiotepa, chlorambucil, or dacarbazine; antimetabolites such as gemcitabine, capecitabine, 5-fluorouracil, cytarabine, 2-fluorodeoxycytidine, methotrexate, idatrexate, tomudex, or trimetrexate; topoisomerase II inhibitors such as rubicin, etoposide, teniposide, or mitoxantrone; topoisomerase I inhibitors such as irinotecan (CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38), or topotecan; antimitotic drugs such as paclitaxel, docetaxel, vinblastine, vincristine, or vinorelbine; platinum derivatives such as cisplatin, oxaliplatin, spiroplatinum, or carboplatinum; and the like.

[0108] Molecularly targeted drugs include, but are not limited to, inhibitors of tyrosine kinase receptors, such as sunitinib and sorafenib; anti-neoplastic antibodies, particularly antibodies that affect the regulation of cell surface receptors (particularly those based on VEGF inhibitory activity), such as trastuzumab, cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, bevacizumab, and ranibizumab; EGFR (epidermal growth factor receptor) inhibitors, such as gefitinib, erlotinib, and lapatinib; and the like.

[0109] Immunomodulatory agents include, but are not limited to, alpha, beta, or gamma interferon, interleukins (particularly IL-2, IL-6, IL-10, or IL-12), or tumor necrosis factors.

[0110] Immunotherapeutic agents include, but are not limited to, checkpoint inhibitors, recombinant chemokines, and the like, such as programmed cell death 1 (PD-1) inhibitors such as nivolumab or pembrolizumab; programmed cell death-ligand 1 (PD-L1) inhibitors such as atezolizumab; cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors such as ipilimumab; T-cell immunoglobulin domain and mucin domain-3 (TIM-3) inhibitors; lymphocyte-activation gene 3 (LAG-3) inhibitors; T-cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors; B-lymphocyte- and T-lymphocyte-associated (BTLA) inhibitors; or suppressor of T-cell activation containing immunoglobulin domain V (VISTA) inhibitors.

[0111] The present invention will be specifically described below with reference to examples, but these are merely examples of the present invention and the scope of the present invention is not limited to these examples.

[0112] Example 1: Induction of expression of antigen-presenting cell-associated T cell stimulatory molecules in melanoma cells overexpressing LMP1 1.1 (A in Figure 1) Using a mouse melanoma cell line (B16F10 melanoma cell line), we established cells (LMP1 B16) that simultaneously express LMP1 and the reporter molecule human CD2 molecule (huCD2) in a doxycycline-inducible manner, and cells (Ctrl B16) that express only huCD2 as a control.

[0113] 1.2 (Figure 1B) The established cancer cell lines were cultured in high-glucose DMEM medium (Fujifilm) supplemented with 10% FBS and 1% penicillin / streptomycin mixture. Gene induction was performed by adding 1 μg / ml doxycycline to the medium, and the cells were cultured for 32 hours before molecular expression analysis. Gene expression was confirmed by staining the cell surface huCD2 with an anti-human CD2 antibody (TS1 / 8 clone), fixing the cells with the BD Cytofix / Cytoperm™ Fixation / Permeabilization Kit (BD Biosciences), staining the intracellular domain of LMP1 with an anti-LMP1 antibody (S12 clone), and then analyzing by flow cytometry.

[0114] 1.3 (Fig. 1C) Flow cytometry analysis confirmed that induction of LMP1 expression increased the expression of MHC class I molecules involved in T cell recognition.

[0115] 1.4 (Figure 1D) Flow cytometry analysis confirmed that induction of LMP1 expression increased the expression of costimulatory molecules involved in T cell activation: ICAM-1, CD80, ICOSL, CD112, and CD155.

[0116] Example 2: Induction of expression of antigen-presenting cell-associated T cell stimulatory molecules in colon cancer cells and pancreatic cancer cells overexpressing LMP1 2.1 (Figure 2A) Using a mouse colon cancer cell line (MC38), we confirmed that induction of LMP1 expression increased the expression of MHC class I, costimulatory molecules ICAM-1, CD80, ICOSL, CD112, and CD155, which are important for T cell activation.

[0117] 2.2 (Figure 2B) Using a mouse pancreatic cancer cell line (Panc02), we confirmed that induction of LMP1 expression increased the expression of MHC class I, costimulatory molecules ICAM-1, CD112, and CD155, which are necessary for T cell activation.

[0118] Example 3: CD8 expression in LMP1-expressing cancer cells + 3.1 T cell activation (Fig. 3A) LMP1 B16 cells and syngenic mouse CD8 +We established a T cell co-culture system and examined whether LMP1 expression induces T cell activation.

