Cytotoxic Lymphocytes Expressing Variable Region-Deleted T Cell Receptors

Abstract

[Subject] An intermediate for the short-term and low-cost production of therapeutic iPS-T cell preparations with reduced allorecognition responses in patients receiving them and a cell bank containing the intermediate is provided. [Solution]　A cytotoxic lymphocyte expressing a variable region-deleted T cell receptor (TCR) comprising a first polypeptide and a second polypeptide, the first polypeptide including a constant region of a human TCRα chain or a fragment thereof, and not including a variable region of the human TCRα chain, the second polypeptide including a constant region of a human TCRβ chain or a fragment thereof, and not including a variable region of the human TCRβ chain, wherein, the cytotoxic lymphocyte lacks antigen specificity due to the expression of the variable region-deleted TCR; the cytotoxic lymphocyte is differentially induced from a pluripotent stem cell into which a nucleic acid encoding the variable region-deleted TCR is introduced from outside the cell; and the cytotoxic lymphocyte is an intermediate for producing antigen-specific iPS-T cells by introducing a nucleic acid encoding a receptor that recognizes an antigen from outside the cell and expressing the receptor; and a cell bank containing the cytotoxic lymphocytes.

Description

Cytotoxic Lymphocytes Expressing Variable Region-Deleted T Cell ReceptorsThe present invention relates to pluripotent stem cell-derived T cells for use in immuno-cell therapy. Specifically, the invention relates to T cells derived from induced pluripotent stem cells (iPS cells) which have been differentially induced from iPS cells and endowed with various antigen specificities by introducing additional genes of desired T cell receptors (TCR) or chimeric antigen receptors (CAR), or the both (TCR / CAR) into cytotoxic lymphocytes (CL) lacking antigen specificity (TCR-iPS-T, CAR-iPS-T, or dual-type TCR / CAR-iPS-T, respectively). Furthermore, the invention relates to a cell bank composed of the cytotoxic lymphocytes for use as an intermediate for productions of the TCR-iPS-T, CAR-iPS-T, or dual-type TCR / CAR-iPS-T that recognize antigens derived from tumors or pathogens and are used in immuno-cell therapy.Background TechnologyT cells are one type of immune cells that have antigen recognition sites called T cell receptors (TCRs) on their surface. The T cells express TCRs, which are receptors used by T cells to recognize antigens, on their cell surfaces. TCRs are receptors used by T cells to recognize antigens and are composed of a heterodimer of α chain (TCRα) and β chain (TCRβ), or γ chain (TCRγ) and δ chain (TCRδ). Among TCRs, TCRαβ heterodimers formed by TCRα and TCRβ each consist of a variable region and a constant region with sequence diversity, specifically recognize the major histocompatibility complexes (MHCs) and peptide complexes on target cells using the antigen recognition sites formed by the variable region of TCRαβ heterodimers. In addition, it is considered that the antigen recognition sites of TCRαβ heterodimers also recognize whether a target cell is autologous or not, and is also involved in allorecognition responses in vivo.TCRαβ heterodimers are expressed on the cell membrane by forming complexes with CD3 subunits (CD3ε, CD3δ, CD3γ and CD3ζ). TCRαβ heterodimers themselves are specialized for recognizing and binding antigens and have no signal transduction ability by themselves. Instead of that, antigen stimulation signals are transduced further downstream due to phosphorylation and activation of intracellular region of CD3 subunits by aggregation of various adapter molecules, co-receptors, or enzyme molecules triggered by TCR-mediated antigen recognition. This TCR / CD3 complex signal not only functions as a survival or exclusion signal in the positive or negative selection that immature T cells undergo during the T cell differentiation process in the thymus, but also functions as an activation or proliferation signal in T cells that have migrated to the periphery after maturation in the thymus.T cells are broadly classified into helper T cells and cytotoxic T cells. T cell therapy methods that utilize the antitumor effects of cytotoxic T cells have been developed. CAR-T therapy, which expresses chimeric antigen receptors (CARs) on T cells to treat tumors and infectious diseases, has been put into practical use as a T cell therapy using autologous T cells, and has already been approved as a cell medicine in Europe, America, and Japan. In the future, therapies using allogeneic T cells will be required to reduce the burden on patients associated with T cell collection, improve the quality of T cells, and reduce manufacturing costs.　　However, in the case of allogeneic T cell therapy, various TCRαβ heterodimers which possess allogeneic T cells induce strong allorecognition responses, resulting in tissue injury, i.e., the on-set of graft versus host disease (GVHD), which is a problem. Currently, in order to solve this problem, attempts are being made to reduce or eliminate allorecognition responses by knocking down or knocking out TCR genes (Non-Patent Documents 1 and 2).In recent years, a method for differential induction of T cells from induced pluripotent stem cells (iPSCs) established from allogeneic T cells has been proposed as a means of producing homogeneous and highly functional T cell preparations without relying on donors (Patent Document 1, Non-Patent Documents 3 to 5).Furthermore, in order to differentially induce T cells efficiently from non-T cell-derived iPSCs, it is considered that it is important to express a functional TCR by gene transfer and stimulate the TCR / CD3 complex formed thereby in vitro to promote proliferation and survival (Non-Patent Document 6). When the TCR gene is knocked down or knocked out, the CD3 subunit is no longer retained on the surface of cell membrane, and therefore proliferation and survival signals via the TCR / CD3 complex are no longer received. As an example of this, it is known that CD3 agonist antibodies and the like used to expand donor-derived T cell preparations become ineffective ex vivo. In order to proliferate and survive desired T cells by CD3 signals at appropriate times, it is desirable that the TCRαβ complex be expressed on the cell surface. In addition, in order to prevent allorecognition responses by allogeneic T cells, it is necessary that the TCRαβ complex does not recognize allo-antigens such as allo-HLA-peptide complexes. For this reason, a TCR with a modified structure has been proposed so that it does not induce antigen responsiveness, retains the CD3 subunit in the cell membrane, and can transduce the CD3 signals (Patent Document 2).In order to provide a more versatile allogeneic iPS cell-derived T cell (iPS-T) preparation, a method has also been proposed that utilizes an iPS cell bank established from blood cells of donors (non-T cells) that have homogeneous human leukocyte antigen (HLA) haplotypes, which are human MHC (Non-Patent Document 7).In order to provide iPS-T preparations, it is necessary to use a master cell bank of iPSCs and go through various manufacturing processes such as gene editing (genome editing) process, differential induction and maturation process, and expansion culture process. Furthermore, it is necessary to carry out the above processes while meeting the Good Manufacturing Practice (GMP) standards set by each country. It is known that it takes a long time to produce iPS-T preparations from the iPSC stage. Therefore, in order to commercialize iPS-T preparations, the necessity of facilitating and shortening the manufacturing time has been studied (Patent Documents 3 to 9).Since iPSCs are multipotent, in order to manufacture uniform cells in all of the above-mentioned processes, high-quality manufacturing and quality control are required each time, and as a result, various costs such as manufacturing period and manufacturing cost become extremely high. Besides, each of the above-mentioned costs may accumulate in the manufacturing processes of the T cell preparations, resulting in serious economic problems. Therefore, in order to provide iPS-T cell therapy to large number of patients, it is desired to construct a supply method that complies with GMP, supplies high-quality, low-cost iPS-T cell preparations, and can manufacture multiple types of such preparations in a short period of time (Patent Document 10).The enhanced cytotoxicity of the iPS-T cell preparations is important. Regarding iPSC-derived CAR-iPS-T preparations, it has been reported that the cytotoxic activity is increased in cells that simultaneously express the fusion protein IL-15 / IL-15Rα compared to the case when only CAR is expressed (Patent Documents 7 and 9). Similarly, in research and development aimed at enhancing the cytotoxicity, reports have been made in recent years on hybrid T cell therapy that takes advantage of the strengths of both CAR-T therapy and TCR-T therapy. This aims to support TCR while weakening the effect of CAR while retaining its cancer specificity, thereby maintaining the antigen specificity of the TCR and enhancing the therapeutic effect. In addition, it is believed that this hybrid T cell therapy may be an effective treatment method with few side effects.It is proposed that iPSCs are induced from human T cells, the resulting iPSCs are differentially induced into T cells again while retaining the recombinant structure of the TCR gene of the original human T cells, and then used for immune cell therapy (Non-patent Documents 3 and 4). Also, reports have been made on additional expression of TCRα due to TCRα somatic recombination along with differentiation into mature T cells, when obtaining T cell-derived iPSCs by the method described in the above documents and differentially inducing into mature T cells again (Non-Patent Document 3). Expression of this additional TCRα not only causes the antigen specificity of the TCR to be lost, but also may generate autoreactive T cells due to unintended mispairing with TCRα. When autoreactive T cells are generated, an autoimmune reaction that damages normal tissues may occur, which is extremely dangerous (Non-Patent Document 8). Regarding immune rejection, in order to generate iPSCs with low risk of immune rejection during allotransplantation, methods which use genome editing technology to knock out the β2-microglobulin (B2M) gene or the MHC class II transactivator (CIITA) gene, which are important for the existence of HLA molecules have been developed (Non-patent Documents 9 and 10). When introducing exogenous TCRαβ into peripheral blood T cells, by mispairing with the transgenic TCR and the endogenous TCRα or TCRβ originally possessed by T cells and expressing it on the cell surface, TCR may causes unexpected GVHD, and T cells may be activated by signals from exogenous TCR and may excessively exhibit the antigen reactivity of endogenous TCR (Non-Patent Document 11).Prior Art DocumentsPatent Documents1. Pamphlet of WO 2016 / 0101542. Pamphlet of WO 2020 / 1383713. Pamphlet of WO 2017 / 1797204. Pamphlet of WO 2013 / 1761975. Pamphlet of WO 2022 / 2166246. Pamphlet of WO 2011 / 0964827. Pamphlet of WO 2020 / 0133158. Pamphlet of WO 2017 / 0880129. Pamphlet of WO 2020 / 03217910. Pamphlet of WO 2021 / 106832Non-Patent Documents1. Okamoto S. et al, Cancer Res. 69: 9003-9011, 2009.2. Qasim W. et al, Sci Transl Med. 9, 2017.3. Nishimura T. et al, Cell Stem Cell. 12: 114-126, 2013.4. Vizcardo R. et al, Cell Stem Cell. 12: 31-36, 2013.5. Themeli M. et al, Nat. Biotechnol. 31: 928-933, 2013.6. Minagawa et al, Cell Stem Cell. 23: 850-858, 2018.7. Kaneko Shin, Current Medicine, 69: 724-733, 2014.8. Bendle GM et al, Nat Med. 16: 565-570, 2010.9. Torikai H. et al., Blood. 122, 1341-1349.10. Sugita S. et al, Stem Cell Reports. 7, 619-634.11. Akatsuka Yoshiki, Journal of the Japanese Society of Internal Medicine,108: 1384-1390, 2019.12. Robbins P. et al., J. Immunol. 180: 6116-6131, 2008.Summary of the InventionProblems that the Invention Aims to SolveBased on the above-mentioned background technology, one aspect of manufacturing iPS-T cell preparations is to differentiate iPS cells by the processes of introducing and expressing an antigen-specific TCR into iPS cells and inputting TCR activation signals into the cells. In order to treat a larger number of patients, iPS-T cell preparations that are tailored to each patient's different tumor or pathogen antigens are required. However, considering that manufacturing iPS-T cells that individually correspond to the antigens of the patient to be treated requires a long period of time, this is unrealistic in terms of manufacturing period and manufacturing cost. One possible method for solving the problems of manufacturing period and manufacturing cost is as follows. That is, some kind of TCR for differentiation is introduced into iPS cells and expressed, and iPS-T cells produced by differentially inducing from these cells are made into a cell bank as production intermediate cells and temporarily stored. Then, a desired therapeutic TCR corresponding to the antigen of the patient to be treated is additionally expressed in the stored stock cells, and various iPS-T cell preparations for final treatment are manufactured with a high degree of variation. This makes it possible to shorten the manufacturing period and reduce manufacturing costs.A concern with the methods described above is the generation of mispaired TCRs that can occur due to the simultaneous expression of two types of TCRs, a differentiation TCR and an additional therapeutic TCR, in one iPS-T cell. For example, if a mispaired TCR occurs due to the association of the differentiating TCRα chain and the therapeutic TCRβ chain, or the association of the differentiating TCRβ chain and the therapeutic TCR chain α, the actual problems that may arise include (1) the possibility of a decrease in therapeutic efficacy due to a decrease in the expression level of the therapeutic TCR, and (2) the possibility of the onset of GVHD due to mispaired TCR.In order to solve the problem caused by mispaired TCR, the properties of the differentiation TCR expressed at the iPS cell stage are important. Desirable properties which are required include (1) that the generation of mispaired TCRs can be avoided or reduced by eliminating or reducing the expression of differentiation TCRs at a stage prior to gene introduction of therapeutic TCRs; and (2) that even if mispaired TCRs occur, they do not function as TCRs. Therefore, it is necessary to differentiate cells using the TCR for differentiation with the above properties to produce iPS-T cells, and generate intermediate cells and cell stocks thereof for the production of therapeutic iPS-T cell preparations at the stage when the expression of TCR for differentiation disappears or decreases. If this problem can be solved, iPS-T cell preparations with low risk of reduced therapeutic efficacy and developing GVHD can be produced quickly and at low cost. Therefore, a method for producing various iPS-T cell preparations as described above can be developed, a variety of highly variable iPS-T cell preparations that are considered to have high therapeutic efficacy in response to neoantigens specific to each individual patient can be produced quickly and inexpensively, and it is expected that on-demand personalized medical care that is optimal for each patient can be provided.Another problem with iPS-T cell preparations is that iPS-T cell preparations derived from other people's cells are rejected by the patient's immune system, reducing their therapeutic efficacy. In order to avoid this problem, the challenge is to create low-immunogenic iPS cells by knocking out the gene using gene editing at the iPS cell stage, and then differentiate the cells using a differentiation TCR to create a manufacturing intermediate cell stock, so that the iPS-T cell preparation does not express protein molecules that could be targeted by the patient's immune cells. If this problem is resolved, it will be possible to rapidly produce iPS-T cell preparations with higher therapeutic efficacy at low cost. Furthermore, it is also a problem to introduce cytokines and various receptor proteins that enhance the functions of iPS-T cell preparations in advance at the iPS cell stage, differentiate the cells using a differentiation TCR to create a manufacturing intermediate cell stock. By resolving this problem, it will be possible to rapidly produce iPS-T cell preparations with even higher therapeutic efficacy at lower cost. One of the objectives of this application is to avoid the immune rejection response in patients undergoing the cell therapy. To achieve this, it is also possible to prepare a manufacturing intermediate cell stock by introducing a TCR for differentiation into iPS cells derived from the patient's own cells. Furthermore, it is possible to produce iPS-T cell preparations with the patient's own therapeutic TCRs using the manufacturing intermediate cell stock.Means to Solve the ProblemsIn order to solve the above-mentioned problems, the present inventors used the technology described in Patent Document 2 mentioned above to first completely delete the variable regions of each of TCRα and TCRβ of wild-type full-length TCR (wtTCR), focus on technology related to TCRαβ that retains at least part of the constant region, verify whether CD3 complex expression could be observed on the cell membrane surface by newly introducing a combination of these deletions into iPS cells (iPSCs).Here, when a gene-edited T cell receptor (variable region-deleted TCR) that does not contain the variable regions of TCRα and TCRβ but contains at least part of the constant region is introduced into iPSCs, it was confirmed that cells differentiate into CD4CD8 double-positive (DP) cells, after differentiation into hematopoietic stem cells (HPC) (Hereinafter, variable region deleted TCR will be referred to as "d TCR", and iPSCs expressing dTCR will be referred to as "dTCR iPSC".). Furthermore, it was confirmed that CD8αβ monopositive (SP) cells could be induced from the CD4CD8 DP cells.FIGS. 1A and 1B are schematic diagrams showing the structures of wtTCR and dTCR, respectively. wtTCR and CD3 molecules (ε, δ, γ, and ζ chains) are expressed on the cell membrane by forming a complex. CD3 alone or TCR alone is not expressed on the cell membrane. The TCR variable region exists outside the cell and recognizes the antigen HLA / peptide complex. When the variable region and antigen bind with appropriate binding force, an activation signal is induced and T cells are activated (FIG. 1A). On the other hand, dTCR forms a complex with CD3 molecules and is expressed on the cell membrane, but cannot recognize antigens because it does not have a variable region that recognizes antigens. However, by cross-linking the dTCR / CD3 complex using an anti-CD3ε antibody (OKT3), it is possible to induce T cell activation signals (FIG. 1B).As an additional validation test, the inventors analyzed cells that were CD8αβ SP by flow cytometry. As a result, it was seen that in the maturation process in which cells become CD8αβ SP, the expression levels of CD3 molecules retained on the cell membrane surface and dTCR on the cell membrane surface tended to decrease as maturation progressed in the maturation process. It is described in the section of problem to be solved by the invention in the above-mentioned Patent Document 2 that "in order to proliferate and survive desired T cells by CD3 signals at appropriate times, it is desirable that the TCRα / TCRβ complex be expressed on the cell surface". Based on this description, the cells having the above characteristics obtained by the present inventors are different from the cells used as a means for solving the problem in the invention of Patent Document 2 above. That is, the present inventors discovered a cell with new characteristics that have not been confirmed so far.The above-mentioned cells have different characteristics from conventionally known cytotoxic T cells in that (1) dTCR has lost antigen recognition ability, and (2) expression levels of dTCR and CD3 molecules on the cell membrane surface are reduced, which is completely different from the previously known phenotypes of cytotoxic T cells. Therefore, in this application, cells having the above characteristics are defined as "cytotoxic lymphocytes without antigen specificity" (Hereinafter, iPSC-derived cytotoxic lymphocytes expressing dTCR will be referred to as "dTCR iPS-CL."). Furthermore, in the present application, the cytotoxic lymphocytes are positioned as an intermediate for producing a cytotoxic iPS-T cell (also referred to as killer T cell in the present invention) preparation.Furthermore, we additionally introduced a TCR gene that recognizes tumor-associated antigens into cytotoxic lymphocytes, which are intermediates for producing cytotoxic iPS-T cell preparations. The cytotoxic lymphocytes are equipped with dTCR in which at least the variable region of TCRαβ has been deleted. However, even if mispairing occurs between the additionally introduced TCR and the dTCR, the TCR subunit derived from the dTCR does not have at least a variable region. Accordingly, a mispaired TCR formed by combining a dTCR-derived subunit and an additional TCR subunit is considered to be incapable of antigen recognition within a patient's body. In addition, in the process of maturation into CD8αβ SP cells, as mentioned above, the expression levels of CD3 molecules and dTCR on the cell membrane surface of the cytotoxic lymphocytes decrease. Accordingly, it is considered that even if a TCR gene is additionally introduced, mispairing with the dTCR is unlikely to occur, and the probability of forming a mispaired TCR is low. In fact, even when the cytotoxic lymphocytes have additionally introduced a TCR gene that recognizes an antigen into the intermediate, it was found that the antigen recognition ability of the obtained cytotoxic T cells was reduced to such an extent that the possibility of causing GVHD due to mispaired TCR was not a problem.FIG. 2 shows the processes involved in clinical application of iPS-T cells expressing dTCR. In this process, first, dTCR is introduced into low-immunogenic iPSCs created by gene editing. After creating dTCR iPSCs expressing dTCR, the cells are stimulated with anti-CD3 antibody to induce differentiation into T cells, and a dTCR iPS-CL cell bank is created. Next, the dTCR iPS-CL stored in this cell bank was used as a manufacturing intermediate. A CAR and / or TCR that recognizes an arbitrary antigen is introduced into the dTCR iPS-CL and expressed, and finally various genetically modified therapeutic iPS-T cells are produced. By using the dTCR iPS-CL stored in the cell bank as an intermediate for the production of therapeutic iPS-T cells, low immunogenic antigen-specific iPS-T cells with TCR appropriate for individual patient tumor-associated antigens, antigen-specific iPS-T cells with CAR, or antigen-specific iPS-T cells with dual TCR / CAR can be delivered to many patients on-demand and at low cost.　That is, the present inventors have discovered that personalized medicine using genetically modified iPS-T cells is possible at reduced cost, and have completed the present invention.That is, the present invention provides the following.[1] A cytotoxic lymphocyte expressing a variable region-deleted T cell receptor (TCR) comprising a first polypeptide and a second polypeptide, the first polypeptide including a constant region of a human TCRα chain or a fragment thereof, and not including a variable region of the human TCRα chain, the second polypeptide including a constant region of a human TCRβ chain or a fragment thereof, and not including a variable region of the human TCRβ chain, wherein:the cytotoxic lymphocyte lacks antigen specificity due to the expression of the variable region-deleted TCR;the cytotoxic lymphocyte is differentially induced from a pluripotent stem cell into which a nucleic acid encoding the variable region-deleted TCR is introduced from outside the cell; andthe cytotoxic lymphocyte is an intermediate for producing an antigen-specific killer T cell by expressing one or more receptors that may recognize an identical or different antigen.[2] The cytotoxic lymphocyte according to [1], wherein the cytotoxic lymphocyte comprises the nucleic acid encoding the variable region-deleted TCR introduced from outside the cell.[3] The cytotoxic lymphocyte according to [1], wherein the cytotoxic lymphocyte comprises an expression vector containing the nucleic acid encoding the variable region-deleted TCR.[4] The cytotoxic lymphocyte according to [1], wherein the pluripotent stem cell is a human induced pluripotent stem cell.[5] The cytotoxic lymphocyte according to [1], wherein one of the one or more receptors that may recognize an identical or different antigen is a TCR containing a variable region recognizing an antigen (an antigen-recognizing TCR) and the cytotoxic lymphocyte expresses CD8α and CD8β chains double positively functioning as co-receptors for the antigen-recognizing TCR.[6] A cell bank comprising the cytotoxic lymphocyte according to any one of [1] to [5].[7] The cell bank according to [6], wherein the cytotoxic lymphocyte is a cell to be introduced with one or more nucleic acids, each encoding a different receptor that may recognize an identical or different antigen.[8] The cell bank according to [6], wherein the cytotoxic lymphocyte is cryopreserved.[9] The cell bank according to [7], wherein one or more nucleic acids, each encoding a different receptor that may recognize an identical or different antigen, are introduced into the cytotoxic lymphocyte, wherein the cytotoxic lymphocyte serves as an intermediate for producing the antigen-specific killer T cell used for prophylaxis and / or treatment of cancer.

