DUAL ANTIGEN-RECOGNIZING iPS CELL-DERIVED CHIMERIC ANTIGEN RECEPTOR-T-CELL THERAPY

Abstract

To provide a novel chimeric antigen receptor-T cell (CAR-T cell) therapy, and to provide a medicament using the same.SOLUTION: Provided are: a method for producing T cell-derived induced Pluripotent Stem Cells (T-iPSCs) with two antigen receptors, comprising introducing, into cytotoxic T lymphocyte (CTL)-derived iPS cells (T-iPSCs) specific to a tumor antigen or viral antigen expressed on target cells, a chimeric antigen receptor (CAR) specific for an antigen different from the antigen; T-iPSCs with a T cell receptor (TCR) specific to a tumor antigen or viral antigen expressed on target cells and a CAR specific to an antigen different from the antigen; and furthermore, CTLs expressing a TCR specific to a tumor antigen or viral antigen expressed on target cells and a CAR specific to an antigen different from the antigen.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to novel chimeric antigen receptor-expressing T cells (CAR-T cells) and pharmaceuticals using the same. [Background technology]

[0002] Chimeric antigen receptor-expressing T cells (CAR-T cells) are T cells that have been transfected with a chimeric antigen receptor and are attracting attention as a groundbreaking cancer treatment. Currently approved CAR-T cell therapy involves transfecting T cells derived from the peripheral blood of cancer patients with a chimeric antigen receptor to produce CAR-T cells, which are then administered to the patient. This autologous CAR-T cell therapy has the drawback of being extremely expensive. Meanwhile, allogeneic CAR-T cell therapy, currently in clinical trials, has also been developed, which has the advantage of reducing production costs, but the drawback of reduced anti-tumor efficacy due to immune rejection.

[0003] Meanwhile, a technology has been developed to establish T-iPS cells from antigen-specific cytotoxic T cells (CTLs) and induce their differentiation into functionally rejuvenated CTLs while maintaining their antigen specificity (Patent Document 1, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6164746 [Non-patent literature]

[0005] [Non-Patent Document 1] Experimental Hematology 2017,47; 2-12 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the anti-cancer effects of CTL-iPS cells or CAR-T cell therapy are still insufficient depending on the antigen or cancer type, and there is a need for immune cell therapy that can proliferate more efficiently, exert better anti-tumor effects, and survive for a long period in the body. [Means for solving the problem]

[0007] The present inventors have therefore discovered that if antigen-specific cytotoxic T cells (CTLs) are used as raw materials, these are converted into iPS cells, and then a chimeric antigen receptor (CAR) is introduced and differentiated, the anti-tumor effect is synergistically enhanced, particularly by the recognition of two antigens by the iPS cell-derived CTLs and CAR, and iPS cells that survive in the body for a long period of time can be obtained, thereby completing the present invention.

[0008] That is, the present invention provides the following [1] to [8].

[0009] [1] iPS cells derived from antigen-specific cytotoxic T cells (CTLs) into which a chimeric antigen receptor (CAR) has been introduced. [2] The iPS cells according to [1], wherein the antigen of the antigen-specific cytotoxic T cells is a viral antigen or a tumor antigen. [3] The iPS cell according to [1] or [2], wherein the chimeric antigen receptor is a CAR that recognizes a tumor surface antigen. [4] A pharmaceutical comprising, as an active ingredient, the iPS cell according to any one of [1] to [3]. [5] A pharmaceutical comprising, as an active ingredient, the iPS cell according to any one of [1] to [3]. A method for producing antigen-specific cytotoxic T cell (CTL)-derived iPS cells with chimeric antigen receptor (CAR) introduced therein, characterized by introducing genes for ox2, Klf4, c-Myc, and SV40 large T antigen to obtain antigen-specific cytotoxic T cell (CTL)-derived iPS cells (T-iPSCs), and then introducing a chimeric antigen receptor (CAR) into the obtained T-iPSCs to induce differentiation. [6] iPS cells derived from antigen-specific cytotoxic T cells (CTLs) into which a chimeric antigen receptor (CAR) has been introduced for the treatment of cancer or viral infections. [7] Use of iPS cells derived from antigen-specific cytotoxic T cells (CTLs) into which a chimeric antigen receptor (CAR) has been introduced for the manufacture of a therapeutic drug for cancer or viral infection. [8] A method for treating cancer or viral diseases, characterized by administering iPS cells derived from antigen-specific cytotoxic T cells (CTLs) into which a chimeric antigen receptor (CAR) has been introduced to a patient in need thereof. [Effects of the Invention]