[0119] 3.2 (Figure 3B) CD8 + T cells were extracted from mouse spleens by negative selection using the BD™ IMag Cell Separation System (BD Biosciences). The extracted cells were suspended in PBS and incubated with the cell proliferation detection reagent CellTrace Violet (Thermo Fisher Scientific) for 20 minutes at 37°C. These T cells were irradiated with 10 Gy and co-cultured with LMP1-B16, which had been growth-arrested, for 3 days. For co-culture, RPMI medium was supplemented with 10% FBS, 1% penicillin / streptomycin solution, sodium pyruvate, non-essential amino acids, β-mercaptoethanol, and 2 ng / ml recombinant mouse IL-2. T cell activation was assessed by a decrease in CellTrace signal associated with cell division, confirming that co-culture with LMP1-B16 induced cell division associated with T cell activation.

[0120] 3.3 (Figure 3C) Similarly, we confirmed that co-culture of LMP1 with MC38 also induced cell division associated with T cell activation.

[0121] Example 4: Cytotoxic CD8 activated by LMP1 + T cell-mediated tumor cell cytotoxicity. 4.1 (Figure 4A) Wild-type B16F10 cells or MHC class I-deficient B16F10 cells (B16F10 β2m - / -) were treated with 100 ng / ml IFNγ for 2 days and then labeled with the cell proliferation detection reagent CellTrace Violet (Thermo Fisher Scientific) in PBS for 20 minutes at 37°C. These cells were cocultured with LMP1-B16-activated T cells for 3 hours. T cell-mediated cytotoxicity to B16 was detected with propidium iodine. T cells exhibited cytotoxic activity against wild-type B16F10 cells, which was dependent on MHC class I expression.

[0122] The results of 4.2 (Figure 4B) (Figure 4A) suggest that tumor antigens are presented on MHC molecules induced by LMP1, and that T cells that recognize these antigens are activated and damage cancer cells.

[0123] Example 5: NF-κB pathway-dependent immunogenicity enhancement by LMP1 and induction of MHC expression in IFN-γ-refractory cancer cells 5.1 (Fig. 5A) LMP1-B16 was treated with 1 μg / ml doxycycline for 10 hours, followed by the addition of 5 μM NF-κB inhibitor BMS-345541 (MedchemExpress) and further incubation for 14 hours. Expression of MHC class I molecules in these cells was analyzed by flow cytometry, confirming that LMP1-induced enhancement of MHC class I expression is dependent on the NF-κB pathway.

[0124] 5.2 (Figure 5B) JAK1, a molecule that regulates the IFN pathway, was knocked out using LMP1 B16, and we investigated whether LMP1 could induce MHC class I expression in IFN-γ-refractory cancers. Knockout cells were treated with 100 ng / ml IFNγ for 2 days or with 1 μg / ml doxycycline for 32 hours. The former treatment did not result in MHC class I expression, whereas the latter treatment induced increased expression of MHC molecules.

[0125] Example 6: Induction of Transcription Factor Spi-B Expression and T Cell Activation by LMP1 6.1 (Figure 6A) Transcription factors whose expression is induced by LMP1 were investigated by RNA sequencing. Total RNA was extracted from LMP1 B16 cells cultured for 24 hours after gene expression was performed with 1 μg / ml of doxycycline. A library was prepared from the extracted RNA using the rRNA depletion method and sequenced using DNBSEQ (BGI). Transcription factors were extracted using the GO term DNA-binding transcription factor activity (GO:0003700), and genes whose expression was increased by LMP1 were examined. As a result, the Spib gene, which is characteristically expressed in immune cells that exhibit antigen-presenting function, showed particularly high expression levels upon LMP1 expression.

[0126] 6.2 (Figure 6B) Two sgRNAs targeting the Spib gene were cloned into pX330 t2a mCherry plasmid, and 2 μg of this plasmid was transfected into LMP1 B16 using 6 μl of Turbofect (Thermo Fisher Scientific). 24 hours after plasmid transfection, mCherry-positive cells were sorted to obtain knockout cells. When these cells were co-cultured with T cells, T cell proliferation was suppressed. These results demonstrate that gene expression regulation by Spi-B is involved in the immunogenicity enhancement effect of LMP1.

[0127] Example 7 Increased MHC class I expression in B16F10 cells using LMP1 mRNA 7.1 (A in Figure 7) The LMP1 t2a GFP sequence was cloned into the Cloning Kit for mRNA Template (Takara) to prepare a plasmid for mRNA synthesis. For mRNA synthesis, the plasmid was linearized by cleavage with the restriction enzyme Hind III, and reverse transcription was performed using the Takara IVT pro™ T7 mRNA Synthesis Kit (Takara) with the addition of modified bases N1-methyl pseudouridine (Tokyo Chemical Industry Co., Ltd.) and Clean Cap M6 (Trilink).