[0010] Use of the cell bank according to [6] for producing the antigen-specific killer T cell used for prophylaxis and / or treatment of cancer.

[0011] An antigen-specific killer T cell produced from the cytotoxic lymphocyte stored in the cell bank according to [6].

[0012] A pharmaceutical composition comprising the antigen-specific killer T cell according to

[0011] .

[0013] A method for prophylaxis and / or treatment of cancer, using the pharmaceutical composition according to

[0012] .

[0014] The cytotoxic lymphocyte according to any one of [1] to [5], wherein the cytotoxic lymphocyte is capable of expressing one or more receptors that may recognize an identical or different antigen.

[0015] The cytotoxic lymphocyte according to

[0014] , wherein the cytotoxic lymphocyte comprises one or more nucleic acids, each encoding a different receptor that may recognize an identical or different antigen.

[0016] The cytotoxic lymphocyte according to

[0014] , wherein the cytotoxic lymphocyte comprises one or more expression vectors, each including a different nucleic acid encoding a different receptor that may recognize an identical or different antigen.

[0017] The antigen-specific killer T cell induced by expressing one or more receptors that may recognize an identical or different antigen in the cytotoxic lymphocyte according to

[0014] .

[0018] The antigen-specific killer T cell according to

[0017] , wherein one of the one or more receptors that may recognize an identical or different antigen is a TCR containing variable region recognizing antigen (an antigen-recognizing TCR).

[0019] The antigen-specific killer T cell according to

[0018] , wherein the antigen-recognizing TCR recognizes a single antigen.

[0020] The antigen-specific killer T cell according to

[0019] , wherein the single antigen is a viral antigen or a tumor-associated antigen.

[0021] The antigen-specific killer T cell according to

[0020] , wherein the viral antigen is selected from the group consisting of viral proteins and fragments thereof produced in cells infected with influenza virus, EBV, HPV, HBV, HCV, HIV, coronavirus, CMV, dengue virus, West Nile virus, hantavirus, Ebola virus, and HTLV-1.

[0022] The antigen-specific killer T cell according to

[0020] , wherein the tumor-associated antigen is selected from the group consisting of GPC3, WT1, XAGE1, LMP2, NY-ESO-1, KRAS, PI3K, MUC1, EGFR, HER-2 / neu, MAGEA-3, PSMA, CEA, MART1, gp100, bcr-abl, hTERT, p53, BCMA, MUC5A1, MUC6, MAGE-A1, PRAME, SSX2 / 4, PSCA, CTLA-4, GD2, GD3, fucosyl GM1, GM3, sLe(a), glycolipid F77, mesothelin, PD-L1, trp1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, ETV6-AML, PSA, AFP, EpCAM, ALK, androgen receptor, EphA2, CYP1B1, OY-TES-1, MAD-CT-2, survivin, Ras, EGR, XBP-1, neoantigens due to genetic mutations, neoantigens due to splicing abnormalities, and fragments thereof.

[0023] The antigen-specific killer T cell according to

[0020] , wherein the tumor-associated antigen is HLA-A24-restricted GPC3 peptide EYILSLEEL (Seq. No. 1) or HLA-A2-restricted GPC3 peptide FVGEFFTDV (Seq. No. 2).

[0024] A pharmaceutical comprising the antigen-specific killer T cell according to

[0018] .

[0025] The pharmaceutical according to

[0024] , for use in prophylaxis and / or treatment of cancer.

[0026] A cytotoxic agent for cells expressing a viral antigen, the cytotoxic agent comprising the antigen-specific killer T cell according to

[0018] .

[0027] The cytotoxic agent according to

[0026] , wherein the viral antigen is selected from the group consisting of viral proteins and fragments thereof produced in cells infected with influenza virus, EBV, HPV, HBV, HCV, HIV, coronavirus, CMV, dengue virus, West Nile virus, hantavirus, Ebola virus, and HTLV-1.