[0010] CTL-derived iPS cells into which the CAR of the present invention has been introduced have an efficient CAR gene introduction efficiency of approximately 100%, and have an extremely high antitumor effect due to the synergistic effect of the antigen-specific cytotoxicity of CTLs and the antitumor effect of CAR. Furthermore, because they survive in the body for a long period of time, they are useful as therapeutic agents for cancer and viral infections. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows tetramer analysis of induced CTLs to established CTL clones. [Figure 2] FIG. 1 shows tetramer analysis of induced CTLs to established CTL clones. [Figure 3] FIG. 1 shows the vector structure of LMP1-CAR. [Figure 4] Figure 10 shows the LMP1-CAR expression rate in LMP1-CAR-LMP2-T-iPS cells. [Figure 5] FIG. 10 shows the LMP1-CAR expression rate in LMP1-CAR / LMP2-rejT. [Figure 6] Shows the cytotoxicity of LMP1-CAR / LMP2-rejT and peripheral blood-derived LMP1-CAR against EB virus-associated lymphoma cell lines. [Figure 7] FIG. 1 shows the tumor growth inhibitory effect of LMP1-CAR / LMP2-rejT on EB virus-associated lymphoma cell lines. [Figure 8] This figure shows a comparison of the survival rates of LMP1-CAR / LMP2-rejT and LMP2-rejT in a mouse model. [Figure 9] FIG. 1 shows the apoptosis rate of LMP1-CAR / LMP2-rejT by CID administration. DETAILED DESCRIPTION OF THE INVENTION

[0012] The cells of the present invention are iPS cells derived from virus-specific or tumor antigen-specific CTLs into which a CAR has been introduced. These iPS cells can be obtained by artificially reprogramming CTLs to produce iPS cells possessing a TCR, then transducing the iPS cells with a CAR gene and allowing them to differentiate. More specifically, the iPS cells can be obtained by transducing Oct3 / 4, Sox2, Klf4, c-Myc, and SV40 large T antigen genes into a CTL clone to obtain CTL-derived iPS cells, and then transducing the resulting T-iPS cells with a CAR and allowing them to differentiate.

[0013] The CTLs used as raw materials are T cells that have cytotoxicity due to their antigen specificity. Viruses that many adults are already infected with are particularly effective as CTLs. These include the LMP1 antigen, LMP2 antigen, and EBNA antigen of Epstein-Barr virus, as well as the penton antigen and hexon antigen of adenovirus antigens. Examples of antigen-specific CTLs targeting these antigens include the CMVpp65, IE1, and IE2 antigens of cytomegalovirus, the IE61, IE62, IE63, and ORF10 antigens of varicella-zoster virus (VZV), and herpes simplex virus (HSV). Virus-specific CTLs targeting viruses related to viral tumors, such as the E6 and E7 antigens of human papillomavirus (HPV) and the Tax antigen of HTLV-1, are also effective for generating iPSC-derived CAR-rejT (rejuvenated CTLs) targeting these antigens. However, CTLs specific for other antigens, such as WT1, NY-ESO-1, MAGE-1, and MART-1, can also be generated.

[0014] The CTLs used in the present invention are preferably human CTLs. The human source of these T cells may be a healthy individual or a human suffering from a viral infection. Furthermore, a preferred human source of T cells is one whose HLA type matches that of the patient to whom the CAR-loaded iPS cells produced by the present invention will be administered, and preferably the same individual as the person to whom the CAR-loaded iPS cells will be administered. When allogeneic cells are used, CTLs induced from human iPS cells whose HLA has been genome-edited can also be used, as long as they have an HLA that matches the HLA restriction of the CTL epitope. Preferred viruses include, but are not limited to, EB virus and cytomegalovirus, which infect most adults.

