Modified cancer cell and cancer vaccine composition containing same

Abstract

The purpose of the present invention is to provide a cancer vaccine composition effective for treating or preventing cancer. The present invention provides a genetically modified cancer cell comprising: a nucleic acid encoding a cytotoxic T cell-inducing antigen and a nucleic acid encoding IL-2. This genetically modified cancer cell can be used as an active ingredient of a cancer vaccine composition. The cytotoxic T cell-inducing antigen in this genetically modified cancer cell is preferably a pathogen-derived antigen or a virus-derived antigen.

Description

Modified cancer cells and cancer vaccine compositions containing the same REFERENCE TO RELATED APPLICATIONS

[0001] This application benefits from the priority of an earlier Japanese application, Patent Application No. 2024-76029 (filing date: May 8, 2024), the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to modified cancer cells and cancer vaccine compositions comprising the same.

[0003] Anti-PD-1 antibodies that inhibit the immune checkpoint, one of the immune evasion mechanisms of cancer, have been developed and are expected to be effective against more than 20 types of cancer. However, only a little over 20% of patients respond to anti-PD-1 antibodies, and only a few percent of cases achieve complete remission (Non-Patent Document 1).

[0004] Against this background, technologies have been developed to circumvent the immune escape mechanism of cancer and induce anti-cancer cytotoxic T cells (CTLs). For example, a technology to specifically increase MHC class I molecules has been developed as such a technology (Non-Patent Document 2). However, no recombinant cancer cell vaccine formulations have been known to date that express antigens derived from foreign pathogenic microorganisms that induce sensitized CTLs against peptides derived from unidentified neoantigens to attack cancer cells.

[0005] Peng S, et al., Sci Rep, 9:4278(2020)Proc. Natl. Acad. Sci. USA 121(6) e2310821121(2024)

[0006] An object of the present invention is to provide modified cancer cells that are useful for treating or preventing cancer.

[0007] The present inventors have now found that when modified mouse cancer cells expressing a CTL-inducing antigen are administered to mice, the administered modified cancer cells disappear, that when cancer cells are transplanted into mice that have already lost the administered modified cancer cells, the cancer cells disappear, and that similar results are obtained when modified cancer cells treated with an antitumor agent are administered.The present inventors have also found that similar results are obtained when HLA-expressing cancer cells are used as the cancer cells and HLA-expressing knock-in mice are used as the recipient mice.The present invention is based on these findings.

[0008] The present invention provides the following inventions. [1] Genetically modified cancer cells comprising a nucleic acid encoding a cytotoxic T cell-inducing antigen and a nucleic acid encoding IL-2. [2] Genetically modified cancer cells according to [1] above, wherein the cytotoxic T cell-inducing antigen is an antigen derived from a pathogenic bacterium or virus. [3] Genetically modified cancer cells according to [1] or [2] above, comprising the nucleic acid encoding the cytotoxic T cell-inducing antigen and the nucleic acid encoding IL-2 in the form of an expression vector. [4] Genetically modified cancer cells according to any of [1] to [3] above, wherein the nucleic acid encoding the cytotoxic T cell-inducing antigen and the nucleic acid encoding IL-2 are operably linked to a promoter having transcriptional activity in cancer cells. [5] Genetically modified cancer cells according to any of [1] to [4] above, derived from a subject suffering from cancer. [6] Genetically modified cancer cells according to any of [1] to [5] above, wherein the proliferation rate after 24 hours is suppressed to 95% or less. [7] A method for producing genetically modified cancer cells, comprising the steps of: (1) introducing a nucleic acid encoding a cytotoxic T cell (CTL)-inducing antigen and a nucleic acid encoding IL-2 into cancer cells isolated from a subject suffering from cancer, thereby obtaining modified cancer cells expressing the CTL-inducing antigen and IL-2. [8] The production method according to [7] above, further comprising the step of (2) contacting the modified cancer cells with an anticancer drug. [9] A cancer vaccine composition comprising the genetically modified cancer cells according to any of [1] to [6] above, or the genetically modified cancer cells produced by the production method according to [7] or [8] above.

[10] The cancer vaccine composition according to [9] above, for use in treating or preventing cancer.

[11] The cancer vaccine composition according to [9] or

[10] above, for treating metastatic and / or recurrent cancer, or for preventing metastasis and / or recurrence of cancer.

[12] The cancer vaccine composition according to any of [9] to

[11] above, for preventing metastasis and / or recurrence of cancer after surgical therapy.

[13] A combination pharmaceutical comprising the cancer vaccine composition according to any one of [9] to

[12] above and another drug.

[14] The combination pharmaceutical according to

[13] above, wherein the other drug is an immunostimulant (preferably an anti-CTLA-4 antibody preparation).

[15] A method for treating or preventing cancer, comprising administering to a subject suffering from cancer in need thereof the genetically modified cancer cells described in any of [1] to [6] above, the genetically modified cancer cells produced by the production method described in [7] or [8] above, or the cancer vaccine composition described in any of [9] to

[12] above.

[16] The method for treating or preventing cancer described in

[15] above, in which the genetically modified cancer cells or the cancer vaccine composition are administered to the subject once or multiple times.

[17] The method for treating or preventing cancer described in

[15] or

[16] above, in which the administration is subcutaneous, intradermal, or topical to the subject.

[18] The method for treating or preventing cancer described in any of

[15] to

[17] above, in which another drug is administered simultaneously or sequentially.

[19] The method for treating or preventing cancer described in

[18] above, in which the other drug is an immunostimulant.

[20] The method for treating or preventing cancer described in

[19] above, in which the immunostimulant is an anti-CTLA-4 antibody preparation.

[0009] According to the present invention, modified cancer cells are provided that induce sensitized cytotoxic T cells against neoantigen-derived peptides and cause them to attack cancer cells. The modified cancer cells of the present invention and cancer vaccine compositions containing the same are advantageous in that they can induce tumor immunity even in subjects in whom sufficient cytotoxic immunity against cancer is not induced.

[0010] FIG. 1 shows the timing of transplantation of etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells) and antigen-non-expressing cancer cells (Colon26-Luc cells) into experimental animals and the timing of IVIS imaging (see Example 1 (1-3)). FIG. 2 shows the sequences targeted for disruption by the CRISPR-Cas9 method in the production of LLC-Luc cells in which the β2M gene has been disrupted (Δmβ2M LLC-Luc cells) (see Example 2 (2-1) B). The boxed sequences are the sequences targeted for disruption. The underlined sequences are protospacer adjacent motifs (PAM). FIG. 3 shows the enrichment of β2M-negative cells together with the results of flow cytometry analysis (see Example 2 (2-1) B). Cell groups transfected with the CRISPR-Cas9 / Exon1, Exon2 plasmid were enriched in negative fractions by H2Db-PE staining. Figure 4 shows the results of flow cytometry analysis of mouse MHC class I expression in wild-type LLC-Luc cells and β2M gene knockout (Δmβ2M) LLC-Luc cells (see Example 2 (2-1) c). Figure 5(A) shows the construction procedure for the human HLA-A2 expression unit DNA used to generate HLA-expressing Δmβ2M LLC-Luc cells (see Example 2 (2-2)). Figure 5(B) is a schematic diagram of a chimeric MHC class I molecule in which the mouse α1 and α2 domains and mouse β2M in the mouse MHC class I molecule are replaced with human α1 and α2 domains and human β2M, respectively, and its intracellular behavior. Figure 6 shows the results of flow cytometry analysis of HLA expression on the cell surface of the HLA-expressing cancer cells prepared in Example 2. Figure 7 shows the results of Western blotting analysis of HLA expression on the HLA-expressing cancer cells prepared in Example 2.

[0011] <<Genetically Modified Cancer Cells>> The genetically modified cancer cells of the present invention are cancer cells modified by gene transfer manipulation, and comprise a nucleic acid encoding a cytotoxic T cell-inducing antigen and a nucleic acid encoding IL-2 in an expressible manner. That is, the genetically modified cancer cells of the present invention are cancer cells into which a nucleic acid encoding a cytotoxic T cell-inducing antigen and a nucleic acid encoding IL-2 have been introduced. Cancer cells to be subjected to gene transfer manipulation can be, for example, cancer cells derived from a subject suffering from cancer. Further, non-limiting examples of cancer cells include cancer cells derived from cancer tissue surgically removed from a subject suffering from cancer. The genetically modified cancer cells of the present invention may be isolated or purified cells.