[0028] A cytotoxic agent for cells expressing a tumor-associated antigen, the cytotoxic agent comprising the antigen-specific killer T cell according to

[0018] .

[0029] The cytotoxic agent according to

[0028] , wherein the tumor-associated antigen is selected from the group consisting of GPC3, WT1, XAGE1, LMP2, NY-ESO-1, KRAS, PI3K, MUC1, EGFR, HER-2 / neu, MAGEA-3, PSMA, CEA, MART1, gp100, bcr-abl, hTERT, p53, BCMA, MUC5A1, MUC6, MAGE-A1, PRAME, SSX2 / 4, PSCA, CTLA-4, GD2, GD3, fucosyl GM1, GM3, sLe(a), glycolipid F77, mesothelin, PD-L1, trp1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, ETV6-AML, PSA, AFP, EpCAM, ALK, androgen receptor, EphA2, CYP1B1, OY-TES-1, MAD-CT-2, survivin, Ras, EGR, XBP-1, neoantigens due to genetic mutations, neoantigens due to splicing abnormalities, and fragments thereof.

[0030] A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the antigen-specific killer T cell according to

[0018] to the mammal.

[0031] A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the pharmaceutical according to

[0024] to the mammal.

[0032] A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the cytotoxic agent according to

[0026] to the mammal.

[0033] A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the cytotoxic agent according to

[0028] to the mammal.

[0034] The antigen-specific killer T cell according to

[0018] , for use in prophylaxis and / or treatment of cancer.

[0035] The antigen-specific killer T cell according to

[0018] , for producing an agent for prophylaxis and / or treatment of cancer.

[0036] The antigen-specific killer T cell according to

[0017] , wherein the receptors comprise both an antigen-recognizing TCR and a chimeric antigen receptor (CAR), each of which may recognize an identical or different antigen.

[0037] A pharmaceutical comprising the antigen-specific killer T cell according to

[0036] .

[0038] The pharmaceutical according to

[0037] , for use in prophylaxis and / or treatment of cancer.

[0039] A cytotoxic agent for cells expressing a viral antigen, the cytotoxic agent comprising the antigen-specific killer T cell according to

[0036] .

[0040] A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the antigen-specific killer T cell according to

[0036] to the mammal.

[0041] A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the pharmaceutical according to

[0037] to the mammal.

[0042] A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the cytotoxic agent according to

[0039] to the mammal.

[0043] The antigen-specific killer T cell according to

[0036] , for use in prophylaxis and / or treatment of cancer.