[0015] Such CTLs can be isolated from, for example, human tissues by known techniques. Examples of human tissues include tissues containing the T cells, such as peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and lesion tissues. Among these, peripheral blood is preferred because it is less invasive to humans and easier to prepare. Known techniques for isolating human T cells include, for example, magnetic selection using magnetic beads for cell separation, flow cytometry using a cell sorter and antibodies against cell surface markers such as CD4 or CD8, and activated T cell induction methods using anti-CD3 antibodies and anti-CD28 antibodies. When isolating T cells from human tissues containing antigen-specific T cells, T cells with the desired antigen specificity can be purified from human tissues using multimerized MHC (major histocompatibility complex) bound to the desired antigen (e.g., "MHC tetramer" or "Pro5 (registered trademark) MHC class I pentamer").

[0016] The genes used to initialize CTLs are Oct3 / 4, Sox2, Klf4, c-Myc, and SV40 large T antigen.

[0017] In the present invention, the method for introducing the gene into CTLs is not particularly limited, and any known method can be appropriately selected and used. For example, when the gene is introduced into the CTLs in the form of a nucleic acid encoding the gene, the nucleic acid encoding the gene (e.g., cDNA, RNA) can be inserted into an appropriate expression vector containing a promoter that functions in T cells, and the expression vector can be introduced into cells by infection, lipofection, liposome method, electroporation, calcium phosphate co-precipitation, DEAE-dextran method, microinjection, electroporation, or genome editing such as CRISPR / Cas9.

[0018] Among these expression vectors, it is more preferable to use a stealth RNA expression vector containing the above gene from the viewpoints of reducing the risk of canceration and improving transfer efficiency. Stealth RNA expression vectors are designed to avoid chromosomal incorporation and to achieve sustained and stable gene expression in the cytoplasm rather than the nucleus. They can be used to introduce large genes of 13,000 base pairs or more, or even 10 genes at a time, without harming cells. They can be removed when the introduced gene is no longer needed, and their stealth properties mean that cells cannot recognize the vector as a foreign body. Such stealth RNA expression vectors include a complex that does not activate innate immune structures and consists of a minus single-stranded RNA (A) containing the RNA sequences (1) to (8) below, a single-stranded RNA-binding protein (B), and an RNA-dependent RNA polymerase. (1) an RNA sequence for the gene; (2) a human mRNA-derived RNA sequence that constitutes a non-coding region; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA synthetase; (4) a transcription termination signal sequence recognized by the RNA-dependent RNA synthetase; (5) an RNA sequence containing a replication origin recognized by the RNA-dependent RNA synthetase; (6) an RNA sequence encoding the RNA-dependent RNA synthetase; (7) an RNA sequence encoding a protein that regulates the activity of the RNA-dependent RNA synthetase; (8) An RNA sequence encoding the single-stranded RNA-binding protein.

[0019] Furthermore, when establishing T-iPS cells, the CTLs are preferably activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in the presence of interleukin-2 (IL-2), interleukin-7 (IL-7), or interleukin-15 (IL-15) before the gene is introduced. Alternatively, the CTLs may be activated by stimulation with at least one substance selected from the group consisting of phytohemagglutinin (PHA), interleukin-2 (IL-2), alloantigen-expressing cells, anti-CD3 antibody, anti-CD28 antibody, CD3 agonist, and CD28 agonist. Such stimulation can be performed, for example, by adding PHA, IL-2, anti-CD3 antibody, and / or anti-CD28 antibody to a medium and culturing the T cells for a certain period of time. Alternatively, the anti-CD3 antibody and / or anti-CD28 antibody may be bound to magnetic beads, and instead of adding these antibodies to the medium, the CTLs may be stimulated by culturing them for a certain period on a culture dish having anti-CD3 antibody and / or anti-CD28 antibody bound to its surface.Furthermore, the CTLs may be stimulated by adding an antigen peptide recognized by the CTLs to the medium together with feeder cells.