[0012] Non-limiting examples of cancer include solid cancers (e.g., colon cancer, lung cancer, mesothelioma, pancreatic cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, breast cancer, ovarian cancer, testicular tumor, prostate cancer, liver cancer, thyroid cancer, kidney cancer, uterine cancer, brain tumor, retinoblastoma, skin cancer, sarcoma, malignant bone tumor, bladder cancer), and blood cancers (e.g., leukemias such as acute myeloid leukemia and acute lymphocytic leukemia, and multiple myeloma).

[0013] The cytotoxic T cell-inducing antigen to be expressed in the modified cancer cells is not particularly limited as long as it is an antigen capable of inducing cytotoxic T cells in a mammal, and can be selected, for example, from antigens derived from pathogenic bacteria or viruses (preferably pathogenic viruses). Antigens capable of inducing cytotoxic T cells in mammals are well known to those skilled in the art, and include, for example, antigens that can be presented by MHC class I or HLA class I. Non-limiting examples of antigens capable of inducing cytotoxic T cells include antigens derived from Mycobacterium tuberculosis (e.g., 85A antigen, 85B antigen, MPB51 antigen, Mtb72f antigen) and antigens derived from the novel coronavirus (e.g., spike antigen, nucleocapsid protein antigen, membrane protein antigen). The amino acid sequences of these antigens and the nucleotide sequences encoding them are available in public databases. The base sequence and amino acid sequence of the 85A antigen can also be the base sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6, and the base sequence and amino acid sequence of the Spike antigen can also be the base sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8, respectively.

[0014] The IL-2 expressed in the modified cancer cells is mammalian IL-2, and when the modified cancer cells are human, human IL-2 can be preferably used. The amino acid sequence of wild-type IL-2 and the nucleotide sequence encoding it are known. The nucleotide sequence and amino acid sequence of human wild-type IL-2 registered in the National Library of Medicine under ACCESSION: BC066257 can be used, and the nucleotide sequence and amino acid sequence of mouse wild-type IL-2 registered in the National Library of Medicine under ACCESSION: NM_008366 can be used. The nucleotide sequence and amino acid sequence of human wild-type IL-2 can also be the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4. The nucleotide sequence and amino acid sequence of mouse wild-type IL-2 can also be the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.

[0015] The IL-2 expressed in the modified cancer cells may be, in addition to wild-type IL-2, modified IL-2 in which several (e.g., 1 to 10, 1 to 6, 1 to 4, 1 to 3, or 1 or 2) amino acids have been substituted, inserted, added, and / or deleted, and which has the immune response-modulating function exhibited by wild-type IL-2, and a nucleic acid encoding the same. The modified IL-2 may also consist of an amino acid sequence that is 80% or more identical (preferably, 85% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98% or more, or 99% or more) to the amino acid sequence of wild-type IL-2. As used herein, "identity" refers to the degree of identity when the sequences to be compared are appropriately aligned, and refers to the percentage of exact amino acid matches between the sequences, for example. Identity is determined by taking into consideration, for example, the presence of gaps in the sequences and the nature of the amino acids (Wilbur, Natl. Acad. Sci. USA 80:726-730 (1983)). The alignment can be performed, for example, using any algorithm, and specifically, homology search software such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Peasron et al., Methods in Enzymology 183:63-69 (1990)), and Smith-Waterman (Meth. Enzym., 164, 765 (1988)) can be used. Furthermore, identity can be calculated using, for example, a known homology search program such as those described above, for example, by using the default parameters in the homology algorithm BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information (NCBI).

[0016] In the present invention, the nucleotide sequences encoding the antigen and IL-2 to be expressed in the modified cancer cells may be codon-optimized to enable expression in the cancer cells and / or to increase the amount of expression in the cancer cells. Codon optimization is well known to those skilled in the art and can be performed by known methods commonly used in this field.

[0017] Introduction of nucleic acids encoding cytotoxic T cell-inducing antigens and nucleic acids encoding IL-2 into cancer cells can be achieved by gene transfer manipulation. Gene transfer manipulation of cancer cells can be achieved by any of chemical, physical, and biological methods. Chemical methods include transfection using cationic polymers, cationic lipids, calcium phosphate, etc. Physical methods include electroporation, microinjection, etc. Biological methods include techniques using viral vectors. The nucleic acids to be introduced into cancer cells can be in the form of recombinant nucleic acid constructs to ensure stable expression in cancer cells, and expression vectors such as plasmids or viral vectors can be used. When a plasmid is used as the expression vector, gene transfer into cancer cells can be achieved by chemical methods or physical methods such as electroporation. When a viral vector is used as the expression vector, gene transfer into cancer cells can be achieved by infecting cancer cells with viral particles. Viral vectors that can be used include, but are not limited to, retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Expression vectors, reagents, and devices used for gene transfer are well known to those skilled in the art, and commercially available ones can be used. Gene transfer into cancer cells can be carried out in vitro or ex vivo.

[0018] The nucleic acid molecules encoding the cytotoxic T cell-inducing antigen and IL-2 can be operably linked to a promoter having transcriptional activity in cancer cells so as to be expressed in the cancer cells into which they are introduced. Promoters having transcriptional activity in cancer cells are well known to those skilled in the art, and an appropriate one can be selected for the present invention. For example, a promoter having cancer cell-specific transcriptional activity can be used. Non-limiting examples of promoters having cancer cell-specific transcriptional activity include the TERT promoter, the AFP promoter (liver cancer-specific), the CEA promoter (pancreatic cancer and colorectal cancer-specific), and the PSA promoter (prostate cancer-specific). Furthermore, the design and construction of expression vectors containing a nucleic acid encoding a cytotoxic T cell-inducing antigen and a nucleic acid encoding IL-2 can be carried out according to methods well known to those skilled in the art.

[0019] In the present invention, nucleic acids include DNA and RNA, as well as modified forms thereof and artificial nucleic acids, but DNA is preferred. DNA also includes cDNA, genomic DNA, and chemically synthesized DNA. Note that the terms nucleic acid (molecule) and polynucleotide can be used interchangeably.

[0020] When the modified cancer cells are used as a cancer treatment vaccine (described later), viable modified cancer cells are preferred from the viewpoint of efficacy, but from the viewpoint of safety, modified cancer cells whose proliferation ability is inhibited are desirable. Examples of means for inhibiting the proliferation ability of modified cancer cells include contact with an anticancer drug and irradiation. Non-limiting examples of anticancer drugs include those that inhibit cell proliferation and exhibit antitumor effects, such as etoposide, mitoxantrone, docetaxel, and mitomycin C.

[0021] The type and concentration of the anticancer agent to be contacted with the modified cancer cells can be determined based on the proliferation rate of the cancer cells. For example, the modified cancer cells can be contacted with a certain concentration of the anticancer agent, and the anticancer agent can be contacted with the modified cancer cells at a concentration such that the proliferation rate (%) after 24 hours is 60 to 95%, preferably 70 to 90% (see Example 1(1-1)K). The proliferation rate (%) after 24 hours indicates the ratio of the number of surviving cancer cells 24 hours after treatment to the number of surviving cancer cells before treatment (i.e., the number of surviving untreated cancer cells).

[0022] Another aspect of the present invention provides a method for producing modified cancer cells, comprising the steps of: (1) introducing a nucleic acid encoding a cytotoxic T-cell-inducing antigen and a nucleic acid encoding IL-2 into cancer cells isolated from a subject suffering from cancer, thereby obtaining modified cancer cells expressing the cytotoxic T-cell-inducing antigen and IL-2. The production method of the present invention may further comprise, after step (1), (2) a step of immersing the modified cancer cells in an anticancer agent. The production method of the present invention may also further comprise a step of isolating cancer cells from a subject suffering from cancer prior to step (1). The production method of the present invention can be carried out according to the description of the modified cancer cells of the present invention. When modified cancer cells are produced using cancer cells isolated from a subject suffering from cancer, the modified cancer cells can be referred to as autologous gene-modified cancer cells. The production method of the present invention may further comprise administering the modified cancer cells obtained by step (1) or (2) to a subject suffering from cancer in need thereof (preferably the subject from which the cancer cells were isolated). In this case, the production method of the present invention can be referred to as a method for treating or preventing cancer.