[0044] The antigen-specific killer T cell according to

[0036] , for producing an agent for prophylaxis and / or treatment of cancer.Effects of the InventionThe cytotoxic lymphocytes of the present invention comprise at least a portion of a constant region, and express exogenous dTCR, which is a TCRαβ that does not contain a variable region and does not recognize an antigen, and CD8αβ, which strengthens the binding between the antigen and the TCR that recognizes the antigen. Therefore, the cytotoxic lymphocytes of the present invention do not themselves have antigen specificity; and can avoid or reduce mispairing with the dTCR even when a TCR that recognizes an antigen is introduced into the cytotoxic lymphocytes of the present invention. The iPS-T cell preparation produced by gene-transferring a TCR that recognizes an antigen using the cytotoxic lymphocytes of the present invention as an intermediate can reduce the risk of developing GVHD in patients receiving the formulation. Furthermore, because of the cytotoxic lymphocytes of the present invention, iPS-T cells for various treatments can be produced by introducing a nucleic acid encoding a receptor that selectively recognizes an antigen and expressing the receptor.By using the cytotoxic lymphocytes of the present invention as an intermediate for producing iPS-T cell preparations, the intermediate can be stored as a cell bank, and various genetic modifications can be performed for the purpose of imparting low immunogenicity and enhancing cytotoxic function in the differentiation / maturation stage of the process of producing the intermediate. Furthermore, by preserving the intermediate as a cell bank, it is possible to reduce the manufacturing cost of cytotoxic iPS-T cell preparations, and on-demand personalized medicine will become possible from the perspective of medical economics.FIG. 1A is a schematic diagram showing the structure of wtTCR.FIG. 1B is a schematic diagram showing the structure of dTCR.FIG. 2 is a diagram showing the processes for clinical application of iPS-T cells expressing dTCR.FIG. 3 is a schematic diagram showing the cell differential induction process after introducing the TCR gene into iPSCs (upper FIG.) and a diagram showing the results of flow cytometry analysis of the expression of CD34, CD43, CD8, CD4, CD8β, and CD8α in cells at each differentiation stage in the above process (lower FIG.).FIG. 4 is a diagram showing the results of flow cytometry analysis of the expression of CD8α, CD19, TCRαβ, and CD3 over time up to Day 14 in CD8αβ SP cells during the expansion culture process.FIG. 5 is a diagram showing the cell proliferation rate of each cell differentially induced from HPC.FIG. 6 is a diagram showing the final manufacturing process in processes of manufacturing iPS-T cell medicine by using dTCR iPS-CL cells with immune reduction function stored in a cell bank as a manufacturing intermediate, and introducing / expressing TCR and / or CAR genes capable of recognizing antigens corresponding to each patient's antigen into the cells in a short period of time.FIG. 7 is a diagram showing the results of flow cytometry analysis of tEGFR expression in dTCR iPS-CL cells into which CD20 CAR and tEGFR genes have been introduced.FIG. 8 is a diagram showing the results of analyzing the reactivity of CD20 CAR-expressing dTCR iPS-CL cells to target cells.FIG. 9 is a diagram showing the results of analyzing the reactivity of CD20 CAR-expressing dTCR iPS-CL cells to target cells.FIG. 10 is a diagram showing the results of analyzing the reactivity of CD20 CAR-expressing dTCR iPS-CL cells to target cells.FIG. 11 is a diagram showing the analysis results of the cytotoxic activity of CD20 CAR-expressing dTCR iPS-CL cells against target cells.FIG. 12 is a diagram showing the results of flow cytometry analysis of GFP expression in dTCR iPS-CL cells into which TCR82 and GFP genes were introduced.FIG. 13 is a diagram showing the results of analyzing TCR expression in TCR82-transduced dTCR iPS-CL using a TCR82 antigen tetramer and a TCRαβ antibody.FIG. 14 is a diagram showing the results of analyzing the antigen reactivity of TCR82-expressing dTCR iPS-CL cells.FIG. 15 is a diagram showing the analysis results of cytotoxic activity of TCR82-expressing dTCR iPS-CL cells against target cells.FIG. 16 is a diagram showing that the TCR82 gene additionally transduced into dTCR iPS-CL cells is stably expressed.FIG. 17 is a diagram showing amino acid sequence information of ba2 and ba9.FIG. 18 is a diagram showing that 1G4 TCR is functionally expressed in iPS-T cells differentiated from iPSCs transduced with 1G4 TCR.FIG. 19 is a diagram showing that TCR Clone1 obtained from a healthy individual is an HPV E7-specific TCR.FIG. 20 is a diagram explaining that a variety of cancer-specific iPS-T cells can be produced from the dTCR iPS-CL cell bank.Embodiments for Carrying out the InventionVariable Region-Deleted TCRThe variable region-deleted TCR (dTCR) in the present invention comprises a polypeptide including a constant region of a human TCRα chain or a fragment thereof and not including a variable region of the human TCRα chain, and a polypeptide including a constant region of a human TCRβ chain or a fragment thereof, and not including a variable region of the human TCRβ chain.The TCRα gene locus is composed of a variable region (V region), a joining region (J region), and a constant region (C region). On the other hand, the TCRβ gene locus is composed of a V region, a diversity region (D region), a J region, and a C region. The TCR gene locus undergoes somatic recombination during the T cell differentiation process. The V region and J region in TCRα and the V region, D region, and J region in TCRβ are connected in various combinations. In addition, variable exons with rich diversity are formed by randomly inserting or deleting bases at each linkage site. During the gene transcription process, variable region exons are joined to C region exons by RNA splicing, thereby forming full-length TCR gene mRNA. Subsequently, the TCRα and TCRβ chains translated as proteins form a heterodimer and are presented on the cell surface as a TCR.The constant region in the present invention refers to sequences excluding C region sequences in genes, and sequences excluding variable domains in polypeptides (non-variable regions). In the present invention, a polypeptide containing a fragment of the constant region of the TCRα chain and TCRβ chain may be a polypeptide having any amino acid sequence as long as it is a partial sequence of the amino acid sequence of the constant region. Furthermore, in the partial sequence, it may also be a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, inserted, deleted, and / or added.Cytotoxic Lymphocyte (CL)In general, CD8-positive T cells, which are cytotoxic lymphocytes, kill target cells based on the antigen specificity of TCR. On the other hand, the cytotoxic lymphocytes of the present invention are CD8 positive but express TCR without antigen specificity. Accordingly, they are different from general cytotoxic T cells because they do not exhibit or exhibit little TCR-dependent cytotoxic activity. Therefore, although the cytotoxic lymphocytes of the present invention are cells that do not exhibit or exhibit little cytotoxicity, in the future, these cells will be able to selectively add cytotoxic activity with antigen specificity.The cytotoxic lymphocytes of the present invention can be used as intermediates for producing cytotoxic antigen-specific iPS-T cells by introducing a nucleic acid encoding a TCR that recognizes a desired antigen from outside the cell and expressing the TCR. "A TCR that recognizes a desired antigen" means a receptor consisting of a heterodimer of TCRα chain and TCRβ chain that recognizes an antigen or the antigen-HLA (human leukocyte antigen) complex (MHC; major histocompatibility complex) and transmits a stimulating signal to T cells. Each TCR chain is composed of a variable region and a constant region formed by somatic recombination consisting of a V region, a D region, and a J region, which are arranged on the chromosome as a number of fragments. There are three complementarity determining regions (CDR1, CDR2 and CDR3) in the variable region. TCRs that recognize antigens include those lacking part or all of the constant region, and those with recombinant amino acid sequences.A nucleic acid encoding a chimeric antigen receptor (CAR) that recognizes a desired antigen may be introduced extracellularly into the cytotoxic lymphocytes of the present invention, and the CAR may be expressed. CAR is a fusion protein between the antigen recognition portion of an antibody that specifically recognizes an antigen and the intracellular domains of 4-1BB (CD137) and CD3ζ. CAR-T cells can recognize antigens expressed on the cell surface in a non-HLA-restricted manner.Nucleic acids encoding a TCR and a CAR that recognize a desired antigen may be introduced individually into the cytotoxic lymphocytes of the present invention, and each may be expressed independently. Furthermore, nucleic acids encoding both TCR and CAR may be introduced into the cytotoxic lymphocytes of the present invention, and both TCR and CAR may be expressed. In the present invention, TCR and CAR may both recognize the same antigen, or each may recognize different antigens. In cases where both the TCR and CAR recognize the same antigen, each of these receptors may recognize different epitopes on the same antigen.Pluripotent Stem CellIn the present invention, pluripotent stem cells are stem cells that have both the ability to proliferate almost infinitely (self-propagation ability) and the ability to differentiate into almost all cells constituting an individual (pluripotency). Pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ cells (EG cells), multipotent germline stem cells (mGS cells), nuclear transfer embryonic stem cell (ntES cells), and multi-lineage differentiating stress enduring cells (Muse cells) isolated from fibroblasts and bone marrow mesenchymal cells, etc. As pluripotent stem cells, iPS cells may be used, taking note that they do not involve destruction of embryos, eggs, etc., and that they have high differentiation potential.Production of iPS CellsiPS cells are produced by reprogramming mammalian somatic cells. Mammals include humans, monkeys, pigs, dogs, cats, rats, and mice, etc., however, humans are preferred. The somatic cells are not particularly limited, however, cells isolated from peripheral blood can be used.In one embodiment of the present invention, the somatic cells may be peripheral blood mononuclear cells from which B cells and T cells have been removed. The peripheral blood mononuclear cells from which B cells and T cells have been removed are obtained by isolating mononuclear cells from whole blood with mononuclear cell separation solution, and then removing B cells and T cells using surface antigens expressed on B cells and T cells. Examples of mononuclear cell separation solutions include Lymphoprep (registered trademark). To remove B cells and T cells from mononuclear cells, for example, magnetic beads such as flow cytometry or MACS (registered trademark) beads may be used, utilizing CD19, CD20, CD22 which are surface antigens possessed by B cells, or B cell receptors, and antibodies against CD3, CD4, or CD8, which are surface antigens possessed by T cells.Methods for producing iPS cells are known in the art. (for example, Takahashi K, Yamanaka S. Cell. 2006; 126:663-676. Takahashi K, et al. Cell. 2007; 131:861-872. Nakagawa M, et al. Nat Biotechnol. 2008;26:101-106.) In one embodiment of the invention, iPS cells can be induced by introducing cell reprogramming factors into peripheral blood mononuclear cells depleted of B cells and T cells. Examples of cell reprogramming factors include gene or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, β-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1 etc. These cell reprogramming factors may be used alone or in combination. From the viewpoint of efficient establishment of iPS cells, Oct3 / 4, Sox2, Klf4, and c-Myc (so-called Yamanaka 4 factors) among these cell reprogramming factors may be introduced into the peripheral blood mononuclear cells.There is no particular limitation on the method for introducing cell reprogramming factors into the peripheral blood mononuclear cells, and methods known in the art can be employed. For example, when introducing a gene encoding the cell reprogramming factor into the peripheral blood mononuclear cells, a gene (e.g., cDNA) encoding the cell reprogramming factor can be inserted into an expression vector containing a promoter that functions in the peripheral blood mononuclear cells, and introduced into the peripheral blood mononuclear cells. When the cell reprogramming factor is in the form of a protein and the protein is introduced into the peripheral blood mononuclear cells, a method using a protein introduction reagent, a method using a protein transduction domain fusion protein, electroporation method, and microinjection method can be exemplified. When the cell reprogramming factor is in the form of messenger RNA (mRNA) and the mRNA is introduced into the peripheral blood mononuclear cells, a method using mRNA introduction reagent and electroporation method can be exemplified.As expression vectors, viral vectors and non-viral vectors can be used. Examples of the viral vectors include lentivirus, retrovirus, adenovirus, adeno-associated virus, herpesvirus, and Sendai virus, etc. When using a transposon vector as a non-viral vector, piggyBac (registered trademark) vectors are preferred. The transposon method is a next-generation gene introduction method that is cheaper and safer than the conventional viral vector method. Examples of gene introduction methods using non-viral vectors include methods of lipofection, liposome, calcium phosphate coprecipitation, DEAE-dextran, microinjection, and electroporation. It is preferable to use electroporation to introduce the transposon vector into cells. As the electroporation device, the gene introduction device ExPERT (registered trademark) system (MaxCyte) can be used.Examples of promoters used in the expression vector used when introducing the gene encoding the cell reprogramming factor into the peripheral blood mononuclear cells include, for example, EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, and ubiquitin promoter, etc. These promoters may be capable of controlling the expression of genes inserted downstream of the promoter, depending on the presence or absence of drugs such as tetracycline. In addition to the promoter, the expression vector can contain an enhancer, a poly A addition signal, a selection marker gene (e.g., neomycin resistance gene), and an SV40 origin of replication, etc.Introduction of Variable Region-Deleted TCR (dTCR) into iPS CellsNucleic acids encoding dTCR can be incorporated into expression vectors. For example, a cDNA encoding a TCR may be incorporated into a viral or non-viral vector (transposon vector) using the Gibson assembly system. Specifically, a gene in which cDNA encoding the dTCR α chain and β chain is linked via a T2A sequence is linked downstream of the EF1α promoter (human polypeptide chain elongation factor gene promoter) and the ubiquitin promoter, etc., and furthermore, downstream thereof, a marker gene such as EGFR (tEGFR, truncated EGFR), which excludes the ligand binding site and intracellular domain, or CD19, which lacks the intracellular domain, is linked to the IRES (internal ribosome entry site) sequence. This construct may be incorporated into a viral or non-viral vector. The cDNAs encoding the dTCR α chain and β chain may be incorporated into separate expression vectors.As vectors, viral vectors and non-viral vectors can be used. Examples of the viral vectors include lentivirus, retrovirus, adenovirus, adeno-associated virus, herpesvirus, and Sendai virus, etc. When using a transposon vector as a non-viral vector, piggyBac (registered trademark) vectors are preferred. The transposon method is a next-generation gene introduction method that is cheaper and safer than the conventional viral vector method. Examples of gene introduction methods using non-viral vectors include methods of lipofection, liposome, calcium phosphate