[0020] To provide such stimulation to the CTLs, the concentration of PHA added to the medium is not particularly limited, but is preferably 1 to 100 μg / mL. The concentration of IL-2 added to the medium is not particularly limited, but is preferably 1 to 200 ng / mL. Furthermore, the concentration of anti-CD3 antibody and / or anti-CD28 antibody added to the medium is not particularly limited, but is preferably 1 to 10 times the culture volume of the CTLs. To provide such stimulation to the CTLs, the concentration of anti-CD3 antibody and / or anti-CD28 antibody bound to the surface of the culture dish is not particularly limited, but the concentration at the time of coating is preferably 0.1 to 100 μg / mL, preferably 1 to 100 μg / mL, for anti-CD3 antibody and 0.1 to 10 μg / mL for anti-CD28 antibody.

[0021] The culture period for such stimulation is not particularly limited, as long as it is a period sufficient to provide such stimulation to the CTLs and to allow the CTLs to proliferate to the number of cells required for gene transfer, and is usually 2 to 7 days, but from the viewpoint of gene transfer efficiency, it is preferably 3 to 5 days. Infection is preferably carried out by mixing the T cells and the vector in a 15 mL tube, or, from the viewpoint of increasing gene transfer efficiency, culture is preferably carried out on a culture dish coated with Retronectin.

[0022] Examples of the medium for culturing the CTLs and adding PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody, etc. include known media suitable for culturing the CTLs (more specifically, Roswell Park Memorial Institute (RPMI) 1640 medium, AIM V medium, etc., which contain other cytokines and human serum). TM The medium may contain, in addition to PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody, amino acids necessary for culture (e.g., L-glutamine), antibiotics (e.g., streptomycin, penicillin), etc. ) may be added to the medium. It is also preferable to add IL-7 and IL-15 to the medium instead of IL-2. There are no particular limitations on the concentrations of IL-7 and IL-15 added, but it is preferable that each be added at 1 to 100 ng / mL.

[0023] Furthermore, there are no particular limitations on the conditions for or after introducing the gene into the CTLs, but the CTLs into which the gene has been introduced are preferably cultured under feeder-free conditions. Examples include wells coated with iMatrix-511 solution, which is a laminin 511E8 fragment, or vitronectin or matrigel. CTLs can also be established by culturing under feeder cell conditions. Examples of feeder cells include mouse embryonic fibroblasts (MEFs), STO cells, and SNL cells whose cell division has been arrested by irradiation or antibiotic treatment.

[0024] Furthermore, during the process of inducing T-iPS cells from the CTLs, it is preferable to add an iPS cell medium from the next day, and then replace the medium by half every other day, gradually replacing the CTL medium with iPS medium.

[0025] Furthermore, it is preferable to culture the CTLs while gradually replacing the known medium suitable for CTL culture with a medium suitable for iPS cell culture as the CTLs transition to iPS cells. The medium suitable for iPS cell culture can be appropriately selected from known media, and examples of suitable media include StemFit AK03N for iMatrix-coated cells or Essential 8 Medium for vitronectin-coated cells, mTeSR for Matrigel, and Dulbecco's modified Eagle's medium / F12 medium (human iPS cell culture medium) containing knockout serum substitute, L-glutamine, non-essential amino acids, 2-mercaptoethanol, b-FGF, and the like for feeder cells such as MEF cells.

[0026] In this way, T-iPS cells can be selected by appropriately selecting known techniques. Examples of such known techniques include selection by observing the morphology of ES cell / iPS cell-like colonies under a microscope. On the other hand, in the case of T-iPS cells established from single-cell CTL clones, their properties are often similar, so an alternative method is to simply passage all established colonies without selecting each T-iPS cell colony.

[0027] The identity of the selected cells as T-iPS cells can be confirmed by, for example, detecting the expression of undifferentiated cell-specific markers (SSEA-4, Tra-1-60, Tra-1-81, etc.) in the selected cells by RT-PCR or by ALP staining, or by transplanting the selected cells into mice and observing the formation of teratomas. Furthermore, the identity of the selected cells as CTL-derived cells can be confirmed by detecting the state of TCR gene rearrangement by genomic PCR.