[0023] <<Cancer Vaccine>> The present invention provides a cancer vaccine composition comprising the modified cancer cells of the present invention. The present invention also provides a pharmaceutical composition for treating or preventing cancer comprising the modified cancer cells of the present invention. The modified cancer cells of the present invention also include modified cancer cells produced by the production method of the present invention (the same applies hereinafter). Because the cancer vaccine composition of the present invention contains the modified cancer cells as an active ingredient, in the present invention, the term "cancer vaccine composition" is used interchangeably with "cancer vaccine cell preparation."

[0024] As shown in the Examples below, administration of the modified cancer cells of the present invention to a subject induces cytotoxic T cell-mediated antitumor immunity in the subject. Without being bound by the following theory, it is believed that this is because the modified cancer cells of the present invention induce cytotoxic immunity against antigens (neoantigens) specific to the cancer cells. Therefore, in the present invention, the term "cancer vaccine composition" is used interchangeably with "cancer immunotherapy formulation." Here, "cancer immunotherapy" refers to a method for treating or preventing cancer by immunologically identifying cancer cells present in the body as foreign substances and activating or proliferating immune cells involved in their elimination. In other words, the cancer vaccine composition of the present invention can be used for the treatment and / or prevention of cancer.

[0025] In cancer surgical therapy, it may not be easy to remove all cancer cells by surgery. The cancer vaccine composition of the present invention is advantageous in that it can remove cancer cells that were not removed in such cases. Furthermore, cancer surgical therapy may have a certain probability of causing cancer metastasis and / or recurrence. The cancer vaccine composition of the present invention is also advantageous in that it can prevent such postoperative cancer metastasis and / or recurrence. That is, the cancer vaccine composition of the present invention can preferably be used for treating metastatic and / or recurrent cancer or for preventing cancer metastasis and / or recurrence, and more preferably for preventing cancer metastasis and / or recurrence after surgical therapy. According to the present invention, there are provided compositions for preventing postoperative cancer metastasis and / or recurrence in cancer patients and compositions for treating metastatic and / or recurrent cancer in cancer patients, each comprising the modified cancer cells of the present invention or the cancer vaccine composition of the present invention as an active ingredient. These compositions can be provided as pharmaceuticals.

[0026] In the present invention, "treatment" includes, but is not limited to, therapeutic benefit, and is used to mean measures to obtain beneficial or desired results. Furthermore, therapeutic benefit means complete cure or amelioration of the disease being treated. In the present invention, "prevention" is used to mean reducing the probability of contracting a disease or the probability of disease recurrence. In the present invention, "treatment" and "prevention" are not limited to complete treatment and prevention, but may be treatment and prevention to the extent that a person skilled in the art recognizes as having potential therapeutic and preventive effects (e.g., delaying the progression of a disease, inhibiting the worsening of a disease, delaying the onset of a disease).

[0027] In the present invention, the "subject" refers to a mammal, including a human, and examples of non-human mammals include mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, and monkeys.

[0028] The cancer vaccine composition of the present invention can be used in combination with other drugs. Here, "combination" means administering multiple active ingredients simultaneously or separately to the same subject for treatment or prevention. In combination, the multiple active ingredients may be contained in the same composition, or may be contained separately in different compositions. Examples of other drugs that can be used in combination with the cancer vaccine composition of the present invention include immunostimulants. Examples of immunostimulants include antibody preparations such as anti-CTLA-4 antibodies, anti-PD-1 antibodies, or anti-PD-L1 antibodies.

[0029] When the cancer vaccine composition of the present invention is used in combination with another drug, the two may be administered simultaneously or sequentially. When the cancer vaccine composition of the present invention and the other drug are administered sequentially, they may be administered continuously or at intervals. When administered sequentially, the order of administration is not particularly limited, but when an immunostimulant is used in combination, it is preferable to administer the cancer vaccine composition of the present invention first from the viewpoint of inducing killer T cells against neoantigens.

[0030] Another aspect of the present invention provides a combination drug comprising the cancer vaccine composition of the present invention and another drug. Here, the term "combination drug" refers to a combination of different compositions each containing multiple active ingredients separately. The combination drug of the present invention can be used for the treatment and / or prevention of cancer. The combination drug of the present invention can also be implemented as described for the cancer vaccine composition.

[0031] The administration route of the cancer vaccine composition of the present invention can be selected from routes suitable for administering the cell preparation, and examples thereof include subcutaneous administration, intradermal administration, topical administration, intraperitoneal administration, intravenous administration, and intramuscular administration. Non-limiting examples of administration sites for subcutaneous or intradermal administration include the back, arm, thigh, etc. As described below, when administering multiple times, the compositions may be administered at the same site or at different sites. Furthermore, examples of administration sites for local administration include the site where cancer has occurred or its vicinity, and the site where cancer is expected to occur, recur, or metastasize or its vicinity.

[0032] Dosage forms suitable for administering the cancer vaccine composition of the present invention include, for example, injections. These preparations can be formulated using pharmaceutically acceptable carriers by methods commonly used in the art (e.g., known methods described in the General Provisions for Preparations of the Japanese Pharmacopoeia, 18th Edition, etc.). Pharmaceutically acceptable carriers include, for example, excipients, binders, diluents, stabilizers, buffers, colorants, emulsifiers, dispersants, suspending agents, preservatives, soothing agents, etc. The cancer vaccine composition of the present invention may be in a form that is dissolved or suspended in water or other suitable solvents prior to use.

[0033] When the cancer vaccine composition of the present invention is administered to a human, the dose can be determined depending on the sex, age, and weight of the recipient, symptoms, dosage form, and administration route. The dose of the cancer vaccine composition of the present invention administered to an adult is, for example, 1.0 × 10 modified cancer cells of the present invention. ６ ~1.0 x 10 １１ pieces (preferably 1.0 x 10 ９ ~1.0 x 10 １０For treatment or prevention, the above-mentioned dose of the active ingredient can be administered in multiple divided doses.

[0034] The cancer vaccine composition of the present invention can be administered multiple times (e.g., 2 to 4 times, preferably 2 to 3 times) to enhance the therapeutic or preventive effect. When administered multiple times, the interval between administrations can be, for example, 1, 2, 3, 4, 5, or 6 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or 1, 2, 3, 4, 5, or 6 months, but is not limited to these.

[0035] The modified cancer cells or cancer vaccine compositions of the present invention can be administered to subjects at risk of developing cancer, thereby reducing the risk of developing cancer. Here, "subjects at risk of developing cancer" refers to subjects who have no subjective symptoms of cancer or have not been diagnosed with cancer, but who are at risk of developing cancer in the future, such as those who have not been diagnosed with cancer and cancer patients who have undergone surgery to remove cancer tissue. Furthermore, "reducing the risk of developing cancer" means reducing the probability of developing cancer, and reducing the probability of developing cancer improves cancer prognosis.

[0036] That is, according to another aspect of the present invention, there are provided a composition for reducing the risk of developing cancer and a composition for improving prognosis in cancer treatment, each of which comprises the modified cancer cell of the present invention or the cancer vaccine composition of the present invention as an active ingredient. These compositions can be provided as pharmaceuticals.

[0037] Another aspect of the present invention provides a method for treating or preventing cancer, comprising administering a therapeutically or prophylactically effective amount of the modified cancer cells of the present invention or the cancer vaccine composition of the present invention to a subject suffering from cancer in need thereof. The therapeutic or preventive method of the present invention can be carried out according to the description of the modified cancer cells of the present invention and the cancer vaccine composition of the present invention.

[0038] Another aspect of the present invention provides a method for reducing the risk of developing cancer or a method for improving prognosis in cancer treatment, the method comprising administering a therapeutically or prophylactically effective amount of the modified cancer cells of the present invention or the cancer vaccine composition of the present invention to a subject in need thereof. Another aspect of the present invention also provides a method for preventing postoperative cancer metastasis and / or recurrence in a cancer patient or a method for treating metastatic and / or recurrent cancer in a cancer patient, the method comprising administering a therapeutically or prophylactically effective amount of the modified cancer cells of the present invention or the cancer vaccine composition of the present invention to a patient in need thereof. The method for reducing the risk of developing cancer, the method for improving prognosis in cancer treatment, the method for preventing postoperative cancer metastasis and / or recurrence in a cancer patient, and the method for treating metastatic and / or recurrent cancer in a cancer patient can be carried out according to the description of the modified cancer cells of the present invention and the cancer vaccine composition of the present invention.