coprecipitation, DEAE-dextran, microinjection, and electroporation. It is preferable to use the method of electroporation to introduce the transposon vector into cells. As the electroporation device, the gene introduction device ExPERT (registered trademark) system (MaxCyte) can be used.iPS Cell CultureThe medium used for culturing iPS cells is not particularly limited. It can be prepared by using a medium used for culturing animal cells as a basal medium, and adding cytokines thereto to maintain the undifferentiated ability of iPS cells. Examples of basal media include Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle Minimum Essential Medium (αMEM), Dulbecco's modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, Fischer's Medium, Neurobasal Medium (Life Technologies), StemFit (registered trademark) AK03N (Ajinomoto Healthy Supply, Inc.), and mixed media thereof. The medium may be supplemented with serum or may be serum-free. As the cytokines, bFGF may be exemplified, and concentration thereof in the medium is, for example, 1 to 100 μg / mL.The method for culturing iPS cells may be adhesive culture or suspension culture, however, adhesive culture is preferred. Examples of methods for isolating iPS cells include a method of physical isolation using a cell scraper etc. and an isolation method using a dissociation solution having protease activity; a dissociation solution having collagenase activity, and a dissociation solution with protease and collagenase activities (such as Accutase (registered trademark) and Accumax (registered trademark)).In one embodiment of the invention, iPS cells are passaged into separate culture vessels preferably, when a cell density of 1×103to 1×104cells / cm2, 1×104to 1×105cells / cm2, or 1×105to 1×106cells / cm2is reached. The number of passages may be any number as long as the required amount of iPS cells can be obtained, preferably 1 to 5 times or 5 to 10 times.Induction of Hematopoietic Stem Cells (HPCs) from iPS CellsIn the present invention, "hematopoietic stem cells" are cells capable of differentiating into hematopoietic cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes, etc. Note that hematopoietic stem cells and hematopoietic progenitor cells are not distinguished and are referred to the same cells unless otherwise specified. The hematopoietic stem / progenitor cells are recognized, for example, by being double positive for the surface antigens CD34 and CD43.HPCs are preferably induced by culturing iPS cells in a culture medium supplemented with vitamin C. Here, "vitamin C" refers to L-ascorbic acid and its derivatives, and "L-ascorbic acid derivative" refers to one that is converted into vitamin C by an enzymatic reaction in vivo. Examples of the derivatives of L-ascorbic acid include vitamin C phosphate, ascorbic acid glucoside, ascorbyl ethyl, vitamin C ester, ascorbyl tetrahexyldecanoate, ascorbyl stearate, and ascorbic acid-2-phosphate-6-palmitic acid. The derivative of L-ascorbic acid is preferably vitamin C phosphate, including, for example, phosphate-L-ascorbate salts such as sodium phosphate-L-ascorbate or magnesium phosphate-L-ascorbate. Vitamin C is contained in a culture medium at a concentration of, for example, 5 to 500 μg / mL.The medium used for inducing HPCs is not particularly limited. It can be prepared by using a medium used for culturing animal cells as a basal medium and adding vitamin C, etc. thereto. Examples of the basal medium include IMDM, Medium 199, EMEM, αMEM, DMEM, Ham's F12, RPMI 1640, Fischer's Medium, Neurobasal Medium (Life Technologies), StemPro34 (Life Technologies), and mixed media thereof. The medium may contain serum or may be serum-free. As needed, the medium may contain one or more substances selected from, for example, albumin, insulin, transferrin, selenium, fatty acid, trace elements, 2-mercaptoethanol, thiol glycerol, monothiol glycerol, lipids, amino acid, L-glutamine, non-essential amino acids, vitamin, growth factors, low molecular weight compounds, antibiotics, antioxidant, pyruvic acid, buffering agent, inorganic salts, and cytokines etc.Cytokine selected from the group consisting of BMP4 (bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), SCF (stem cell factor), TPO (thrombopoietin), and FLT3L (Flt3 ligand) may be further added to the medium used for inducing HPCs. More preferably, a medium supplemented with VEGF, SCF and FLT3L is used. The concentrations thereof are, for example, 1 to 100 ng / mL for BMP4, 1 to 100 ng / mL for VEGF, 1 to 100 ng / mL for bFGF, and 10 to 100 ng / mL for SCF, 1 to 100 ng / mL for TPO, or 1 to 100 ng / mL for FLT3L.A TGFβ inhibitor may be added to the culture solution of HPCs. "TGFβ inhibitor" is a small molecule inhibitor that interferes with TGFβ family signal transduction, such as SB431542 and SB202190 (R.K.Lindemann et al., Mol. Cancer 2: 20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908 and SD208 (Scios), and LY2109761, LY364947 and LY580276 (Lilly Research Laboratories). The concentrations thereof when added to the medium is preferably 0.5 to 100 μM.To induce HPCs, iPS cells may be cocultured with feeder cells such as C3H10T1 / 2 (Takayama N., et al. J Exp Med. 2817-2830, 2010) or xeno-derived stromal cells (Niwa A et al. J Ce11 Physiol. 221:367-77, 2009).The method for culturing iPS cells during production of HPCs may be adhesive culture or suspension culture, however, suspension culture is preferred. For example, iPS cells can be cultured to 80% confluence in the dish used, then released, dissociated into single cells, and then subjected to suspension culture. In the case of adhesive culture, a culture container coated with a coating agent may be used. An example of the coating agent is Matrigel (Niwa A et al. J Ce11 Physiol. 221:367-77, 2009). Examples of methods for isolating iPS cells include a method of physical isolation using cell scraper etc. and an isolation method using a dissociation solution with protease and collagenase activities (such as Accutase (registered trademark) and Accumax (registered trademark)), or a dissociation solution with collagenase activity.Suspension culture refers to culturing cells in a non-adherent state in a culture container. Suspension culture is not particularly limited. In order to improve adhesion with cells, it can be carried out using a culture vessel that has not been subjected to artificial treatments (e.g., coating treatment with extracellular matrix, etc.), or a culture vessel that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylic acid (po1y-HEMA) or nonionic surfactant polyol (Pluronic F-127, etc.)). When carrying out suspension culture, it is preferable to form embryoid bodies (EBs) before culturing. When HPCs are obtained by suspension culture of embryoid bodies, it is preferable to dissociate them into single cells and then carry out adhesive culture.Hematopoietic stem cells can also be prepared from cyst-like structures (also referred to as iPS-sac) obtained by culturing iPS cells. Here, the "cyst-like structure" refers to a three-dimensional sac-like (with an internal space) structure derived from iPS cells, which is formed from a population of endothelial cells, etc., and contains HPCs inside.　Temperature conditions for culturing iPS cells to produce HPCs are not particularly limited, but are, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. Furthermore, the culture period can be appropriately determined by those skilled in the art while monitoring the number of HPCs, etc. The number of culture days is not particularly limited as long as HPCs are obtained, for example, at least 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, or 14 days or more, preferably 14 days. Long culture periods are not a problem in the production of HPCs. Furthermore, the culture may be performed under hypoxic conditions, and in one embodiment of the present invention, the hypoxic conditions include, for example, oxygen concentrations of 15%, 10%, 9%, 8%, 7%, 6%, 5% or less.Induction of CD4CD8 Double-Positive (CD4CD8 DP) Cells from Hematopoietic Stem Cells (HPCs)Induction of differentiation from HPCs to CD4CD8 DP cells can be performed in the presence of T cell differentiation-inducing factor and cell adhesion factors. As the T cell differentiation-inducing factor, a Notch ligand for Notch molecules expressed on the cell membrane can be used. Notch molecules, Notch 1 to 4, exist in mammals, and Notch signals are induced by the binding of Notch molecules and Notch ligands, and are involved in the control of cell survival, proliferation, and differentiation (Artavanis-Tsakonas S, et al. Science. 1999; 284:770-776). Notch ligands are not particularly limited as long as they induce Notch signals, and include, for example, Delta-Like-Protein (DLL) 1, 3, and 4, and Jagged 1 and 2. In the present invention, DLL1, DLL4 and Jagged 1 may be used. In the present invention, DLL4, which is an endogenous Notch1 ligand of mammals, particularly humans, may be used. The DLL4 may be a partial peptide of DLL4 as long as it induces a Notch signal. For example, a recombinant protein (Fc-DLL4) in which the Fc domain of human IgG1 (residue numbers 1 to 524 or 27 to 529 (including mutations)) is added to the C-terminus of the extracellular domain of human DLL4 can also be used.Fibronectin or a fragment thereof can be used as a cell adhesion factor. Any region of fibronectin can be selected as the fibronectin fragment as long as it retains the functions required by the present invention. Fibronectin may be of human origin or may be a recombinant protein. In the present invention, RetroNectin (registered trademark) (Takara Bio Inc.) may be used as the cell adhesion factor. RetroNectin is a recombinant protein containing cell adhesion domain that binds to α5β1integrin (VLA-5) in human fibronectin (C-domain), heparin-binding domain (H-domain), and the CS-1 site that binds to α4β1integrin (VLA-4).In cell culture for inducing differentiation of HPCs into CD4CD8 DP cells, the T cell differentiation-inducing factor and the cell adhesion factor may be present as coated on a cell culture substrate. The above-mentioned "present as coated" refers to that the T cell differentiation-inducing factor and the cell adhesion factor are adsorbed or bonded to the surface of the cell culture substrate and are in an immobilized state (coated state). The coating can be carried out by diluting the T cell differentiation-inducing factor and the cell adhesion factor to 1 to 100 μg / mL with for example, phosphate buffer or Hanks balanced salt solution, or culture medium without serum, Ca and Mg, and contacting with cell culture substrate for a certain period of time. As the cell culture substrate, commercially available cell culture beads (microcarriers) or cell culture plates can be used.Examples of basal media used for cell culture to induce differentiation from HPCs to CD4CD8 DP cells include IMDM, Medium 199, EMEM, αMEM, DMEM, Ham's F12, RPMI1640, Fischer's Medium, Neurobasal Medium (Life Technologies), and mixed media thereof. The medium may be supplemented with serum or may be serum-free. For example, 5 to 20% (v / v) fetal bovine serum (FBS) may be added to the serum. One or more substances selected from, for example, albumin, insulin, transferrin, selenium, fatty acid, trace elements, 2-mercaptoethanol, thiol glycerol, monothiol glycerol, lipids, amino acid, L-glutamine, non-essential amino acids, vitamin, growth factors, low molecular weight compounds, antibiotics, antioxidant, pyruvic acid, buffering agent, inorganic salts, and cytokines etc. may be added to the basal medium as necessary. As the cytokine, one selected from the group consisting of FLT3L and IL-7 is preferred. The concentration of IL-7 in the medium is, for example, 0.01 ng / mL to 100 ng / mL, preferably 0.1 ng / mL to 10 ng / mL.(Induction of CD8αβ Single-positive (SP) Cells from CD4CD8 double-positive (CD4CD8 DP) Cells)CD4CD8 DP cells refer to cells that are positive for both surface antigens CD4 and CD8. CD8αβ SP cells refer to cells that are positive for both CD8α and CD8β and negative for CD4.CD8αβ SP cells can be differentially induced by culturing CD4CD8 DP cells in a medium containing interleukin-7 (IL-7) and a TCR activator. This differential induction process is preferably performed by adhesive culture, but preferably cultured without using feeder cells. CD4CD8 DP cells may be cultured in direct contact with a culture vessel coated with a T cell differentiation-inducing factor and / or a cell adhesion factor. As a cell adhesion factor, fibronectin or a fragment thereof can be used. Any region of fibronectin can be selected as the fibronectin fragment as long as it retains the functions required by the present invention. As a cell adhesion factor, retroNectin may also be used. As the T cell differentiation-inducing factor, the aforementioned Notch ligand may be used. Examples of Notch ligands include Fc-DLL4. The concentration of IL-7 added to the medium in the process of differentially inducing from CD4CD8 DP cells to CD8αβ SP cells is preferably higher than the concentration of IL-7 added to the medium in the process of "inducing CD4CD8 DP cells from HPCs" described above, for example, 0.05 ng / mL to 500 ng / mL, preferably 0.1 ng / mL to 100 ng / mL, and more preferably 0.5 ng / mL to 50 ng / mL.Examples of the TCR activator include PHA (phytohemagglutinin), anti-CD3 antibody, anti-CD28 antibody, PMA (Phorbol 12-Myristate 13-Acetate), and ionomycin, etc. For example, CD4CD8 DP cells can be TCR-stimulated by culturing in a medium containing anti-CD3 antibodies. The anti-CD3 antibody may be bound to magnetic beads, etc. Alternatively, TCR may be stimulated by culturing CD4CD8 DP cells on a culture dish coated with an anti-CD3 antibody. In order to coat the surface of a culture dish with anti-CD3 antibody, the culture dish may be treated with, for example, 0.1 to 100 μg / mL of anti-CD3 antibody. The anti-CD3 antibody is not particularly limited as long as it recognizes CD3 specifically, and includes, for example, an antibody produced from an OKT3 clone.After the initiation of differential induction from CD4CD8 DP cells to CD8αβ SP cells, culture is preferably carried out for 2 to 4 days. Thereafter, the cells may be further cultured, preferably in the presence of IL-7 and in the absence of T cell differentiation-inducing factors and cell adhesion factors.Examples of the basal medium used in the process of differentially inducing from CD4CD8 DP cells to CD8αβ SP cells include IMDM, Medium 199, EMEM, αMEM, DMEM, Ham's F12, RPMI 1640, Fischer's Medium, Neurobasal Medium (Life Technologies), and mixed media thereof. The basal medium may be supplemented with serum or may be serum-free. The medium may contain one or more substances selected from, for example, albumin, insulin, transferrin, selenium, fatty acid, trace elements, 2-mercaptoethanol, thiol glycerol, lipids, amino acid, L-glutamine, non-essential amino acids, vitamin, growth factors, low molecular weight compounds, antibiotics, antioxidant, pyruvic acid, buffering agent, inorganic salts, and cytokines etc. A preferred medium is αMEM containing serum, transferrin, selenium, L-glutamine, and ascorbic acid.The medium used in the process of differentially inducing into CD8αβ SP cells may further contain other cytokines, such as IL-21 and FLT3L, and ascorbic acid. Note that the medium preferably does not contain IL-15 (for example, the IL-15 concentration is less than 1 ng / mL to below the detection limit). By using a medium that does not contain IL-15, it is possible to suppress the maturation of innate immune system lymphocytes, which have a higher requirement for IL-15, while promoting their maturation into CD8αβ SP cells that have characteristics of acquired immune system lymphocytes. The medium used in the process of differentially inducing into CD8αβ SP cells may contain a caspase inhibitor. As the caspase inhibitor, Z-VAD-FMK, which is a pan-caspase inhibitor, may be used.