[0028] These cells are selected and collected while observing the growth of the colonies. If the Sendai virus factor vector contains a label such as GFP, the cells are preferably collected when the disappearance of the GFP can be confirmed under a fluorescent microscope. This is generally 10 to 40 days, preferably 14 to 28 days, after the gene is introduced into the T cells. Unless otherwise specified above, the culture environment is preferably 5% CO2, 35 to 38°C, and more preferably 37°C.

[0029] Methods for introducing CAR into the obtained CTL-derived iPS cells include using viral vectors such as lentiviral vectors, genome editing such as CRISPR / Cas9, and using transposons.

[0030] CARs are chimeric antigen receptors (CARs) that are specific to tumor cell surface antigens and have the ability to activate T cells. CAR-T therapy involves transfecting T cells with nucleic acids encoding these chimeric antigen receptors, and then expanding the resulting transgenic T cells ex vivo and injecting them into the body.

[0031] As used herein, "CAR" refers to a fusion protein comprising an extracellular domain that binds to an antigen, a transmembrane domain derived from a polypeptide different from the extracellular domain, and at least one intracellular domain. A "chimeric antigen receptor (CAR)" is also called a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." The "extracellular domain that binds to an antigen" refers to any oligopeptide or polypeptide that can bind to a certain antigen, and the "intracellular domain" refers to any oligopeptide or polypeptide that is known to function as a domain that transmits a signal that activates or inhibits a biological process within a cell.

[0032] As used herein, the term "tumor antigen" refers to an antigenic biological molecule whose expression is newly observed as cells become cancerous. Detection of tumor antigens, for example, immunological detection, is useful for distinguishing between cancerous cells and their parent cells. Tumor antigens in the present invention include tumor-specific antigens (antigens that are present only in tumor cells and not found in other normal cells) and tumor-associated antigens (antigens that are also present in other organs / tissues or heterologous normal cells, and antigens that are expressed during development / differentiation).

[0033] As used herein, the term "single-chain antibody (scFv)" refers to a single-chain polypeptide derived from an antibody that retains its antigen-binding ability. For example, an example is an antibody polypeptide produced by recombinant DNA technology, in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for producing scFv are known, including those described in U.S. Pat. No. 4,694,778; Science, Vol. 242, pp. 423-442 (1988); Nature, Vol. 334, pp. 54454 (1989); and Science, Vol. 242, pp. 1038-1041 (1988).

[0034] As used herein, the term "domain" refers to a region within a polypeptide that folds into a specific structure independently of other regions.

[0035] A typical CAR structure consists of a single-chain antibody (single chain variable fragment: scFv) that recognizes a surface antigen on tumor cells, a transmembrane domain, and the intracellular domain of the TCR complex CD3ζ, which activates T cells. CARs with this structure are called first-generation CARs. The gene encoding the single-chain antibody portion is isolated, for example, from a hybridoma that produces a monoclonal antibody that recognizes the target antigen. CAR-expressing T cells directly recognize tumor cell surface antigens, regardless of the expression of major histocompatibility complex class I on tumor cells, and simultaneously activate T cells, enabling efficient tumor cell killing.

[0036] To enhance the T cell activation ability of first-generation CARs, second-generation CARs have been developed that link the intracellular domain of CD28, a T cell costimulatory molecule, or CD137 (4-1BB), a tumor necrosis factor (TNF) receptor superfamily. Further improvements are also being made to third-generation CARs that link two costimulatory molecules, CD28, 4-1BB, or CD134 (OX40), in tandem (Current Opinion in Immunology, Vol. 21, p. 215-223 (2009)). Furthermore, CARs incorporating the suicide gene inducible caspase-9 are also useful as a safety system, allowing for the induction of cell death in CAR-T cells in the event of side effects. .

[0037] Specific methods for introducing CAR include gene transfer using a viral vector such as lentivirus, using a transposon such as piggy bac, or genome editing using CRISR / Cas9 or the like.