[0039] Another aspect of the present invention provides use of the modified cancer cells of the present invention for the manufacture of a cancer vaccine composition, a pharmaceutical composition for treating or preventing cancer, a cancer immunotherapy preparation, a composition for reducing the risk of developing cancer, a composition for improving prognosis in cancer treatment, a composition for preventing cancer metastasis and / or recurrence after surgery in cancer patients, or a composition for treating metastatic and / or recurrent cancer in cancer patients. The use of the present invention can be carried out according to the descriptions of the modified cancer cells of the present invention and the cancer vaccine composition of the present invention.

[0040] The present invention will be described in more detail based on the following examples, but is not limited to these examples. Example 1: Analysis of antitumor effect and cancer vaccine effect using cancer cells expressing a CTL-inducing antigen In this example, we investigated whether cancer cells genetically modified to express IL-2 and an antigen that induces cytotoxic T cells against cancer induce antitumor immunity mediated by cytotoxic T cells.

[0041] (1-1) Materials and Methods A. Animal Experiments All experiments using animals in this study were conducted in accordance with the guidelines of the Teikyo Heisei University Animal Welfare Committee (approval number: 01-098). All animals were kept under a 12-hour / 12-hour light / dark cycle, and food and water were available ad libitum. Female CB6F1 mice (6-9 weeks old, weighing 18-25 g) obtained from Japan SLC were inoculated with 1 x 10 ６ Cancer cells were administered by subcutaneous transplantation. The tumor volume was calculated using the following formula: Tumor volume (mm ３ ) = a × b × b × 0.5 (In the above formula, a is the longest diameter, b is the shortest diameter, and 0.5 is a constant used to calculate the volume of the ellipsoid.)

[0042] The endpoint of all experiments was when any animal lost 10% or more of its body weight from the weight measured on the first day of administration, or when the tumor volume reached 1,000 mm ３ The time point was defined as the time point when the tumor formation was observed. After subcutaneous injection, tumor formation was monitored every week. Meth-A cells were diluted with PBS and mixed with Matrigel (Corning, #356234, NY, USA) (1:1) on ice for later use. For Meth-A cells, the mixture of cells and Matrigel was injected subcutaneously using a 1 mL syringe. For cells other than Meth-A cells, the cells were diluted with PBS and injected subcutaneously using a 1 mL syringe.

[0043] B. Plasmid Construction <85A-mIL2 Plasmid> The mIL2-encoding DNA fragment (SEQ ID NO: 1) containing BstXI at the 5' end and NotI at the 3' end was amplified by polymerase chain reaction (PCR) using the mIL2 cDNA ORF clone (Sino Biological) as a template. The amplified mIL2-encoding DNA fragment was substituted for the GFP-encoding DNA in pIRES-AcGFP1 (Takara Bio) to obtain pIRES-mIL2. A DNA fragment encoding the human telomerase promoter (TERTp) (578 bp upstream from the first ATG) linked to 85A (SEQ ID NO: 5) containing EcoRI at the 5' end and NotI at the 3' end (891 bp consisting of the first ATG and stop codon) was chemically synthesized and cloned into pEX plasmid (Eurofin genomics). A DNA fragment encoding TERTp-85A was inserted into pIRES-mIL2 to obtain TERTp-85A / pIRES-mIL2. A DNA fragment encoding TERTp-85A-IRES-mIL2-polyA, containing EcoRI at the 5' end and XhoI at the 3' end, was amplified by PCR using the TERTp-85A / pIRES-mIL2 plasmid as a template. The amplified DNA fragment was inserted into pBluescript SK(+) (Stratagene / Agilent) to obtain the plasmid vector (TERTp-85A-IRES-mIL2).

[0044] <Spike-mIL2 Plasmid> A human telomerase promoter (TERTp) containing a BamHI site at the 5' end and an EcoRI site containing an ATG at the 3' end was used as a template to amplify the plasmid using polymerase chain reaction (PCR). The amplified DNA fragment was inserted into pBluescript SK(+) (Stratagene / Agilent) to obtain the TERTp / pBluescript SK(+) plasmid. A Spike-encoding DNA fragment containing an EcoRI site at the 5' end and a SalI site excluding the ATG at the 3' end was amplified using PCR using a Spike cDNA ORF clone (Sino Biological) as a template. The amplified DNA fragment encoding Spike (SEQ ID NO: 7) was inserted into the TERTp / pBluescript SK(+) plasmid to obtain TERTp-Spike / pBluescript SK(+). A DNA fragment encoding IRES-mIL2-polyA having SalI at the 5' end and XhoI at the 3' end was amplified by PCR using the pIRES-mIL2 plasmid as a template. The amplified DNA fragment was inserted into TERTp-Spike / pBluescript SK(+) to obtain the plasmid vector (TERTp-Spike-IRES-mIL2).

[0045] <Plasmid for mIL2 alone> A DNA fragment encoding TERTp (including the first ATG) having EcoRI at the 5' end and BstXI at the 3' end was amplified by PCR using a chemically synthesized DNA / pEX plasmid as a template. The amplified DNA fragment was inserted into the EcoRI and BstXI cleavage sites of pIRES-mIL2 (see the procedure for constructing the 85A-mIL2 plasmid) to obtain a plasmid vector (plasmid containing mIL2 alone).

[0046] C. Cell culture and transfection Colon26-Luc cells (JCRB1496) were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition. Meth-A cells (TKG 0158) were purchased from the Cell Resource Center, Institute of Development, Aging and Cancer, Tohoku University. LLC-Luc cells (JCRB1716) were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition. Renca cells (CRL-2947) were purchased from the American Type Culture Collection. Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone), 50 U / mL penicillin, and 50 μg / mL streptomycin (Invitrogen) at 5% CO ２ The cells were cultured at 37°C in a humidified atmosphere containing 100 μg / mL hygromycin B (Thermo Fisher Scientific). Cell transfection was performed using Targefect-F1 (Nacalai Tesque) according to the manufacturer's protocol. After 48 hours of culture, the medium was replaced with fresh medium. After 48 hours, the cells were transferred to a 10-cm dish and cultured in medium containing 300 μg / mL hygromycin B (Thermo Fisher Scientific). All cell lines were tested for mycoplasma using a mycoplasma detection kit (Southern Biotech, Birmingham, AL).

[0047] The following antibodies were used: Anti-β-actin (A5316, Sigma, 1:5000 dilution), anti-V5-tag (#R960-25, Invitrogen, 1:2000 dilution), anti-HLA A (EP1395Y, abcam, 1:2000 dilution), anti-HLA class I (W6 / 32, abcam, 1:100 dilution), anti-β2-microglobulin (B1G6, BECKMAN COULTER, 1:100 dilution), goat anti-mouse IgG H&L (FITC) (ab6785, abcam, 1:2000 dilution), PE anti-mouse H-2Ld / H-2Db (28-14-8, BioLegend, 1:100 dilution), APC anti-mouse H-2Kb / H-2Db Antibody (28-8-6, BioLegend, 1:100 dilution), anti-mouse IgG, HRP-conjugated whole sheep antibody (NA931, Cytiva, 1:5000 dilution).

[0048] Measurement of the amount of mIL2 secreted in the supernatant of each cell. Cells were plated in a 12-well plate at 1 × 10 per well. ５ The supernatant was collected 48 hours after cell seeding and measured using an mIL2 ELISA kit (Invitrogen, 88-7024) according to the manufacturer's instructions.

[0049] Luciferin (LK10000, OZ Biosciences), a luciferase substrate, was subcutaneously administered to mice at a dose of 30 mg. Luciferin was administered in a modified D-PBS (WAKOKCl 0.02 w / v%, KH ２ P.O. ４ 0.02w / v%, NaCl 0.8w / v%, Na ２ HPO ４ The luciferin was dissolved in 0.115% (w / v) of 100% ethanol. Mice were then anesthetized with isoflurane and placed in a supine position on the imaging stage of an IVIS device. Images were collected 10 minutes after luciferin injection using an IVIS Imaging System (Caliper Life Sciences, PerkinElmer). Photons emitted from the tumor and its surrounding area were quantified using Living Image software (PerkinElmer).