(Expansion Culture of CD8αβ SP Cells)By expansion culture of CD8αβ SP cells differentially induced to from CD4CD8 DP cells, a sufficient amount of CD8αβ SP cells to constitute a cell bank can be secured. These CD8αβ SP cells are iPSC-derived dTCR-expressing cytotoxic lymphocytes (dTCR iPS-CL). Expansion culture can be carried out, for example, in the presence of IL-7, IL-15, and IL-21. Alternatively, it can be carried out in the presence of one or more selected from IL-21, IL-18, IL-12 and TL1A (TNF-like ligand 1A: also known as vascular endothelial growth inhibitor (VEGI)) or TNF superfamily member 15 (TNFSF15), in addition to IL-7 and IL-15.Expansion culture of CD8αβ SP cells may be carried out using CD8αβ SP cells sorted by FACS, affinity column, etc. from a cell group differentially induced from CD4CD8 DP cells. This sorting can be carried out using, for example, one or more indicators selected from CD8β positive, CD5 positive, CD336 negative, and CD1a negative. Since CD1a is an immature T cell marker, immature cells can be removed by sorting using CD1a negativity as an indicator. Furthermore, since CD336 is a marker expressed only in innate immune system cells, abnormal NK-like cells can be removed by sorting using CD336 negativity as an indicator.The medium used for expansion culture of CD8αβ SP cells can be prepared by adding cytokines such as IL-7, IL-15, and IL-21 to the basal medium used for culturing animal cells. Examples of the basal medium include IMDM, Medium 199, EMEM, αMEM, DMEM, Ham's F12, RPMI 1640, Fischer's Medium, Neurobasal Medium (Life Technologies), and mixed media thereof. The basal medium may be supplemented with serum or may be serum-free. The medium may contain one or more substances selected from albumin, insulin, transferrin, selenium, fatty acid, trace elements, 2-mercaptoethanol, Thiol glycerol, lipids, amino acid, L-glutamine, non-essential amino acids, vitamin, growth factors, low molecular weight compounds, antibiotics, antioxidant, pyruvic acid, buffering agent, inorganic salts, and cytokines etc., as necessary. A preferred medium is αMEM containing serum, transferrin, selenium, L-glutamine, and ascorbic acid. The medium may also contain a caspase inhibitor. As the caspase inhibitor, Z-VAD-FMK, which is a pan-caspase inhibitor, may be used. The concentrations of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A in the medium used for expansion culture of CD8αβ SP cells are, for example, 1 ng / mL to 100 ng / mL, respectively.In expansion culture, CD8αβ SP cells may be cultured in direct contact with a culture vessel coated with a cell adhesion factor. As a cell adhesion factor, fibronectin or a fragment thereof can be used. Any region of fibronectin can be selected as the fibronectin fragment as long as it retains the functions required by the present invention. As a cell adhesion factor, retroNectin may also be used.In expansion culture, CD8αβ SP cells may be cultured in the presence of a TCR activator. Examples of TCR activators include PHA, anti-CD3 antibody, anti-CD28 antibody, PMA, and ionomycin, etc. For example, TCR can be stimulated by culturing CD8αβ SP cells in a medium containing an anti-CD3 antibody. The concentration of anti-CD3 antibody in the medium is, for example, 10 ng / mL to 10 μg / mL. The anti-CD3 antibody may be bound to magnetic beads, etc. Alternatively, TCR may be stimulated by culturing CD8αβ SP cells on a culture dish coated with an anti-CD3 antibody. In order to coat the surface of a culture dish with anti-CD3 antibody, the culture dish may be treated with, for example, 0.1 to 100 μg / mL of anti-CD3 antibody.After the initiation of expansion culture of CD8αβ SP cells, culture is preferably carried out for 2 to 4 days, and then further culture may be performed in the absence of cell adhesion factors and / or TCR activators. The culture period can be determined as appropriate while checking the state of cell proliferation. In the expansion culture process, IL-7 and IL-15 are preferably present in the medium throughout the expansion culture process. On the other hand, IL-21, IL-18, IL-12, and TL1A may not be contained in the medium in the absence of cell adhesion factors and / or TCR activators.A Cell BankIn the present invention, a "cell bank" refers to CD8αβ SP cells cultured for expansion, which is a collection of cytotoxic lymphocytes (dTCR iPS-CL) that express variable region deleted TCR (dTCR) derived from iPS cells, and involves storage facilities that maintain the cells under conditions favorable for cell survival. The cell bank can include a lot record for each manufactured lot of stored dTCR iPS-CL. The lot record includes manufacturing records that record the details of the manufacturing process of dTCR iPS-CL, performance test records of cells to be stored, test management record, quality control record, judgment letter, packaging record, label record, warehousing / in / out management record, warehousing instructions / records, shipping instructions / records, and temperature control records, etc., which can guarantee the quality of stored dTCR iPS-CL. In the cell bank of the present invention, intermediates (dTCR iPS-CL) for the production of antigen-specific iPS-T cells used for prophylaxis and / or treatment of diseases (e.g., cancer) are stored in a highly controlled and ready-to-use state. Therefore, by using the cell bank of the present invention, it is possible to quickly produce a wide variety of high-quality iPS-T cell preparations (antigen-specific iPS-T cell preparations), and it is also possible to provide on-demand personalized medicine that is optimal for each patient. (see FIG. 2 and 6). The period required to produce antigen-specific iPS-T cells using dTCR iPS-CL stored in the cell bank of the present invention is about 1 to 4 weeks.The dTCR iPS-CLs cultured for expansion are collected from the medium, washed with a buffer solution or culture solution, counted for cell numbers, concentrated by centrifugation etc., suspended in a freezing medium (e.g., a culture medium containing 10% DMSO), dispensed into appropriate containers such as freezing vials and freezing bags according to the required amount and then can be cryopreserved as a cell bank of the present invention. The number of cells to be dispensed can be set as the number of cells that can be used up in one TCR and / or CAR transfection procedure. The number of cells to be dispensed can be 1×105to 1×1010cells, but may also be 1×107to 1×109cells. When cryopreserving dTCR iPS-CL, the storage temperature is not particularly limited as long as it is suitable for preserving the cells. For example, the temperature may be -150°C to -196°C, but preferably -150°C or lower. In order to minimize the risk of cell damage when freezing dTCR iPS-CL cells, it is preferred to store the cells in suitable containers, such as cryovials and cryobags. In addition, the risk of cell damage during freezing can be reduced by placing the container in a freezer (e.g., ultra-low temperature freezer) or by contacting the cells with a low-temperature medium (e.g., liquid nitrogen, etc.) and storing them in a freezer or cryopreservation system (e.g., Locator, etc.). However, it is not limited thereto.FIG. 6 shows an outline of the final manufacturing process for producing iPS-T cell medicines as necessary using dTCR iPS-CL cells constituting the cell bank of the present invention as production intermediates. By preparing in advance TCR and / or CAR genes (TCRs / CARs) tailored to each patient's target antigen in the final manufacturing process, and introducing / expressing the desired gene into dTCR iPS-CL cells with low immunogenicity, the TCR-iPS-T cells (TCR-introduced antigen-specific iPS-T cells) of FIG.6-(a), the dual type TCR / CAR-iPS-T cells (dual receptor-introduced antigen-specific iPS-T cells) of FIG.6-(b), and the CAR-iPS-T cells (CAR-introduced antigen-specific iPS-T cells) of FIG.6-(c) can be manufactured.Introduction of a Nucleic Acid Encoding a Receptor that Recognizes an Antigen into dTCR iPS-CLBy introducing a nucleic acid encoding a receptor that selectively recognizes an antigen from outside the cells into dTCR iPS-CL and expressing the receptor, various antigen-specific iPS-T cells can be produced with a high degree of variation. Examples of nucleic acids encoding receptors that recognize antigens include TCR-encoding nucleic acids and CAR-encoding nucleic acids, and may be naturally derived or artificially synthesized nucleic acids. Sequence information on these nucleic acids is available from known literature and databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ).If the receptor that recognizes the antigen is TCR, a cDNA encoding a wtTCR (wild-type TCR) that recognizes an antigen may be prepared from T cells of a patient targeted for prophylaxis and / or treatment of cancer. Preferably, cDNAs encoding the TCR α chain and β chain are prepared for each single T cell. T cells collected from patients are a T cell population with genetic diversity as a whole, and the antigen specificity of individual T cells differs. In order to select the optimal TCR for each tumor-associated antigen or viral antigen described below, i.e., the TCRs with high reactivity to individual tumor-associated or viral antigens, cDNA can be prepared for each single cell. A tumor-associated antigen, which is TCR gene that recognizes HLA-A24-restricted GPC3 peptide and HLA-A2-restricted GPC3 peptide described below may be isolated from peripheral blood lymphocytes collected from patients who have received these GPC3 peptide vaccines.After T cells obtained from a patient are cultured with the antigen of interest, single T cells may be isolated with a cell sorter or the like using an activation marker from the T cell population that responds to the antigen used. Examples of activation markers include cell surface CD137 and CD107a, etc. Examples of known techniques for isolating human T cells include flow cytometry using antibodies against T cell surface markers such as CD3 and CD137 and a cell sorter. Gene cloning is carried out using the RT-PCR method from the single T cell obtained, and cDNAs encoding the TCR α chain and β chain, respectively, can be amplified.In order to obtain single T cells, single cell sorting can be carried out with a cell sorter by binding of MHC Dextramer (registered trademark) that forms a complex with antigen peptide to antigen-specific CD8 single positive T cells obtained from peripheral blood etc. The MHC Dextramer is a compound composed of a dextran polymer skeleton in which MHC and fluorescent dye molecules are bonded. MHC tetramer may also be used instead of the MHC Dextramer. The MHC tetramer is a complex of an antigen peptide and an MHC molecule made into a tetramer using biotin and avidin. In another embodiment, CD8 single-positive T cells specific for an antigen obtained from peripheral blood, etc. may be cultured for expansion in the presence of the antigen, and then CD3 / CD137 double-positive cells may be single-cell sorted with a cell sorter. In another embodiment, a cell population that binds to MHC dextramer that forms a complex with an antigen peptide may be single-cell sorted from CD3 / CD137 double-positive cells with a cell sorter. RNA is extracted from the obtained single cell, and the TCR gene pair (TCRα chain gene and TCRβ chain gene) can be isolated by PCR using cDNA obtained by reverse transcription reaction. Sequence analysis is carried out on the isolated TCR gene pairs, and the types of antigen-reactive T cells (TCR repertoire) and their appearance frequency can be analyzed.cDNAs encoding TCR α chain and β chain can be incorporated into expression vectors. These cDNAs may be incorporated into viral or non-viral vectors (transposon vectors) using, for example, the Gibson assembly system. Specifically, a gene in which cDNA encoding the dTCR α chain and β chain is linked via a T2A sequence is linked downstream of ubiquitin promoter, CMV promoter and EF1α promoter etc., and furthermore, downstream thereof, a marker gene such as EGFR (tEGFR, truncated EGFR), which excludes the ligand binding site and intracellular domain, or CD19, which lacks the intracellular domain, is linked to the IRES (internal ribosome entry site) sequence. This construct may be incorporated into a viral or non-viral vector. The cDNAs encoding the dTCR α chain and β chain may be incorporated into separate expression vectors.When the receptor that recognizes the antigen is CAR, cDNA encoding CAR can be prepared and incorporated into an expression vector. For example, total RNA from the lymph node of an animal immunized with a desired tumor-related antigen can be extracted and used as a template to synthesize cDNA. The light chain and heavy chain of the variable region of the monoclonal antibody against the tumor-associated antigen can be amplified separately, combined using a flexible linker, and amplified by assembly PCR. A CAR construct can be prepared by combining the sequences encoding the fused light chain and heavy chain with the sequence encoding the intracellular domain of the CAR molecule via the sequence encoding the transmembrane domain of the CAR molecule. In order to confirm the expression of the introduced gene, EGFR without the ligand binding site and intracellular domain (tEGFR) may be linked to this CAR construct via the T2A sequence. The flexible linker that combines the light chain and heavy chain of the variable region of a monoclonal antibody is a linker peptide of about 5 to 20 amino acid residues, and is known in the art (For example, see Ueda T, et al. Cancer Sci. 2020; 111:1478-1490. Tomomi Kawasaki et al., SCEJ 72nd Annual Meeting (Kyoto, 2007) H209.).Antigen-binding domains to be incorporated into CARs may be produced using phage display without immunizing animals with antigens. For example, antibodies that specifically bind to a desired tumor-associated antigen may be screened from a phage display antibody library expressing the Fab regions of a large number of human antibodies.One or more genes encoding a receptor that recognizes an antigen may be introduced into dTCR iPS-CL. For example, a TCR or a CAR reactive with a tumor-associated antigen may be introduced alone, or a TCR and a CAR may be introduced simultaneously. The expression vectors used to simultaneously introduce TCR and CAR may be the same or different. When TCR and CAR are introduced at the same time, TCR and CAR may have reactivity to the same antigen or may have reactivity to different antigens.As expression vectors, viral vectors and non-viral vectors can be used. Examples of the viral vectors include lentivirus, etrovirus, adenovirus, adeno-associated virus, herpesvirus, and Sendai virus, etc. Preferably, lentivirus or retrovirus can be used. When performing retrovirus or lentivirus infection, a spin infection method, etc. may be used. Examples of non-viral vector include piggyBac (registered trademark) vector, which is a transposon vector. The transposon method is a next-generation gene introduction method that is cheaper and safer than the conventional viral vector method. When replacing the TCR inherent in iPS cells with a TCR or CAR introduced from outside the cell, genome editing technology may be used. Examples of such genome editing technology include methods of CRISPR / Cas9, CRISPR / MAD, and CRISPR / Cas3. Examples of guide RNA and donor DNA gene transfer methods for non-viral vectors and genome editing include methods of lipofection, liposome, calcium phosphate coprecipitation, DEAE-dextran, microinjection, and electroporation. It is preferable to use electroporation to introduce the transposon vector into cells. As the electroporation device, the gene introduction device ExPERT (registered trademark) system (MaxCyte) can be used.TCR and / or CAR gene transfer may be performed at the TCR locus or other loci (e.g., the β2-microglobulin locus). In cases when modification or disruption of existing genes, including TCR, by transgenes are not desired, it may also be performed on safe harbor loci. Examples of the safe harbor include AAVS1 (adeno-associated virus integration site 1) region in the human genome. Examples of methods for introducing genes targeting the safe harbor include the methods of CRISPR / Cas9 and TALEN.A TCR that recognizes an antigen comprises an antigen-recognizing variable region and recognizes a single antigen. Furthermore, a CAR that recognizes an antigen comprises the variable region of a monoclonal antibody against the antigen and recognizes a single antigen. The single antigen may be selected from tumor-associated antigens or viral antigens.A "tumor-associated antigen" is an antigen that