[0038] To confirm that CAR has been introduced into cells, the efficiency of CAR gene introduction is analyzed by flow cytometry by staining protein L, which binds to scFv.

[0039] CAR-introduced cells can be differentiated into CAR-rejT cells. The differentiation induction method involves finely crushing CAR-T-iPSCs after CAR gene transduction and culturing them on feeder cells for two weeks, followed by further culturing on feeder cells expressing Notch ligands with cytokines for four weeks. After four weeks, floating cells are identified and stimulated with T cell receptors (TCRs). After stimulation, the CAR gene transduction efficiency and antigen specificity are confirmed using the methods described above. The differentiated CAR-rejT cells have the CAR gene introduced into them and retain their original antigen specificity.

[0040] For final cell collection, if there are few antigen-specific cells, bead selection using tetramers or FACS sorting of antigen-specific cells is performed. If the CAR gene transduction efficiency is low, protein L staining is performed to sort CAR-positive cells. Other methods, such as selection of CD8-positive cells, can be used to select and amplify the necessary cells.

[0041] The CAR-transduced iPS cell-derived CTL cells produced by the method of the present invention target two antigens, making it difficult for tumor cells to escape, and thus exhibiting efficient cytotoxicity. These CTL cells are useful as therapeutic agents for various malignant tumors and virus-related tumors.

[0042] Examples of therapeutic agents for malignant tumors include tumor antigen-specific rejT such as Survivin, NY-ESO-1, WT-1, and MAGE3, combined with GD2-CAR, HER2-CAR, NY-ESO-1-CAR, MUC1-CAR, and CD19-CAR. Examples of virus-related tumor therapeutics include LMP2-specific rejT combined with LMP1-CAR / LMP2-rejT or LMP2-CAR combined with LMP1-specific rejT for EB virus-related tumors such as EB virus-associated lymphoma and nasopharyngeal carcinoma, CD19-CAR / LMP1-rejT and CD19-CAR / LMP2-rejT for EB virus-positive B-cell lymphoma, NY-ESO-1 / Tax-rejT combined with NY-ESO-1-CAR for Tax-specific rejT for adult T-cell leukemia (ATL), and NY-ESO-1 / HBZ-rejT combined with NY-ESO-1-CAR for HBZ-specific rejT. Combining a virus-specific rejT, which is known to have excellent in vivo durability, with a CAR is also useful. For example, combining an LMP2-specific rejT, LMP1-specific rejT, CMV-specific rejT, adenovirus-specific rejT, VZV-specific rejT, or HSV-specific rejT with a CAR such as GD2-CAR, HER2-CAR, NY-ESO-1-CAR, MUC1-CAR, or CD19-CAR is also useful.

[0043] The immune cell therapy drug of the present invention is preferably administered parenterally, for example, intravenously, intraperitoneally, subcutaneously, or intramuscularly, and more preferably intravenously, although not particularly limited thereto. Alternatively, it can be administered locally to the affected area.

[0044] The pharmaceutical compositions of the present invention can be prepared by formulating the T-iPS cells produced by the method of the present invention using known pharmaceutical methods, such as capsules, liquids, film coatings, suspensions, emulsions, injections (intravenous injections, drip infusions, etc.), etc. It can be used primarily parenterally.

[0045] In preparing these formulations, the composition may be appropriately combined with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, solubilizing agent, or other additives, etc. Furthermore, the composition may be used in combination with known pharmaceutical compositions or immunostimulants used in the treatment or prevention of the above-mentioned diseases.

[0046] When administering the pharmaceutical composition of the present invention, the dosage is appropriately selected depending on the age, weight, symptoms, health condition of the subject, type of composition (drug, food, drink, etc.), etc. [Example]