[0128] 7.2 (Figure 7B) Transfection and cell culture. 2 μg of synthesized mRNA was encapsulated in 10 μl of Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) and transfected into B16F10 cells. After 4 hours, the supernatant was replaced, and the cells were cultured for an additional 24 hours. Increased expression of MHC class I molecules was confirmed by flow cytometry analysis.

[0129] Example 8: Intratumoral administration of LMP1 mRNA-LNP inhibits proliferation of murine B16F10 tumor cells 8.1 (Figure 8A) LMP1 mRNA-LNP was prepared by dissolving the lipid SGL0806 (Sougo Pharmaceutical Co., Ltd.), DPPC (Nippon Fine Chemicals Co., Ltd.), DMG PEG2000, and cholesterol in 99.5% ethanol and LMP1 mRNA in 1 mM citrate buffer, and then simultaneously introducing these into a microchannel. The prepared mRNA-LNP was concentrated using an Amicon Ultra15 (Merck) and adjusted to 100 μg / ml with RNase-free water and sucrose. B16F10 cells were subcutaneously implanted in this solution, and 50 μl of this solution was administered into the tumors of mice 7 days later. The long and short diameters of the tumor were measured using digital calipers, and the long diameter x (short diameter) was calculated. 2 The volume was calculated using a 2×100 / 2000 filter and proliferation was examined.

[0130] 8.2 (Figure 8B) Tumor cells were isolated 12 hours after administration of LMP1 mRNA-LNP, and LMP1 protein expression was confirmed by flow cytometry analysis.

[0131] 8.3 (Figure 8C) B16 cells were transplanted into C57BL / 6 wild-type mice, and LMP1-mRNA-LNP was administered intratumorally on days 7, 10, and 13 after transplantation. Tumor growth was monitored by measuring the long and short diameters with a digital caliper and calculating the volume up to day 15, and it was confirmed that LMP1 mRNA suppressed tumor growth.

[0132] Example 9 Identification of IRF1, a transcription factor that regulates MHC expression 9.1 (Fig. 9A) The immune-related transcription factors IRF1, IRF9, STAT1, and STAT2, whose expression is significantly elevated by LMP1 gene transfection, were identified as candidates for MHC regulatory transcription factors. These transcription factors were transfected individually or in combination into Spi-B-expressing B16F10 cells using a lentiviral vector, and MHC class I expression was evaluated by flow cytometry.

[0133] 9.2 (Fig. 9B) Expression of MHC class I molecules was assessed by flow cytometry 56 hours after lentiviral vector infection. Expression of these molecules was significantly elevated in combination with transcription factors including IRF1, and was most strongly induced by IRF1 alone. These results identify IRF1 as a regulator of LMP1-induced MHC class I expression.

[0134] Example 10: Verification of T cell activity by Spi-B / IRF1 cotransfection 10.1 (Fig. 10A) Spi-B and IRF1 were transfected singly or in combination into B16F10 cells, and the expression of MHC class I and CD80 molecules was examined by flow cytometry. As a result, the expression of both molecules was elevated in cells cotransfected with Spi-B and IRF1. Furthermore, the expression level of MHC class I molecules was elevated in cells cotransfected with Spi-B and IRF1 compared to cells transfected with IRF1 alone.

[0135] 10.2 (Fig. 10B) B16F10 cells transfected with transcription factors and CD8 + T cells were co-cultured and T cell proliferation was examined using the cell division detection reagent CellTrace. As a result, the CellTrace signal was significantly reduced in Spi-B / IRF1 co-transfected B16F10 cells compared to control cells. These results suggest that cancer cells co-transfected with the Spib / Irf1 gene are CD8 + Furthermore, this T cell activation was significantly enhanced compared to cancer cells transfected with either Spi-B or IRF1 alone, demonstrating that these transcription factors mutually enhance the T cell activation ability.

[0136] Example 11: Induction of T cell immune surveillance by Spib / Irf1 gene transfer 11.1 (Fig. 11A) B16F10 cells transfected with the transcription factors Spi-B / IRF1 or mock lentiviral vector were mixed with wild-type cells at a 1:1 ratio and subcutaneously implanted into the right flank of C57BL / 6 mice. After 10 days, tumors were excised and the weight and characteristics of tumor-infiltrating immune cells were examined.

[0137] 11.2 (FIG. 11B) When the tumor weight was examined 10 days after subcutaneous implantation in mice, the weight was significantly reduced in the tumors containing a mixture of Spi-B / IRF1-transfected cells compared to the control cells.