is expressed specifically or non-specifically in a tumor, such as wild-type antigens and their variants derived from proteins overexpressed in tumor cells, tumor virus-derived antigen, certain differentiation antigens and genetic mutations, and novel tumor-associated antigens (neoantigens) due to splice abnormalities, etc. In the case of a protein antigen, a peptide obtained by fragmenting the antigen (peptide fragment) may be a tumor-related antigen. Examples of Antigens expressed specifically or nonspecifically on tumors include, but are not limited thereto, GPC3 (Glypican 3), WT1 (Wilms' Tumor 1), XAGE1 (X Antigen Family Member 1), LMP2 (Latent Membrane Protein 2), NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma 1), KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) PI3K (Phosphatidylinositol 3-Kinase), MUC1 (Mucin 1), EGFR (Epidermal Growth Factor Receptor) HER-2 (Human Epidermal Growth Factor Receptor 2) / neu, MAGE-A3 (Melanoma Antigen Family A3), PSMA (Prostate-Specific Membrane Antigen) CEA (Carcinoembryonic Antigen) MART1 (Melanoma Antigen Recognized by T cells 1), gp100 (glycoprotein 100), bcr-abl (breakpoint cluster region-Abelson), hTERT (human telomerase reverse transcriptase), p53, BCMA (B-Cell Maturation Antigen), MUC5A1 (Mucin 5AC), MUC6 (Mucin 6), MAGE-A1 (Melanoma Antigen Family A1), PRAME (Preferentially Expressed Antigen in Melanoma) SSX2 / 4 (Synovial Sarcoma, X Breakpoint 2 / 4), PSCA (Prostate Stem Cell Antigen), CTLA-4 (Cytotoxic T-Lymphocyte Associated Protein 4), GD2 ganglioside, GD3 ganglioside, fucosyl GM1 ganglioside, GM3 ganglioside, sLe(a) (Sialyl Lewis a), Glycolipid F77, mesothelin, PD-L1 (Programmed Death-Ligand 1), trp1 (Tyrosinase-related protein 1), trp2 (Tyrosinase-related protein 2), CD19, CD20, CD22, ROR1 (Receptor Tyrosine Kinase Like Orphan Receptor 1), CD33, c-Met (MET Proto-Oncogene, Receptor Tyrosine Kinase), ETV6-AML (ETS Variant 6-Acute Myeloid Leukemia), PSA (Prostate Specific Antigen) AFP (Alpha-fetoprotein), EpCAM (Epithelial Cell Adhesion Molecule), ALK (Anaplastic Lymphoma Kinase), androgen receptor, EphA2 (Ephrin Type-A Receptor 2), CYP1B1 (Cytochrome P450 Family 1 Subfamily B Member 1), OY-TES-1 (OYantigen Testis-specific Protein 1), MAD-CT-2 (Melanoma Antigen D1, Cancer / Testis Antigen 2), Survivin, Ras, EGR (Early Growth Response) XBP-1 (X-Box Binding Protein 1), Neoantigens due to genetic mutations, and neoantigens due to splice abnormalities and their fragments, etc.In one embodiment of the invention, the tumor-associated antigens are EYILSLEEL (Seq. No. 1), which is an HLA-A24-restricted GPC3 peptide, and FVGEFFTDV (Seq. No. 2), which is an HLA-A2-restricted GPC3 peptide. Amino acid residues in these sequences are indicated by single letter codes."Viral antigens" include antigens derived from characteristic proteins of viruses and variants thereof. Examples of viral antigens include, but are not limited thereto, viral proteins produced by cells infected by influenza virus, EBV (Epstein-Barr Virus) HPV (Human papillomavirus) HBV (Hepatitis B Virus), HCV (Hepatitis C Virus), HIV (Human Immunodeficiency Virus) coronavirus, CMV (Cytomegalovirus), dengue virus, west nile virus, hantavirus, ebola virus, and HTLV-1 (Human T-cell Leukemia Virus type 1), and fragments thereof.Antigen-recognizing TCRs are reactive against tumor-associated antigens and viral antigens. The "being reactive against tumor-associated antigens and viral antigens" means that T cells shows the reaction that occurs when T cells selectively bind / conjugate to epitope peptides derived from tumor-associated or viral antigens presented on major histocompatibility complex (MHC) class I or class II on antigen-presenting cells, via TCR, and that binding / conjugation of T cells to anything other than the epitope peptide does not result in a T cell response. The T cell responses generated by binding / conjugation to epitope peptides derived from tumor-associated or viral antigens presented on MHC class I or class II via TCR include cytotoxic, production of IFN-γ and granzymes; expression of T cell activation markers; and activation of transcription factors such as NF-AT.A Pharmaceutical Comprising the Antigen-Specific iPS-T Cell of the Present InventionThe pharmaceutical comprising the antigen-specific iPS-T cell of the present invention can be used as a preventive and / or therapeutic agent for cancer in mammals. The pharmaceutical of the present invention may be manufactured by a method commonly used in the field of pharmaceutical technology. The pharmaceutical composition of the present invention may also contain pharmaceutically acceptable additives. Examples of the additives include cell culture medium, physiological saline, and appropriate buffers (e.g., phosphate buffers).The pharmaceutical composition of the present invention can be produced by suspending the antigen-specific iPS-T cells of the present invention in physiological saline or an appropriate buffer (e.g., phosphate buffer). A single dose may contain, for example, 1×107or more, 1×108or more, or 1×109or more cells so that the desired therapeutic effect is achieved. The content of cells can be adjusted in consideration of the gender, age, weight, condition of the affected area, cell condition, etc. of the subject to be administered. The pharmaceutical composition of the present invention may contain, in addition to the cells of the present invention, dimethyl sulfoxide (DMSO), and serum albumin, etc. for the purpose of protecting the cells. Furthermore, it may contain an antibiotic, etc. to prevent contamination with bacteria. In addition, vitamins and cytokines, etc. may be contained for the purpose of promoting cell activation and differentiation. Furthermore, the pharmaceutical composition of the present invention may contain other pharmaceutically acceptable ingredients (For example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.).Administration routes for the pharmaceutical composition of the present invention include, for example, infusion, intratumoral injection, intraarterial injection, portal vein injection, and intraperitoneal administration. However, the administration route is not limited to these as long as the antigen-specific iPS-T cells, which are the active ingredients in the pharmaceutical composition of the present invention, are delivered to the affected area. The administration schedule can be a single administration or multiple administrations. As for the period of multiple administrations, for example, a method of repeating administration once every 2 to 4 weeks, or a method of repeating administration once every six months to a year, etc. can be adopted. In creating the administration schedule, the gender, age, weight, pathological condition, etc. of the target patient can be taken into consideration.Pharmaceutical compositions containing cells of the present invention can be cryopreserved. In the case of cryopreservation, the storage temperature is not particularly limited as long as it is suitable for preserving cells. Examples include -20°C, -80°C and -120°C to -196°C, but -150°C or lower is preferred. When cryopreserved, cells may be stored in suitable containers such as cryovials and cryobags.The pharmaceutical of the present invention is used for prophylaxis and / or treatment of cancer. Examples of cancers include, but are not limited thereto, ovarian cancer, hepatoblastoma, hepatocellular carcinoma, stomach cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, malignant melanoma, non-small cell lung cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastic tumor, chronic lymphocytic leukemia, papillary thyroid cancer, Colorectal cancer, head and neck cancer, brain tumor, multiple myeloma, and B-cell non-Hodgkin lymphoma.Since the antigen-specific iPS-T cells of the present invention can kill cells expressing tumor-associated antigens or viral antigens, it can be used as a killing agent for cells expressing tumor-associated or viral antigens. The killing agent can be produced and used in the same manner as the pharmaceutical composition.ExamplesExample 1 Generation of cytotoxic lymphocytes (dTCR iPS-CL)Establishment of dTCR-Introduced iPS CellsGenes of TCR lacking variable region (dTCR) wherein at least all of the variable regions of TCRαβ are deleted and all of the constant regions are present, or part of the constant region is deleted was provided by Kyoto University / Kirin Holdings. For the gene sequences and amino acid sequences of dTCRs, ba2 and ba9 (referred to as dTCRba2 and dTCRba9, respectively), the description in Patent Document 2 (Pamphlet of WO 2020 / 138371) can be referred to. These ba2 and ba9 are polypeptides having 379 (Seq. No. 3) and 322 (Seq. No. 4) amino acid residues, respectively, and information about their amino acid sequence is shown in FIG. 17. Amino acid residues in the amino acid sequence shown in FIG. 17 are shown in single letter notation.As dTCR gene, dTCRba2 vector that deletes all of the sequences of variable region of the TCRα and TCRβ chains but contains all of the sequences of constant region and dTCRba9 vector that deletes all of the sequences of variable region of the TCRα and TCRβ chains, or deletes a part of the sequence of constant region of the TCRα chain, but contains all of the sequences of the constant region of TCRβ chain were used. For dTCRba2 and dTCRba9, a piggyBac vector (vector builder) incorporated with the gene wherein a gene (dTCRb-T2A-dTCRa IRES tCD19) in which the TCRβ chain gene and TCRα chain gene of dTCR with the variable region deleted are connected in this order by a T2A peptide sequence (dTCRb-T2A-dTCRa) was placed downstream of the EF1α promoter (human polypeptide chain elongation factor gene promoter), and internal ribosome entry site (IRES)-tCD19 (CD19 molecule lacking intracellular domain) was placed downstream of dTCRb-T2A-dTCRa was created. In addition, as a control full-length TCR without deletion, TCR82, a full-length TCR with a variable region that recognizes a complex of a peptide derived from the tumor-associated antigen GPC3 (EYILSLEEL, sequence number 1) and HLA-A*24:02 as an antigen (hereinafter referred to as TCR82 or antigen recognition TCR) was used. Regarding the gene sequence and amino acid sequence of TCR82, the description in Japanese patent application No. 2022-101473 can be referred to. Hereinafter, similar to the method for producing dTCR, TCR82 was also transfected into iPSCs and induced to differentiate in order to be used as a control.The research human iPS cell line Ff-I01s04, which is derived from human peripheral blood but is not derived from T cells, provided by the Kyoto University iPS Cell Research Foundation, was used as the iPS cell (iPSC). StemFit AK03N medium (Ajinomoto) was used to maintain iPSCs. Human iPSCs were cultured on a plate culture surface coated with Easy iMatrix-511 silk (MATRIXOME). The iPSCs were detached using a release agent (final concentration 0.5×TrypLE Select, prepared by diluting 1×TrypLE Select (ThermoFisher) and 0.5 moL / L-EDTA solution (Nacalai Tesque) in PBS (Nacalai Tesque), and 0.75 mM EDTA), and subcultured in StemFit AK03N medium added with 10 μM Y-27632 (Fuji Film Wako Pure Chemical Industries, Ltd.) (37°C, 5% CO2). The next day, the medium was replaced with StemFit AK03N and the procedure was repeated once a week to maintain iPSCs.iPSCs were collected using the above-mentioned release agent. A dTCR-expressing piggyBac vector and a plasmid (A-SEEDS) encoding piggyBac transposase were added to the collected human iPSCs, and transfected into iPSCs by an electroporation method using MaxCyte ExPERT ATx (registered trademark).The collected human iPSCs were suspended in MaxCyte electroporation buffer to a concentration of 2×107cells / mL. The cell suspension was applied to a special cuvette, and transfection was performed using the program of Optimization 6.After culturing human iPSCs for 1 week after introducing the dTCR-expressing piggyBac vector, purification and amplification culture of CD19-positive fraction was performed using a BD FACSAria cell sorter (Nippon Becton Dickinson), and a stable CD19-positive cell line (dTCR-introduced strain: dTCR iPSC) was established.Generation of Hematopoietic Stem Cells from dTCR iPSCsHematopoietic stem cells (HPCs) were produced from dTCR iPSCs using the Embryoid Body (EB) differential induction method by following procedures.dTCR iPSCs were cultured in the presence of 10 μM Y-27632 in an environment of 37°C and 5% CO2. Thereafter, dTCR iPSCs were detached using 0.5×TrypLE Select and 0.75 mM EDTA, suspended in StemFit AK03N medium supplemented with 10 μM Y-27632 and 10 μM CHIR99021 (Fuji Film Wako Pure Chemical Industries, Ltd.), and seeded in a Petri dish (Corning, diameter: 60 mm) at 1×106cells / dish. A Petri dish (diameter: 60 mm) seeded with dTCR-introduced iPSCs was cultured on a rotary shaker (orbital diameter: 10 mm, 70 rpm) in an environment of 37°C and 5% CO2.The next day, the cells were cultured on a rotary shaker (orbital diameter: 10 mm, 70 rpm) in an environment of 37°C and 5% CO2　using StemPro34 (Thermo Fisher Scientific) medium (EB medium) containing 1×insulin-transferrin-selenium (Thermo Fisher Scientific), 1× GlutaMAX-l CTS (Thermo Fisher Scientific), 0.2%×L-glutamine-penicillin-streptomycin solution (SHIGMA), and 1× StemPro34 nutrient supplement (Thermo Fisher Scientific), added with 50 ng / mL BMP-4 (Miltenyi Biotec), 50 ng / mL bFGF (Fuji Film Wako Pure Chemical), 50 ng / mL VEGF (Fuji Film Wako Pure Chemical), 50 μg / mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Nacalai Tesque), and 0.4 mM MTG (Fuji Film Wako Pure Chemical) (iPSC_Day1).The next day, 6 μM SB431542 (Fuji Film Wako Pure Chemical Industries) was added to the medium, and the cells were cultured for another 2 days in an environment of 37°C and 5% CO2on a rotary shaker (orbital diameter: 10 mm, 70 rpm) (iPSC_Day2). The medium was changed to EB medium supplemented with 50 ng / mL bFGF, 50 ng / mL VEGF, 50 μg / mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 50 ng / mL SCF (R&D systems), and 0.4 mM MTG, and cultured for an additional 2 days (Day 4). Thereafter, the medium was changed to EB medium supplemented with 50 ng / mL bFGF, 50 ng / mL VEGF, 50 μg / mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 50 ng / mL SCF, 30 ng / mL TPO(PEPROTECH), 10 ng / mL Flt3L(PEPROTECH), and 0.4 mM MTG and cultured (Day 6 onwards). After Day 6, the medium was replaced every other day, and the cells were cultured until Day 14 in an environment of 37°C and 5% CO2and under condition of 70 rpm (orbital diameter: 10 mm). The number of viable cells was confirmed by trypan blue (Nacalai Tesque) staining of the cells cultured until Day 14. The expression of CD34, CD43, CD14, CD235a, and CD19 was analyzed by flow cytometry, and the presence of HPCs was confirmed (FIG. 3-(a)). FIG.3 is a schematic diagram showing each differential induction process in which differentiation progresses into (a) TCR HPCs, (b) CD4CD8 double-positive (DP) cells, and (c) CD8αβ single-positive (SP) cells after transducing the TCR gene into iPSCs (upper FIG.), and analysis results of flow cytometry to confirm the expression of CD34, CD43, etc. for cells at each differentiation stage ((a) to (c) in lower FIG.). Here, regarding the vertical column in the lower part of FIG. 3, (i) shows the results of flow cytometry of the research human iPS cell line Ff-I01s04. (ii), (iii), and (iv) show the results of flow cytometry of cells into which the wtTCR genes TCR82, dTCRba2, and dTCRba9 were transduced into iPSC Ff-I01s04, respectively.Induction of CD4CD8 DP Cells from HPCs (Differential Induction Process)Induction of differentiation to CD4CD8 DP Cells from HPCs was performed by following procedures.On Day 13, the solutions of 5 μg / mL RetroNectin (TaKaRa) and 5 μg / mL Fc-DLL4 (Shino Biological) were added to the culture vessel. The culture vessel was coated with RetroNectin at 4°C overnight.Cells collected on Day 14 were re-seeded into culture vessels coated with RetroNectin / Fc-DLL4 the day before and cultured in an environment of 37°C and 5% CO2using αMEM (Nacalai Tesque) medium (cell medium) supplemented with 15% FBS (Thermo Fisher Scientific), 1% GlutaMAX-I CTS (Thermo Fisher Scientific) and 1% P / S (SIGMA), and further added with 1× insulin-transferrin-selenium, 