[0047] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[0048] Example 1: Establishment of T-iPS cells from human papillomavirus (HPV)-specific CTL clones using Sendai virus vectors. 1) Peripheral blood mononuclear cells were isolated from the peripheral blood of healthy donors, and dendritic cells were induced for antigen presentation. Seven days later, HPV antigen peptides (HPV16-E6, A2402) were added to the induced dendritic cells, which were then incubated in a CO2 incubator for 15 minutes before co-culture with peripheral blood mononuclear cells. Co-culture with dendritic cells was repeated, and after approximately 8-10 days, HPV-specific CTLs were detected by staining with MHC tetramers and confirming tetramer positivity by flow cytometry. After HPV-specific CTLs were identified, single-cell sorting or tetramer / PE bead selection was performed, followed by limiting dilution. The cells were stimulated with PBMCs irradiated with 50 Gy of X-rays, IL2, and PHA. 2) After approximately 3-6 weeks, colonies that had formed were stained with tetramers, and the establishment of CTL clones was confirmed by flow cytometry. After confirming establishment, the CTL clones were stimulated with CD3 / 28 and then transfected with the vector described in A) below. The transfected CTLs were transferred to a 6-well plate coated with iMatrix, and culture was initiated in a CO2 incubator using CTL medium supplemented with IL-2 (Figure 1).

[0049] A) SeV4 vector + SV40 large T antigen

[0050] 3) The day after SeV gene transfection, an equal volume of iPS medium (StemFitAK03N) was added, and thereafter half the volume was replaced with StemFitAK03N every other day. 4) After 7 days, colonies of T-iPS cells were observed, and colonies were then picked.

[0051] Example 2: Establishment of T-iPS cells from Epstein-Barr virus (EBV)-specific CTL clones using Sendai virus vectors. 1) Peripheral blood mononuclear cells were isolated from the peripheral blood of healthy donors, and dendritic cells were induced for antigen presentation. Seven days later, the induced dendritic cells were treated with EBV LMP2 antigen peptide (A2402) and incubated in a CO2 incubator for 15 minutes, after which co-culture with peripheral blood mononuclear cells was initiated. Co-culture with dendritic cells was repeated, and after approximately 8-10 days, LMP2-specific CTLs were detected by staining with MHC tetramers and confirming tetramer positivity by flow cytometry. After LMP2-specific CTLs were identified, single-cell sorting or tetramer / PE bead selection was performed, followed by limiting dilution. The cells were stimulated with PBMCs irradiated with 50 Gy of X-rays, IL2, and PHA. 2) After about 3 to 6 weeks, the clones that emerged were stained with tetramers and analyzed by flow cytometry. Establishment of the LMP2 CTL clone was confirmed. After confirming establishment, the LMP2 CTL clone was stimulated with CD3 / 28 and then transduced with a Sendai virus vector at an MOI of 10. The transduced CTLs were transferred to a 6-well plate coated with iMatrix and cultured in a CTL medium supplemented with IL-2 in a CO2 incubator. 3) Approximately two weeks later, numerous T-iPS colonies derived from LMP2CTL were established, picked, and expanded. They were then infected with a lentivirus-derived LMP1-CAR vector to generate LMP1-CAR / LMP2-T-iPS cells, which were then induced to differentiate into LMP1-CAR / LMP2-rejT cells. 4) The LMP1-CAR expression rate of the differentiated LMP1-CAR / LMP2-rejT was extremely high at 100%. 5) Cytotoxicity tests and co-culture tests showed that tumor cells were efficiently eliminated.

[0052] FIG. 2 shows the results of tetramer staining of LMP2-CTL induced from peripheral blood and the established CTL clones. Figure 3 shows the vector structure of iC9-LMP1-CAR-T-iPSCs. Figure 4 shows the LMP1-CAR expression rate in LMP1-CAR-LMP2-T-iPS cells. Figure 5 shows the LMP1-CAR expression rate in LMP1-CAR / LMP2-rejT.