[0138] 11.3 (Figure 11C) Flow cytometry analysis of tumor cells revealed a significant increase in the proportion of immune cells expressing CD45.2 in tumors containing Spi-B / IRF1-transduced cancer cells. Furthermore, among these immune cells, CD8 + The proportion of T cells significantly increased. Furthermore, these T cells were cytotoxic T cells (CTLs) that express granzyme B, a cytotoxic granule required for eliminating cancer cells. These results suggest that Spib / Irf1 gene transfer increases the cytotoxic CD8 T cells in tumors. + It has been shown to increase T cells and induce immune surveillance.

[0139] As described above, we generated solid cancer cell lines expressing LMP1 in a doxycycline-inducible manner using lentiviral vectors, and confirmed that LMP1-expressing cancer cells upregulated the expression of MHC class I, costimulatory molecules CD80, ICOSL, CD112, CD155, ICAM-1, etc. Furthermore, LMP1-expressing cancer cells upregulated the expression of CD8 + Activated T cells. Cytotoxic CD8 + We confirmed that T cells recognize tumor antigens. Furthermore, we confirmed that LMP1-mediated immunogenicity enhancement is primarily mediated by NF-κB signaling, and that it can induce MHC expression even in cancer cells that have lost IFN-γ responsiveness. Furthermore, we confirmed that LMP1-mediated transformation of cancer cells into antigen-presenting cell-like cells involves the induction of expression of the transcription factors Spi-B and IRF1. Furthermore, we confirmed that LMP1 gene transfer into cancer cells via mRNA can induce MHC class I expression. Furthermore, in a cancer xenograft model, we confirmed the antitumor effect of intratumoral administration of LMP1 mRNA-LNP in vivo. Furthermore, we confirmed that Spib and Irf1 gene transfer into cancer cells via mRNA can induce MHC class I expression and activate T cells. Furthermore, in a cancer xenograft model, we confirmed the antitumor effect of intratumoral administration of Spib and Irf1 mRNA in vivo. These findings demonstrate that antitumor immunity can be induced by introducing virus-derived genes or genes induced by these virus-derived genes into cancer cells and expressing the corresponding proteins.

[0140] The present invention can be used in the fields of cancer treatment for humans and animals (human medical field, veterinary medical field), cell therapy field, cancer prevention for immunocompromised patients, pharmaceutical field, etc.

[0141] <Explanation of the sequences> SEQ ID NO: 1: DNA (ORF) sequence of human gammaherpesvirus 4 gene SEQ ID NO: 2: Amino acid sequence of human gammaherpesvirus 4 protein SEQ ID NO: 3: DNA (ORF) sequence of human Spib gene SEQ ID NO: 4: Amino acid sequence of human Spi-B protein SEQ ID NO: 5: DNA (ORF) sequence of mouse Spib gene SEQ ID NO: 6: Amino acid sequence of mouse Spi-B protein SEQ ID NO: 7: DNA (ORF) sequence of human Irf1 (interferon regulatory factor-1) gene SEQ ID NO: 8: Amino acid sequence of human IRF1 protein SEQ ID NO: 9: DNA (ORF) sequence of mouse Irf1 gene SEQ ID NO: 10: Amino acid sequence of mouse IRF1 protein

Claims

1. A method for inducing anti-tumor immunity, which comprises introducing into cancer cells a virus-derived gene or a gene whose expression is induced by said virus-derived gene.

2. The method of claim 1, wherein the cancer cells are non-B cell cancer cells.

3. The method according to claim 1, wherein the virus-derived gene is an EB virus gene.

4. The method of claim 3, wherein the EB virus gene is LMP1.

5. The method of claim 1, wherein the virus-derived gene is selected from the group consisting of: (A) a gene comprising the base sequence set forth in SEQ ID NO: 1; (B) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (C) a gene encoding a protein comprising an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 2 with substitution, deletion, insertion, and / or addition of one or more amino acid residues, said protein having the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (D) a gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 2, said protein having the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO:

2.

6. The method according to claim 1, wherein the gene whose expression is induced is Spib and / or Irf1.

7. The method of claim 1, wherein the gene whose expression is induced is selected from the group consisting of: (E) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 5; (F) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (G) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6, wherein the amino acid sequence comprises substitution, deletion, insertion, and / or addition of one or several amino acid residues, said protein having the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (H) a gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 4 or 6, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (I) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 7 or 9; (J) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; (K) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10, wherein the amino acid sequence comprises substitution, deletion, insertion, and / or addition of one or several amino acid residues, said protein having the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; (L) A gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence shown in SEQ ID NO: 8 or 10, wherein the protein has the activity of a protein comprising the amino acid sequence shown in SEQ ID NO: 8 or 10.