[0050] Etoposide Treatment: We examined how the proliferation rate of cancer cells changes with etoposide treatment. Specifically, we measured the proliferation rate (%) of cancer cells when Colon26-Luc cells were contacted with etoposide at 10 μM or 50 μM. The results are shown in Table 1.

[0051]

[0052] As is clear from Table 1, it was confirmed that increasing the concentration of the antitumor agent or extending the treatment time with the antitumor agent reduced the proliferation rate of cancer cells. In order to ensure viable cells that could be administered, in the following experiments, cancer cells were treated with 10 μM or 50 μM etoposide for 24 hours.

[0053] Statistical Analysis: In this example, data were expressed as mean values. Treatment groups were compared using a two-tailed Student's t-test. Significance was indicated as *: P<0.05, **: P<0.01, ***: P<0.001.

[0054] (1-2) Verification of the antitumor effect of cancer cells expressing BCG vaccine-derived 85A A. Administration of antigen-expressing cancer cells to experimental animals (1) BCG vaccine-derived 85A was selected as the antigen to be introduced into cancer cells to induce CTL in the body, interleukin-2 (IL2) was linked via an Internal Ribosome Entry Site (IRES), and human telomerase reverse transcriptase (hTERT) was selected as the promoter to express them. The plasmid (TERTp-85A-IRES-mIL2) was constructed according to (1-1) B above. Balb / c mouse-derived colon cancer cells (Colon26-Luc cells) and C57BL / 6 mouse-derived Lewis lung carcinoma cells (LLC-Luc cells), which stably express luciferase (Luc), were selected as cancer cells into which the antigen was introduced. The antigen was introduced into the cancer cells by transfecting the cancer cells with the plasmid constructed as described above according to (1-1) (c) above. The introduction of the antigen into the cancer cells was confirmed by the secretion of IL2 into the cell culture supernatant. The amount of IL2 secreted was measured by ELISA according to (1-1) (e) above. As shown in Table 2, it was confirmed that Colon26-Luc cells expressing 85A and IL2 were established.

[0055]

[0056] Cancer cells transfected with antigen (85A) (85A-mIL2-expressing Colon26-Luc cells) and cancer cells not transfected with antigen (Colon26-Luc cells) were administered to CB6F1 mice, a hybrid of Balb / c and C57BL / 6 mice, and the growth of the cancer cells was analyzed. The tumor volume of the cancer cells was measured by IVIS imaging according to (1-1) (a) above. Animal experiments including subcutaneous administration to animals were performed according to (1-1) (a) above.

[0057] The results are shown in Table 3.

[0058] As is clear from Table 3, in the group administered with cancer cells transfected with antigen (85A), the cancer cells completely disappeared on days 14 and 21, and all mice survived (n = 7), whereas in the group administered with cancer cells not transfected with antigen (85A), it was confirmed that the tumor volume increased over time (n = 7). Note that, because some mice administered with Colon26-Luc cells not transfected with antigen died on day 21, the average tumor volume was not calculated.

[0059] B. Administration of antigen-expressing cancer cells to experimental animals (2) An experiment was conducted in the same manner as in (1-2)A above, except that Meth-A cells were used instead of Colon26-Luc cells as the cancer cells into which the antigen (85A) was introduced. That is, according to the procedure in (1-2)A above, cancer cells into which the antigen (85A) was introduced (85A-mIL2-expressing Meth-A cells) and cancer cells without the antigen (Meth-A cells) were each administered to CB6F1 mice, and the growth of the cancer cells was analyzed.

[0060] The results are shown in Table 4.

[0061] As is clear from Table 4, in the group administered with cancer cells (Meth-A cells) into which antigen (85A) was introduced and expressed, the cancer cells completely disappeared on days 14 and 21, and all mice survived (n = 6), whereas in the group administered with cancer cells not introduced with antigen (85A), it was confirmed that the tumor volume increased over time (n = 4). These experimental results showed that the cancer cells into which the antigen is introduced are not limited to Colon26-Luc cells, and that other cancer cells also exerted effects similar to those of Colon26-Luc cells. Note that the average tumor volume was not calculated because some mice died on day 21 after receiving Meth-A cells without antigen introduction.

[0062] C. Administration of cancer cells expressing only mIL2 to experimental animals. Cancer cells transfected with only mIL2 (mIL2-expressing Colon26-Luc cells) and cancer cells transfected with neither mIL2 nor antigen (85A) (Colon26-Luc cells) were administered to CB6F1 mice, and the growth of the cancer cells was analyzed. The transduction plasmid (mIL2 alone) was constructed according to (1-1) B above. Animal experiments, including the introduction of the plasmid into cancer cells, measurement of IL2 secretion levels, and subcutaneous administration to animals, were performed in the same manner as (1-2) A above.

[0063] The results are shown in Table 5.

[0064] As is clear from Table 5, it was confirmed that the tumor volume increased over time in both the group administered with cancer cells transfected with only mIL2 (n = 5) and the group administered with cancer cells not transfected with mIL2 (n = 5). Note that the mean tumor volume was not calculated for the mice treated with Colon26-Luc cells not transfected with mIL2, as some mice died on day 21.

[0065] D. Verification of vaccine efficacy for antigen-expressing cancer cells (1) Next, cancer cells expressing the antigen (85A) (85A-mIL2-expressing Colon26-Luc cells) were administered, and then cancer cells not expressing the antigen (Colon26-Luc cells) were re-transplanted into mice in which the cancer cells had disappeared (see (1-2)A above), and analysis was performed to determine whether sensitized CTLs against neoantigen-derived peptides against the cancer cells were induced. CB6F1 mice administered cancer cells (85A-mIL2-expressing Colon26-Luc cells) into which the antigen (85A) had been introduced were generated according to (1-2)A above. Animal experiments involving subcutaneous administration of cancer cells (Colon26-Luc cells) not expressing the antigen (85A) to animals were performed according to (1-1)A above.

[0066] The results are shown in Table 6.

[0067] As is clear from Table 6, when wild-type mouse cancer cells (Colon26-Luc cells) were transplanted into mice administered with antigen-expressing cancer cells (85A-mIL2-expressing Colon26-Luc cells), no growth of the cancer cells was observed. All mice lost their cancer cells on days 7, 14, and 21, and all survived (n=7). This indicates that in mice administered with cancer cells expressing the antigen (85A) (85A-mIL2-expressing Colon26-Luc cells) and in which the cancer cells disappeared, killer T cells were induced against neoantigens of the Colon26-Luc cells. In other words, a vaccine effect of inducing immunity against tumors was observed for Colon26-Luc cells expressing the antigen (85A).

[0068] E. Verification of vaccine efficacy for antigen-expressing cancer cells (2) Administering antigen-expressing cancer cells directly to a subject may lead to cancer proliferation. Therefore, we investigated whether similar effects could be achieved using cancer cells that had been treated with an antitumor agent to lose their proliferation ability. Specifically, the 85A-mIL2-expressing Colon26-Luc cells were replaced with 85A-mIL2-expressing Colon26-Luc cells treated with 50 μM etoposide (CAS: 33419-42-0) (ApipoGen (AG-CR1-3572-5100)) for 24 hours, and experiments were conducted according to (1-2) A and D above. That is, experiments were conducted according to (1-2) A and D above, except that etoposide-treated 85A-mIL2-expressing Colon26-Luc cells were used as antigen-expressing cancer cells.

[0069] Table 7 shows the results of administering etoposide-treated antigen-expressing cancer cells (85A-mIL2-expressing Colon26-Luc cells) to experimental animals.

[0070] As is clear from Table 7, in the group administered with etoposide-treated antigen-expressing cancer cells, the cancer cells completely disappeared on days 7, 14, and 21, and all patients survived (n=6).

[0071] Table 8 shows the results of verifying the vaccine effect on etoposide-treated antigen-expressing cancer cells (85A-mIL2-expressing Colon26-Luc cells).