[0053] Example 3 (Cytotoxicity test) 1) To compare the cytotoxicity of LMP1-CAR / LMP2-rejT and peripheral blood-derived LMP1-CART against EBV-associated lymphoma cell lines, we performed a 51Cr release assay. LMP1-CAR / LMP2-rejT or peripheral blood-derived LMP1-CART was used as an effector, and HLA-matched EBV-associated lymphoma cell lines (extranodal NK / T-cell lymphoma, nasal type; ENKL) and HLA-mismatched EBV-infected tumor cell lines (LCL) were co-cultured for 6 hours at effector:target ratios of 5:1 and 2.5:1. 2) After co-cultivation, the culture supernatant was transferred to another counter plate, dried, and then measured using a plate reader. 3) LMP1-CAR / LMP2-rejT showed strong antigen-specific cytotoxicity (70-80%) against HLA-matched EBV-associated lymphoma cell lines, but showed no cytotoxicity (<10%) against HLA-mismatched control tumor cell lines. Peripheral blood-derived LMP1-CART showed approximately 30-50% cytotoxicity against HLA-matched EBV-associated lymphoma cell lines. The cytotoxicity of LMP1-CAR / LMP2-rejT was stronger than that of LMP1-CART (Figure 6).

[0054] Example 4 (Co-culture test) 1) A co-culture experiment was performed to compare the cytotoxicity of LMP1-CAR / LMP2-rejT against EBV-associated lymphoma cell lines with that of peripheral blood-derived LMP1-CAR / LMP2-rejT. LMP1-CAR / LMP2-rejT was used as the effector, and rejT with antigen specificity other than EBV was used as the control effector. An HLA-matched EBV-associated lymphoma cell line (ENKL) was used as the target. The cells were co-cultured at an effector:target ratio of 5:1 for 9 days. 2) After co-culture, the ratio of tumor cells to T cells was analyzed by flow cytometry after staining for CD56 and CD3. 3) LMP1-CAR / LMP2-rejT eliminated HLA-matched EBV-associated lymphoma cells by day 9, whereas the control rejT, which lacked antigen specificity, was unable to eliminate tumor cells and tumor growth was observed (Figure 7).

[0055] Example 5 (Anti-cancer effect) A comparative study of survival rates between LMP1-CAR / LMP2-rejT and LMP2-rejT in a mouse model After the Epstein-Barr virus-associated lymphoma cell line (ENKL) was transplanted into the peritoneal cavity of immunodeficient mice, the survival rates of the mice were compared among three groups: the LMP1-CAR / LMP2-rejT treatment group, the LMP2-rejT treatment group, and the untreated control group. The results are shown in Figure 8.

[0056] Example 6 (Apoptosis due to CID administration) The Annexin / 7-AAD positivity rate was analyzed 48 hours after administration of the dimerizer small molecule compound CID to LMP1-CAR / LMP2-rejT cells to examine the rate of apoptosis. The results are shown in Figure 9.

Claims

1. iPS cells derived from antigen-specific cytotoxic T cells into which a chimeric antigen receptor has been introduced.

2. The iPS cells according to claim 1, wherein the antigen of the antigen-specific cytotoxic T cells is a viral antigen or a tumor antigen.

3. The iPS cell according to claim 1 or 2, wherein the chimeric antigen receptor is a CAR that recognizes a tumor surface antigen.

4. A pharmaceutical comprising the iPS cell according to any one of claims 1 to 3 as an active ingredient.

5. A method for producing antigen-specific cytotoxic T cell (CTL)-derived iPS cells into which a chimeric antigen receptor (CAR) has been introduced, the method comprising: introducing genes for Oct3 / 4, Sox2, Klf4, c-Myc, and SV40 large T antigen into tumor antigen- or viral antigen-specific cytotoxic T cells (CTL) to obtain antigen-specific cytotoxic T cell (CTL)-derived iPS cells (T-iPSCs); and introducing a chimeric antigen receptor (CAR) into the obtained T-iPSCs to induce differentiation.

6. An iPS cell derived from an antigen-specific cytotoxic T cell (CTL) into which a chimeric antigen receptor (CAR) has been introduced for the treatment of cancer or a viral infection disease.

7. Use of iPS cells derived from antigen-specific cytotoxic T cells (CTLs) into which a chimeric antigen receptor (CAR) has been introduced for the manufacture of a therapeutic agent for cancer or a viral infection disease.

8. A method for treating cancer or a viral disease, comprising administering iPS cells derived from antigen-specific cytotoxic T cells (CTLs) into which a chimeric antigen receptor (CAR) has been introduced to a patient in need thereof.

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