8. A composition for inducing anti-tumor immunity by introducing into cancer cells the virus-derived gene or the gene whose expression is induced by the virus-derived gene, the composition comprising a nucleic acid having the sequence of the virus-derived gene or the gene whose expression is induced by the virus-derived gene.

9. The composition of claim 8, wherein the cancer cells are non-B cell cancer cells.

10. The composition according to claim 8, wherein the virus-derived gene is an EB virus gene.

11. The composition of claim 10, wherein the EB virus gene is LMP1.

12. The composition of claim 8, wherein the virus-derived gene is selected from the group consisting of: (A) a gene comprising the base sequence set forth in SEQ ID NO: 1; (B) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (C) a gene encoding a protein comprising an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 2 with substitution, deletion, insertion, and / or addition of one or more amino acid residues, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (D) a gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 2, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO:

2.

13. The composition according to claim 8, wherein the gene whose expression is induced is Spib and / or Irf1.

14. The composition according to claim 8, wherein the gene whose expression is induced is selected from the group consisting of: (E) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 5; (F) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (G) a gene encoding a protein comprising an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6, but with substitution, deletion, insertion, and / or addition of one or more amino acid residues, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (H) a gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 4 or 6, wherein the protein has the activity of a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6; (I) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 7 or 9; (J) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; (K) A gene encoding a protein comprising an amino acid sequence containing the substitution, deletion, insertion, and / or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 8 or 10, wherein the protein has the activity of a protein comprising the amino acid sequence shown in SEQ ID NO: 8 or 10; (L) A gene encoding a protein comprising an amino acid sequence having 80% or more identity to the entire amino acid sequence shown in SEQ ID NO: 8 or 10, wherein the protein has the activity of a protein comprising the amino acid sequence shown in SEQ ID NO: 8 or 10.

15. The composition according to claim 8, wherein the nucleic acid comprising the sequence of the virus-derived gene or the sequence of the gene whose expression is induced by the virus-derived gene is mRNA.

16. The composition of claim 8, wherein the composition further comprises lipid nanoparticles.

17. The composition described in claim 16, comprising an effective amount of mRNA containing the sequence of the virus-derived gene or a gene whose expression is induced by the virus-derived gene to confer antigen-presenting cell-like functions to the traits of cancer cells, and lipid nanoparticles.

18. The composition of claim 16, wherein the lipid nanoparticles comprise, by molar ratio based on the total lipid content in the lipid nanoparticles, 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

19. A method for treating or preventing cancer, comprising administering to a subject an effective amount of the composition according to any one of claims 8 to 18.

20. The method of claim 19, wherein the cancer is a solid cancer or a liquid cancer.

21. The method of claim 20, wherein the solid cancer is one or more selected from the group consisting of nasopharyngeal cancer, gastric cancer, lung cancer, cervical cancer, ovarian cancer, glioblastoma, colorectal cancer, pancreatic cancer, and malignant melanoma.

22. The method of claim 20, wherein the liquid cancer is one or more selected from the group consisting of Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-related B-cell lymphoma, multiple myeloma, central nervous system B-cell lymphoma, post-transplant lymphoproliferative disorder (PTLD), T-cell lymphoma, and chronic lymphocytic leukemia of B-cell origin.

23. The method of claim 19, wherein the administration form of the composition is one or more selected from the group consisting of intratumoral administration, intravenous administration, intraarterial administration, intramuscular administration, intraperitoneal administration, intrathecal administration, epidural administration, subcutaneous administration, intradermal administration, nasal administration, pulmonary administration, and oral administration.

24. The method according to claim 19, wherein the effective amount is 0.1 μg / kg (body weight) to 1000 mg / kg (body weight) of a nucleic acid comprising the sequence of a virus-derived gene or a gene whose expression is induced by said virus-derived gene.

25. The method of claim 19, wherein the composition is administered to a subject once a day, twice a day, three times a day, once a week, twice a week, three times a week, four times a week, five times a week, once a month, twice a month, three times a month, four times a month, five times a month, six times a month, seven times a month, eight times a month, nine times a month, or ten times a month for one day to several years.

26. The method of claim 19, wherein the composition is administered to the subject for a period of from 1 day to 3 years.

27. The method of claim 19, comprising administering the method in combination with one or more additional therapeutic agents or therapies selected from the group consisting of surgery, radiation, chemotherapy, molecularly targeted drugs, immunotherapy, hormone therapy, CAR-T cell therapy, and cancer vaccines.

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