[0072] As is clear from Table 8, when wild-type mouse cancer cells (Colon26-Luc cells) were transplanted into mice administered etoposide-treated antigen-expressing cancer cells (85A-mIL2-expressing Colon26-Luc cells), no growth of the cancer cells was observed. All mice had lost cancer cells by days 14 and 21, and all survived (n=6). This indicates that etoposide-treated antigen-expressing cancer cells (85A-mIL2-expressing Colon26-Luc cells) also had a vaccine effect in inducing immunity against tumors. From the above, it was confirmed that pre-treating antigen-expressing cancer cells with an antitumor agent ensures safety for use as a cancer cell vaccine while maintaining efficacy.

[0073] (1-3) Verification of the antitumor effect of cancer cells expressing novel coronavirus-derived Spike protein (1) The experiments in (1-2) A, D, and E above were performed in the same manner, except that the antigen used was replaced with novel coronavirus-derived Spike (SEQ ID NO: 7) instead of BCG vaccine-derived 85A. Specifically, the experiments were performed in the same manner as in (1-2) A, D, and E above, except that the transduction plasmid (Spike-mIL2) was constructed and used in accordance with (1-1) B above. That is, the animal experiments, including the introduction of the plasmid into cancer cells, measurement of IL2 secretion levels, and subcutaneous administration to animals, were performed in the same manner as in (1-2) A above. In addition to Colon26-Luc cells, LLC-Luc cells were also used as cancer cells to express the antigen (Spike). Furthermore, experiments were conducted in which etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells and Spike-mIL2-expressing LLC-Luc cells) were administered to experimental animals not only once but also multiple times (see Figure 1).

[0074] Table 9 shows the results of administering cancer cells (Colon26-Luc cells) expressing the antigen (Spike) to experimental animals.

[0075] As is clear from Table 9, in the group administered with cancer cells transduced with the antigen (Spike), the cancer cells completely disappeared on day 21 (n = 6), whereas in the group administered with cancer cells not transduced with the antigen (Spike), it was confirmed that the tumor volume increased over time (n = 5). Furthermore, in the group administered with cancer cells transduced with the antigen (Spike), all cases were still alive 50 days after administration, whereas in the group administered with cancer cells not transduced with the antigen (Spike), all cases died on day 37 after administration.

[0076] Table 10 shows the results of verifying the vaccine effect for cancer cells (Spike-mIL2-expressing Colon26-Luc cells) in which an antigen (Spike) was expressed.

[0077] As is clear from Table 10, when wild-type mouse cancer cells (Colon26-Luc cells) were transplanted into mice administered with antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells), no growth of the cancer cells was observed. Cancer cells disappeared in all mice on days 14 and 21, and all mice survived (n=6). This indicates that killer T cells against neoantigens of Colon26-Luc cells were induced in mice administered with cancer cells (Spike-mIL2-expressing Colon26-Luc cells) expressing the antigen (Spike) and in which the cancer cells disappeared. In other words, a vaccine effect of inducing immunity against tumors was observed for Colon26-Luc cells expressing the antigen (Spike).

[0078] Table 11 shows the results of administering cancer cells (LLC-Luc cells) expressing the antigen (Spike) to experimental animals.

[0079] As is clear from Table 11, in the group administered with cancer cells transduced with the antigen (Spike), the cancer cells completely disappeared on days 7, 14, 21, and 28 (n = 6), whereas in the group administered with cancer cells not transduced with the antigen (Spike), it was confirmed that the tumor volume increased over time (n = 5). Furthermore, in the group administered with cancer cells transduced with the antigen (Spike), all cases were still alive 80 days after administration, whereas in the group administered with cancer cells not transduced with the antigen (Spike), all cases died 60 days after administration.

[0080] Table 12 shows the results of verifying the vaccine effect of cancer cells (Spike-mIL2-expressing LLC-Luc cells) expressing an antigen (Spike).

[0081]

[0082] As is clear from Table 12, when wild-type mouse cancer cells (LLC-Luc cells) were transplanted into mice administered with antigen-expressing cancer cells (Spike-mIL2-expressing LLC-Luc cells), no growth of the cancer cells was observed. Cancer cells disappeared in all mice on days 7, 21, and 35, and all mice survived (n=6). This indicates that killer T cells against neoantigens of LLC-Luc cells were induced in mice administered with cancer cells (Spike-mIL2-expressing LLC-Luc cells) expressing the antigen (Spike) and in which the cancer cells disappeared. In other words, a vaccine effect of inducing immunity against tumors was observed for LLC-Luc cells expressing the antigen (Spike).

[0083] Table 13 shows the results of administering etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells) to experimental animals.

[0084] As is clear from Table 13, in both the group receiving a single dose of etoposide-treated antigen-expressing cancer cells and the group receiving multiple doses, the cancer cells completely disappeared by day 7, and all patients survived (n = 5 for single and double doses, n = 6 for triple doses).

[0085] Table 14 shows the results of verifying the vaccine effect on etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells).

[0086] As is clear from Table 14, when mouse cancer cells (Colon26-Luc cells) were transplanted into mice that had received three doses of etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells), no growth of the cancer cells was observed, and the cancer cells disappeared in all mice on days 14 and 21, and all mice survived (n = 6). Furthermore, when mouse cancer cells (Colon26-Luc cells) were transplanted into mice that had received a single or two doses of etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells), no growth of the cancer cells was observed in 80% of the mice, and the cancer cells disappeared (n = 5).

[0087] From the above, it was confirmed that the antigen-expressing cancer cells (85A-mIL2-expressing Colon26-Luc cells) treated with etoposide also had a vaccine effect of inducing immunity against tumors.

[0088] Table 15 shows the results of administering etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing LLC-Luc cells) to experimental animals.

[0089] As is clear from Table 15, in both the group receiving a single dose of etoposide-treated antigen-expressing cancer cells and the group receiving multiple doses, the cancer cells had completely disappeared by day 7, and all patients survived (n=5).

[0090] Table 16 shows the results of verifying the vaccine effect on etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing LLC-Luc cells).

[0091] As is clear from Table 16, when wild-type mouse cancer cells (LLC-Luc cells) were transplanted into mice that had received three doses of etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing LLC-Luc cells), no growth of the cancer cells was observed, and the cancer cells disappeared in all mice by days 28 and 42, and all mice survived (n=5). Furthermore, when wild-type mouse cancer cells (LLC-Luc cells) were transplanted into mice that had received a single dose of etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing LLC-Luc cells), no growth of the cancer cells was observed in 40% of the mice, resulting in their disappearance. When wild-type mouse cancer cells (LLC-Luc cells) were transplanted into mice that had received two doses of etoposide, no growth of the cancer cells was observed in 80% of the mice, resulting in their disappearance (n=5).

[0092] From the above, it was confirmed that antigen-expressing cancer cells (Spike-mIL2-expressing LLC-Luc cells) treated with etoposide also had a vaccine effect of inducing immunity against tumors.

[0093] (1-4) Verification of the antitumor effect of cancer cells expressing novel coronavirus-derived Spike protein (2) Below, the results of an experiment using Renca cells as the cancer cells into which antigen and mIL2 were introduced are shown. The experiments in (1-2) A, C, and D above were similarly performed, except that the antigen used was replaced from BCG vaccine-derived 85A with novel coronavirus-derived Spike (SEQ ID NO: 7). That is, the animal experiments, including the introduction of the plasmid into cancer cells, measurement of IL2 secretion levels, and subcutaneous administration to animals, were performed in the same manner as in (1-2) A above. Furthermore, Renca cells were used as the cancer cells into which the antigen (Spike) and the like were expressed.

[0094] First, cancer cells expressing only mIL2 (mIL2-expressing Renca cells) and wild-type cancer cells (Renca cells) transfected with neither mIL2 nor antigen (Spike) were administered to CB6F1 mice, and the growth of the cancer cells was analyzed. The transduction plasmid (mIL2 alone) was constructed according to the procedure described above in (1-1)B.

[0095] The results are shown in Table 17.

[0096] As is clear from Table 17, in the group administered with cancer cells transfected with only mIL2, the cancer cells completely disappeared on days 21 and 28, and all mice survived (n = 6), whereas in the group administered with cancer cells not transfected with mIL2 (n = 6), it was confirmed that the tumor volume increased over time. Note that, since some mice administered with wild-type Renca cells not transfected with antigen died on day 21, the average tumor volume was not calculated.

[0097] Table 18 shows the results of verifying the vaccine effect on cancer cells in which mIL2 was expressed (mIL2-expressing Renca cells).

[0098] As shown in Table 18, when wild-type mouse cancer cells (Renca cells) were transplanted into mice that had been administered mIL2-expressing cancer cells and had lost the cancer cells, no growth of the cancer cells was observed in half (50%) of the mice on days 21, 28, and 35, and the cancer cells had disappeared (n=6). However, the cancer cells proliferated in the remaining (50%) of the mice.

[0099] Next, the results of administering cancer cells (Renca cells) expressing the antigen (Spike) to experimental animals are shown in Table 19. The transduction plasmid (Spike-mIL2) was constructed according to (1-1) B above.

[0100] As is clear from Table 19, in the group administered with cancer cells transduced with the antigen (Spike), the cancer cells completely disappeared on days 21 and 28 (n = 8), whereas in the group administered with cancer cells not transduced with the antigen (Spike), it was confirmed that the tumor volume increased over time (n = 7). Furthermore, in the group administered with cancer cells transduced with the antigen (Spike), all cases were still alive 60 days after administration, whereas in the group administered with cancer cells not transduced with the antigen (Spike), all cases died on day 55 after administration.

[0101] Table 20 shows the results of verifying the vaccine effect of cancer cells (Spike-mIL2-expressing Renca cells) expressing the antigen (Spike).

[0102] As is clear from Table 20, when wild-type mouse cancer cells (Renca cells) were transplanted into mice administered with antigen-expressing cancer cells (Spike-mIL2-expressing Renca cells), no growth of the cancer cells was observed, and the cancer cells disappeared in all mice on days 14, 21, and 28, and all mice survived (n=8). This indicates that in mice administered with cancer cells (Spike-mIL2-expressing Renca cells) expressing the antigen (Spike) and in which the cancer cells disappeared, killer T cells were induced against neoantigens of Renca cells. In other words, Renca cells expressing the antigen (Spike) were found to have a vaccine effect of inducing immunity against tumors.

[0103] Example 2: Evaluation of the applicability of CTL-inducing antigen-expressing cancer cells to humans using an HLA expression system In this example, an extrapolation experiment to humans was carried out on the antigen-expressing cancer cells prepared in Example 1 using HLA-expressing mouse cancer cells and a cancer-bearing mouse model of an HLA-expressing knock-in mouse, and efficacy in humans was verified by evaluating the antigen reactivity in a situation closer to that of humans.

[0104] (2-1) Generation of Δmβ2M LLC-Luc Cells A. Chromosomal Analysis of the Mouse β2M Gene in LLC-Luc Cells The mouse β2M gene locus is located on mouse chromosome 2, and it is known that chromosome excess or deficiency occurs in cancer cells. To perform chromosome painting, which specifically stains mouse chromosome 2, and confirm the chromosome copy number, Total Mouse DNA for chromosome 2 in Green (Applied Special Imaging Ltd) was purchased and performed according to the accompanying manual. Chromosomal analysis revealed that LLC-Luc cells contained two sets of chromosome 2. Therefore, it was highly likely that there were four copies of the mouse β2M gene locus on chromosome 2.

[0105] (i) Mouse β2M gene disruption using the CRISPR-Cas9 method The target sequence of the mouse β2M gene was searched for on a target sequence search site (http: / / chopchop.cbu.uib.no / ), and highly ranked sequences expected to have few off-target effects were selected. The boxed portions of Exon 1 and Exon 2 of the mouse β2M gene shown in Figure 2 were used as the target sequence for CRISPR-Cas9.

[0106] pX458 (Cas9-2A-GFP) was used as the CRISPR-Cas9 vector (see Addgene: pSpCas9(BB)-2A-GFP(pX458) instructions). A plasmid with the Exon1 or Exon2 target sequence inserted under the guide RNA sequence of pX458 (Cas9-2A-GFP) plasmid DNA was introduced into LLC-Luc cells by electroporation, and GFP (green fluorescent protein) positive fractions (CRISPR-Cas9 / Exon1, Exon2 plasmid-introduced cell group) were collected. The collected GFP positive fractions (CRISPR-Cas9 / Exon1, Exon2 plasmid-introduced cell group) were transferred to mouse H-2D ｂ The cells were stained with a PE antibody (Monoclonal Antibody (28-14-8)-PE-labeled eBioscience™ (Invitotogen / ThermoFisherSciene)), and the PE-negative fraction (M fraction) was collected and cultured. This procedure was repeated five times, and the cell group that became almost a negative fraction was designated as LLC-Luc cells in which the β2M gene was disrupted (Δmβ2M LLC-Luc cells).

[0107] The CRISPR-Cas9 / Exon1, Exon2 plasmid-introduced cell group was cultured under H-2D ｂ The results of obtaining Δmβ2M LLC-Luc cells by concentrating the negative fraction after H-2Db-PE staining are shown in Figure 3. The analysis results showed that the negative fraction (H2Db) to which the mouse H-2Db-PE antibody, which specifically binds to mouse MHC class I, was not bound. ｂ Since a negative fraction / M fraction was observed, it was confirmed that the β2M gene had been disrupted by the CRISPR-Cas9 method.

[0108] c) Analysis of Mouse MHC Class I Expression Using FACS (Flow Cytometry) Δmβ2M LLC-Luc cells and wild-type LLC-Luc cells (LLC-Luc WT cells) were stained with purchased Mouse MHC Class 1 (H-2Db) Monoclonal Antibody (28-14-8)-PE-labeled eBioscience™ (Invitotogen / ThermoFisherSciene) according to the accompanying manual, and mouse MHC Class I expression was analyzed by flow cytometry. The results of mouse MHC Class I expression analysis of Δmβ2M LLC-Luc cells using the FACS Versa are shown in Figure 4.

[0109] Comparing wild-type LLC-Luc cells supplemented with anti-mouse H-2Db-PE antibody and β2M gene knockout LLC-Luc cells supplemented with anti-mouse H-2Db-PE antibody, it was confirmed that the β2M gene knockout LLC-Luc cells lost expression of mouse MHC class I. This confirmed that disruption of the mouse β2M gene can eliminate expression of mouse MHC class I.

[0110] D. DNA analysis of β2M gene in LLC-Luc cells lacking mouse MHC class I expression ISOGENOME (Nippon Gene) was purchased and genomic DNA was extracted from LLC-Luc cells lacking MHC class I expression according to the attached manual.

[0111] A DNA fragment containing the 5' leader sequence region of β2M exon 1, which contains the CRISPR-Cas9 target sequence, to the 3' end of exon 2 was PCR amplified, and the resulting DNA was digested with restriction enzymes BamH1 and Sal1 and purified. The purified DNA fragment was cleaved with EcoRV, and a DNA fragment containing exon 1 (BamHI-EcoRV ~ 300 bp) and exon 2 (SalI-EcoRV ~ 400 bp) was inserted into the pBluescriptSK(+) vector and transformed into E. coli. After insert checking, DNA sequencing of clones containing inserts was outsourced.

[0112] <Exon 1 Analysis Results> Nine types of mutations were observed in genomic DNA extracted from a cell population enriched by FACS that did not express mouse MHC class I. Of the 16 clones analyzed for DNA sequence, 10 clones preserved Exon 1 gene function, with base insertion or deletion before the 1st ATG (9 clones) and an intact sequence (1 clone). Of the 16 clones, Exon 1 gene disruption occurred in 6 clones, with 1st ATG deletion (3 clones), frameshift (2 clones), and amino acid mutation (1 clone) observed.

[0113] <Exon 2 Analysis Results> Eight types of mutations were observed in genomic DNA extracted from a group of cells enriched by FACS that did not express mouse MHC class I. Base deletions (1 to 6 bases) were observed in 11 of 15 clones, and extensive deletions in the upstream region of Exon 2 were observed in 4 clones, resulting in disruption of the Exon 2 gene in 15 of 15 clones. It was confirmed that the loss of MHC class I expression was due to 100% gene disruption in Exon 2 of the β2M gene in the obtained LLC-Luc cells that had lost MHC class I expression.

[0114] (2-2) Preparation of HLA-Expressing Δmβ2M LLC-Luc Cells HLA-expressing Δmβ2M LLC-Luc cells were prepared by introducing an HLA expression unit into Δmβ2M LLC-Luc cells. Regarding human HLA, HLA-A2 (gene A0201), which is compatible with multiple races, was selected.

[0115] Specifically, human HLA-A2 expression unit DNA (PGK promoter + human β2M-cDNA-HLAα1α2-gDNA + mouse H2Dbα3-gDNA) was introduced into the Δmβ2M LLC-Luc cells prepared in (2-1) above to express human HLA class I. The detailed procedure for constructing the human HLA-A2 expression unit DNA is shown in Figure 5(A). Figure 5(B) shows a schematic diagram of the chimeric MHC class I expressed by the human HLA-A2 expression unit and its behavior within the cells.

[0116] Whether the obtained cells expressed HLA was confirmed by flow cytometry and Western blotting. Specifically, confirmation was carried out by the following procedure.

[0117] <Flow cytometry> After washing the cultured cells with PBS, the cells were detached with EDTA-containing trypsin and collected. A primary antibody diluted with PBS containing 2% FBS was added to the collected cells and incubated at 4°C for 1 hour. After centrifugation, the cells were washed three times with PBS. A fluorescently labeled secondary antibody diluted with PBS containing 2% FBS was added and incubated at 4°C for 30 minutes. The cells were washed three times with PBS and analyzed using a FACSCalibur flow cytometer (BD Biosciences).

[0118] <Western blotting method> After removing the supernatant from the cultured cells, the cells were washed with PBS and lysed in 1x Sample Buffer (2% SDS, 80 mM Tris-HCl pH 6.8, 15% glycerol, Brilliant Green, CBB-G250). The cells were disrupted using a sonicator (BRANSON), and 2-mercaptoethanol was added to a final concentration of 2%. The cells were boiled at 100°C for 3 minutes before use in analysis. The purified sample was subjected to 10% acrylamide SDS electrophoresis, and the proteins were transferred from the gel to a PDVF membrane. The cells were blocked with 5% skim milk diluted with PBS containing Tween 20 at room temperature for 1 hour. After washing with PBS containing Tween 20, the primary antibody was diluted with Can Get Signal Solution 1 (TOYOBO) and incubated at room temperature for 1 hour. After washing with PBS containing Tween 20, the secondary antibody was diluted with Can Get Signal Solution 2 (TOYOBO) and incubated at room temperature for 30 minutes. After washing with PBS containing Tween 20, detection was performed using ECL™ Select Western blot detection reagent.

[0119] The results are shown in Figures 6 and 7. The flow cytometry analysis results in Figure 6 confirmed that HLA molecules and human β2M were expressed on the cell surface of HLA-A2-introduced Δmβ2M LLC-Luc cells. The Western blotting analysis results in Figure 7 confirmed that the introduced human β2M and HLA were expressed as a complex.

[0120] (2-3) Verification of the antitumor effect of HLA-expressing cancer cells expressing BCG vaccine-derived 85A. HLA-A2-expressing Δmβ2M LLC-Luc cells prepared in (2-2) above were transfected with a plasmid (TERTp-85A-IRES-mIL2) to prepare HLA-expressing cancer cells expressing BCG vaccine-derived 85A. The plasmid was prepared and transfected according to the procedure in (1-2) above. IL2 secretion levels were measured to confirm the establishment of HLA-expressing Δmβ2M LLC-Luc cells expressing 85A and IL2.

[0121] HLA-A2-expressing Δmβ2M LLC-Luc cells (85A-mIL2-expressing HLA-A2 cells) transfected with antigen (85A) and HLA-A2-expressing Δmβ2M LLC-Luc cells (HLA-A2 cells) without antigen transfection were administered to HLA-A2-expressing knock-in mice (generated in accordance with WO 2015 / 056774), and cancer cell growth was analyzed. Cancer cell tumor volume was measured by IVIS imaging according to (1-1) (a) above. Animal experiments involving subcutaneous administration to animals were performed according to (1-1) (a) above.

[0122] The results are shown in Table 21.

[0123] As is clear from Table 21, cancer cells significantly disappeared in the HLA-expressing knock-in mouse group administered with HLA-expressing mouse cancer cells into which the antigen (85A) had been introduced (n = 4), whereas tumor volume was confirmed to increase over time in the HLA-expressing knock-in mouse group administered with HLA-expressing mouse cancer cells into which the antigen (85A) had not been introduced (n = 4). In other words, the experiments conducted this time confirmed that the 85A antigen can be induced via HLA-A2, indicating that the experimental results of the mice in Example 1 are fully expected to be applicable to humans as well.

Claims

1. A cancer vaccine composition comprising genetically modified cancer cells, each comprising a nucleic acid encoding a cytotoxic T cell (CTL)-inducible antigen and a nucleic acid encoding IL-2, wherein the proliferative capacity of the genetically modified cancer cells is suppressed by an anticancer agent.

2. The cancer vaccine composition according to claim 1, wherein the CTL-inducing antigen is an antigen derived from a pathogen or virus.

3. The cancer vaccine composition according to claim 1, wherein the CTL-inducing antigen is an antigen derived from the novel coronavirus.

4. The cancer vaccine composition according to any one of claims 1 to 3, wherein the gene-modified cancer cells are introduced in the form of an expression vector containing the nucleic acid encoding the CTL-inducible antigen and the nucleic acid encoding IL-2.

5. The cancer vaccine composition according to any one of claims 1 to 3, wherein the nucleic acid encoding the CTL-inducible antigen and the nucleic acid encoding IL-2 are operably linked to a promoter having transcriptional activity in cancer cells.

6. The cancer vaccine composition according to any one of claims 1 to 3, wherein the gene-modified cancer cells are derived from a subject suffering from cancer.

7. The cancer vaccine composition according to any one of claims 1 to 3, wherein the proliferation rate of the gene-modified cancer cells 24 hours after treatment (the ratio of the number of surviving cancer cells 24 hours after treatment to the number of surviving cancer cells before anticancer drug treatment) is 60 to 95%.

8. A cancer vaccine composition according to any one of claims 1 to 3, for use in the treatment or prevention of cancer.

9. A cancer vaccine composition according to any one of claims 1 to 3, for treating metastatic and / or recurrent cancer, or for preventing the metastasis and / or recurrence of cancer.

10. A cancer vaccine composition according to any one of claims 1 to 3, for preventing metastasis and / or recurrence of cancer after surgical treatment.

11. A combination pharmaceutical comprising a cancer vaccine composition according to any one of claims 1 to 3 and another drug.

12. The combination drug according to claim 11, wherein the other drug is an immunostimulant.

13. The combination pharmaceutical according to claim 12, wherein the immunostimulant is an anti-CTLA-4 antibody preparation.

14. A method for producing a cancer vaccine composition containing genetically modified cancer cells as an active ingredient, comprising: (1) introducing nucleic acids encoding a cytotoxic T cell (CTL)-inducible antigen and nucleic acids encoding IL-2 into cancer cells isolated from a subject suffering from cancer to obtain genetically modified cancer cells expressing the CTL-inducible antigen and IL-2; and (2) contacting the modified cancer cells with an anticancer agent.

15. The method for producing the CTL-inducible antigen according to claim 14, wherein the CTL-inducible antigen is an antigen derived from a pathogen or virus.

16. The method for producing the CTL-inducible antigen according to claim 14, wherein the CTL-inducible antigen is an antigen derived from the novel coronavirus.

17. The manufacturing method according to any one of claims 14 to 16, wherein the nucleic acid encoding the CTL-inducible antigen and the nucleic acid encoding IL-2 are operably linked to a promoter having transcriptional activity in cancer cells.

18. The manufacturing method according to any one of claims 14 to 16, wherein the gene-modified cancer cells are derived from a subject suffering from cancer.

19. The manufacturing method according to any one of claims 14 to 16, wherein the proliferation rate of the genetically modified cancer cells obtained by step (2) after 24 hours (the ratio of the number of surviving cancer cells 24 hours after contact with the anticancer drug to the number of surviving cancer cells before contact with the anticancer drug) is 60 to 95%.

20. The manufacturing method according to any one of claims 14 to 16, wherein the cancer vaccine composition is for use in the treatment or prevention of cancer.