55 μM 2-mercaptoethanol (Thermo Fisher Scientific), 50 μg / mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 10 ng / mL SCF, 5 ng / mL Flt3L, 5 ng / mL IL-7 (PEPROTECH), 100 ng / mL SDF1α (PEPROTECH), and 10 μM SB203580 (TOCRIS). The day when the culture started was defined as Day 0 (HPC_Day0). The medium was replaced every 2 to 3 days. A culture vessel coated with RetroNectin / Fc-DLL4 was prepared on HPC Day 6. The cells cultured on HPC_Day7 were collected, and the number of cells was confirmed using trypan blue staining. After counting the number of cells, they were re-seeded into the culture vessels coated the previous day. The medium was replaced every 2 to 3 days, and culture was continued until HPC_Day21. After collecting the cultured cells on Day 21, the number of cells was confirmed by trypan blue staining. The expression of CD4, CD7, CD8α, CD8β, CD45, and CD19 was analyzed for the differentiated cells on Day 21, and the presence of CD4CD8 DP cells was confirmed (FIG. 3-(b)).Induction of CD8αβ SP Cells from CD4CD8 DP Cells (Maturation Process)The induction of CD8αβ SP cells to CD4CD8 DP cells was performed by following procedures.CD4CD8 DP cells were reseeded into culture vessels coated with 5 μg / mL anti-human CD3 antibody (Clone: OKT3) (Invitrogen) and 25 μg / mL RetroNectin. The cells were cultured in cell medium (cell medium-Ma) supplemented with 50 μg / mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 50 ng / mL IL-7, 20 ng / mL IL-21 (PEPROTECH), 20 ng / mL IL-1b (PEPROTECH), 80 ng / mL SDF1a (PEPROTECH), and 10 μM SB203580.The cells were collected from the coated culture vessels on the third day of culture (CD4 / 8_Day3), washed with cell culture medium, suspended in cell medium-Ma, and then reseeded into uncoated culture vessels. The medium was replaced every 2 to 3 days, and the culture was continued until the 7th day of culture (CD4 / 8_Day7).The number of cells collected on CD4 / 8_Day7 was confirmed by trypan blue staining. The recovered cells (CD4 / 8_Day7) were analyzed for expression of CD3, CD4, CD7, CD8α, CD8β, CD28, CD45RO, NKG2D, CCR7, and CD19, and the presence of CD8αβ SP was confirmed (FIG. 3-(c)).Expansion Culture of CD8αβ SP Cells (Expansion Culture Process)CD8αβ SP cells were reseeded into culture vessels coated with 1 μg / mL anti-human CD3 antibody (Clone: OKT3) and 5 μg / mL RetroNectin, and cultured with a medium mixed with cell culture medium (cell medium-ExA) supplemented with 1×insulin-transferrin-selenium, 50 μg / mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 5 ng / mL IL-7, and 5 ng / mL IL-15 (PEPROTECH) and cell culture medium (cell medium-ExB) supplemented with 20 μM Z-VAD FMK (Promega), 100 ng / mL IL-12 (PEPROTECH), 100 ng / mL IL-18 (MBL), and 40 ng / mL IL-21 (PEPROTECH).Cells were collected on the third day of culture (CD8αβ_Day3), washed with cell culture medium, suspended in cell culture medium-ExA, and reseeded into uncoated culture vessels. The medium was replaced every 2 to 3 days, and the culture was continued until the 14th day of culture (CD8αβ_Day14). The number of cells collected on CD8αβ_Day14 was confirmed by trypan blue staining, and the expression of TCRαβ, CD3, CD4, CD7, CD8α, CD8β, CD45, and CD19 was analyzed.Confirmation of Production of Cytotoxic Lymphocytes (dTCR iPS-CL) during the Expansion Culture ProcessFIG. 4 shows the results of flow cytometry analysis of the expression of CD8α, CD19, TCRαβ, and CD3 in CD8αβ SP cells during the expansion culture process over time up to Day 14. For its details, the vertical columns show analysis results of(i) CD4CD8 DP cells (cells on Day 21 in the induction process from HPCs and before the expansion culture process),(ii) CD8αβ SP cells (cells on Day 7 in the expansion culture process of CD8αβ SP cells),(iii) CD8αβ SP cells (cells on Day 9 in the expansion culture process of CD8αβ SP cells), and(iv) CD8αβ SP cells (cells on Day 14 in the expansion culture process of CD8αβ SP cells).The horizontal rows show the results of flow cytometry analysis of cells into which the respective genes of (a) dTCRba9, (b) dTCRba2, and (c) wtTCR TCR82 were transduced into iPSCs.It can be seen from FIG. 4 that in (a) dTCRba9 and (b) dTCRba2, the expression levels of TCRαβ and CD3 decreased as cell proliferation by expansion culture progressed. However, in (c) TCR82, stable TCRαβ and CD3 expression was maintained. On the other hand, the expression level of CD19, which is a gene expression marker, was maintained at the same level in all of (a), (b), and (C). Accordingly, it was considered that the decrease in the expression levels of TCRαβ and CD3 in (a) dTCRba9 and (b) dTCRba2 was due to promoter silencing.These results revealed a phenomenon in which the expression level of dTCR, which does not have antigen recognition ability, on the cell membrane surface significantly decreases from day 7 to day 14 in the expansion culture process of CD8αβ SP cells. Since this characteristic is completely different from the phenotype of cytotoxic T cell known so far, the cells obtained by the expansion culture is defined as cytotoxic lymphocytes (dTCR iPS-CL) in the present application.Proliferation Rate of Each Cell during Differential Induction Process and Expansion Culture ProcessFIG. 5 shows the cell proliferation rate of each cell differentially induced from HPCs during the differential induction process and expansion culture process. The human iPS cell line Ff-I01s04 was defined as parental, and each cell into which the genes of dTCRba9, dTCRba2, and TCR82 were introduced into iPSC Ff-I01s04 was used as a target for measurement of cell proliferation. In FIG. 5, the cell proliferation rates of each cell in the differential induction process and expansion culture process are shown, assuming that the number of cells at HPC_Day0 is 1. As a result, it was recognized that similar to the cells into which the wild-type TCR82 gene was introduced, the cell number increased approximately 104times about 6 weeks after the initiation of HPC culture also in the cells into which the dTCRba9 and dTCRba2 genes were introduced.Example 2 Construction of a cell bank and its utilizationCell Bank and Stock of dTCR iPS-CL CellsStocking of dTCR iPS-CL and iPS-T into which the genes of dTCRba9, dTCRba2 and TCR82 produced in the differential induction process and expansion culture process of Example 1 were introduced, respectively was carried out based on the procedure manual for loading and unloading liquid nitrogen tanks in Shinobi Therapeutics Co. Ltd., and a cell bank was constructed. Each cell was suspended in a TC-Protector (Cell Freezing Media: KAC Corporation) containing DMSO at a concentration of 1×107cells / mL, and 2 mL of the suspension was dispensed into a freezing vial (Sumitomo Bakelite Co., Ltd., Serum tube: MS-4603W). The each of cell was frozen in a -80°C deep freezer and then stored in a liquid nitrogen tank. The inside of the liquid nitrogen tank for storing the cell stock is kept at -150°C or lower constantly.It was first checked that the liquid nitrogen meets the management standards (5 cm or more from the floorboard) when freezing and storing the stock in a liquid nitrogen tank. It was also confirmed that the lid of the liquid nitrogen tank was tightly closed before and after storing the cell stock. For traceability, such as manufacturing records of the stock, performance tests, test management, quality control, etc. of the cells to be stored, for each dTCR iPS-CL and iPS-T dispensing vial to be stored frozen, the sample ID (items written on the label, etc.) and the store address were recorded and stored for each vial.In order to produce cytotoxic iPS-T cells, it was first confirmed that the liquid nitrogen met the management standards when taking out samples of dTCR iPS-CL stored frozen in a cell bank. It was confirmed that the lid of the liquid nitrogen tank was tightly closed before and after taking out the vials containing the cells. Since record management is an important element in construction of a cell bank, also when the vials containing the cells were taken out from the liquid nitrogen tank, necessary information was recorded for each vial, such as the sample ID (items written on the label, etc.) and the address of the storage source, in the same way as when it was stored.Example 3 Production of cytotoxic iPS-T cells using dTCR iPS-CL cells stored in a cell bank (1)Generation of CD20-Expressing dTCR iPS-CL CellsA retroviral vector containing the CD20 CAR was generated, and the retroviral vector was introduced into dTCR iPS-CL cells stored in a cell bank.Confirmation of Gene Transfer of CD20 CAR and tEGFR into dTCR iPS-CL CellsFIG. 7 shows the results of flow cytometry analysis of tEGFR expression in dTCR iPS-CL cells into which CD20 CAR and tEGFR genes have been introduced.The CD20 CAR IRES tEGFR gene was introduced into dTCR iPS-CL cells induced to differentiate from iPSCs expressing dTCRba2 and dTCRba9 using a retrovirus, and CD20 CAR-expressing iPS-CL cells (dTCR iPS CD20 CAR-CL) were generated (FIG. 7(a)). Instead of dTCR, TCR82 iPS-T cells differentiated from iPSCs expressing TCR82, a wtTCR, were used as a control. TCR82 is a full-length TCR with a variable region that recognizes a complex of a peptide derived from the tumor-associated antigen GPC3 and HLA-A*24:02 as an antigen. Similarly, dTCR iPS-CL / TCR82 iPS-T cells into which the CD20 CAR IRES tEGFR gene had not been introduced were generated and used as a control (FIG. 7(b)). The expression of the marker tEGFR was analyzed by flow cytometry, and the percentage of cells into which the gene had been introduced was analyzed using tEGFR expression as an indicator. As a result, 41.9 - 54.6% of the cells were EGFR positive, and the gene transfer was confirmed.Analysis of Reactivity of dTCR iPS CD20 CAR-CL Cells to Target CellsFIG. 8 shows the results of analyzing the reactivity of dTCR iPS CD20 CAR-CL cells to target cells. After coculturing dTCR iPS CD20 CAR-CL cells with B-LCLs (B lymphoblastoid cell lines) target cells expressing CD20, production of CD107a, IFN-γ, and TNF-α in CAR-negative (CAR(-)) and CAR-positive (CAR(+)) dTCR iPS-CL cells was analyzed using flow cytometry. FIG. 8 shows the development of flow cytometry gates. As a result, it is indicated that when the cocultured cell population was separated into B-LCLs and dTCR iPS-CL cells based on the expression of CD20 and tEGFR, and then, after CAR(-) cells and CAR(+) cells were separated using tEGFR expression as an indicator, the production of CD107a, IFN-γ, and TNF-α in each cell can be analyzed by flow cytometry.FIG. 9 shows the results of analyzing the reactivity of dTCR iPS CD20 CAR-CL cells to target cells. After coculturing each dTCR iPS CD20 CAR-CL cell with B-LCLs target cells (#1 and #2) from two different donors, production of CD107a and IFN- γ was analyzed by flow cytometry. It was indicated that compared to CAR(-) cells, CAR(+) cells had a significantly higher proportion of cells expressing CD107a and IFN-γ, indicating that dTCR iPS CD20 CAR-CL cells were responding to target cells. In particular, a large number of cells reacted with dTCRba9 iPS CD20 CAR-CL cells generated from dTCRba9 iPS-CL cells, and the percentage of CD107a- and IFN-γ-positive cells was high.Similarly, FIG. 10 also shows the results of analyzing the reactivity of dTCR iPSC CD20 CAR-CL cells to target cells. Similar to the method described above, each dTCR iPS CD20 CAR-CL cell was cocultured with B-LCLs target cells, and then production of CD107a and TNF-α in CAR(-) and CAR(+) cells was analyzed by flow cytometry. It was indicated that compared to CAR(-) cells, the percentage of cells expressing CD107a and TNFα was significantly higher inCAR(+) cells, indicating that dTCR iPS CD20 CAR-CL cells were responding to target cells. In particular, a large number of cells reacted with dTCRba9 iPS CD20 CAR-CL cells generated from dTCRba9 iPS-CL, and the percentage of CD107a- and TNFα-positive cells was high.Analysis of Cytotoxic Activity of dTCR iPSC CD20 CAR-CL CellsFIG. 11 shows the analysis results of the cytotoxic activity of CD20 CAR-expressing iPS-CL cells against target cells. After coculturing dTCR iPSC CD20 CAR-CL cells with various effector: target ratios using CD20-positive B-LCLs as target cells, the percentage of target cells killed was analyzed using a non-RI cytotoxicity assay kit (Techno Suzuta). dTCR iPS CD20 CAR-CL cells have cytotoxic activity against target cells, in particular, dTCRba9 iPS CD20 CAR-CL cells generated from dTCRba9 iPS-CL showed excellent cytotoxic activity comparable to CD20 CAR-expressing TCR82 iPS-T cells generated from TCR82 iPS-T cells harboring full-length TCR.Example 4 Production of cytotoxic iPS-T cells using dTCR iPS-CL cells stored in a cell bank (2)Generation of dTCR iPS-CL Cells Expressing GPC3 Tumor Antigen-Specific TCRTCR82 gene that connects the TCRβ chain gene sequence and TCRα chain gene sequence for EYILSLEEL (Seq. No. 1), an HLA-A24-restricted peptide derived from the tumor antigen GPC3 with a P2A sequence and pMXs retroviral vector that contains IRES-GFP were generated. This vector was transfected into packaging cells to produce a retrovirus.The virus generated as described above was attached to a 24-well plate coated with 50 μg / mL RetroNectin by centrifugation (Refer to the RBV method described in TaKaRa Bio Catalog "RetroNectin"). Then, dTCR iPS-CL cells stimulated with 1 μg / mL anti-human CD3 antibody (Clone: OKT3) and 5 μg / mL RetroNectin for 3 days were added to the wells for infection. dTCR iPS-CL cells were cultured with cell culture medium A (α-MEM (Nacalai) supplemented with 15% fetal bovine serum (Thermo), 2 mM L-glutamine (Thermo), 100 U / mL penicillin, and 1G streptomycin) supplemented with 1 ng / mL rhIL-7 (Wako), 50 μg / mL ascorbic acid 2-phosphate, and insulin-transferrin-selenium, and allowed to grow for about 10 days, while replacing the medium every few days.Confirmation of TCR82 IRES GFP Gene Introduction into dTCR iPS-CL CellsFIG. 12 shows the results of flow cytometry analysis of GFP expression in dTCR iPS-CL cells into which TCR82 and GFP genes have been introduced.The TCR82 IRES GFP gene or GFP gene was introduced into iPS-CL cells induced to differentiate from iPSCs expressing dTCRba2 and dTCRba9 using a retrovirus, and TCR82-expressing dTCR iPS-CL cells (TCR82-transduced antigen-specific iPS-T cells) were generated. Instead of dTCR, TCR82 iPS-T cells differentiated from iPSCs expressing TCR82, a wtTCR, were generated as a control (FIG. 12(a)). Cells into which only GFP was introduced were also generated (Fig. 12(b)). The expression of the marker GFP was analyzed by flow cytometry, and the percentage of cells into which the gene had been introduced was analyzed using GFP expression as an indicator. As a result, 37.5-58.6% of the cells were GFP positive, and the gene transfer was confirmed.Analysis of Expression of TCR82 in TCR82-Transduced-dTCR iPS-CL cellsFIG. 13 shows the results of analyzing TCR expression in TCR82-transduced-dTCR iPS-CL cells using a TCR82 antigen tetramer and a TCRαβ antibody. dTCR iPS-CL cells into which the GFP gene or TCR82 IRES GFP gene has been introduced using a retrovirus were stained using GPC3 peptide / HLA-A*24:02 tetramer and TCR PAN α / β antibody, and analyzed by flow cytometry. As a result, GPC3 tetramer / TCRαβ double positive cells were observed in TCR82-transduced-dTCR iPS-CL cells.Analysis of Antigen Reactivity of TCR82-transduced-dTCR iPS-CL cellsFIG. 14 shows the results of analyzing the antigen reactivity of TCR82-transduced-dTCR iPS-C cells. After coculturing HLA-A*24:02-positive B-LCLs (target cells) pulsed with GPC3 peptide and TCR82-transduced-dTCR iPS-CL cells, expression of IFN-γ, CD107a and TNF-α was analyzed by flow cytometry. Furthermore, cells into which TCR82 had been introduced were gated as GFP-positive cells, and the percentages of IFN-γ, CD107a, and TNF-α positive cells in these cells were analyzed. In both types of cells, cells expressing IFN-γ, CD107a, and TNF-α were observed. Particularly, results in which TCR82-transduced-dTCRba9 iPS-CL cells had a high percentage of cells expressing IFN-γ, CD107a, and TNF-α were obtained.Analysis of Cytotoxic Activity of TCR82-transduced-dTCR iPS-CL cellsFIG. 15 shows the analysis results of the cytotoxic activity of TCR82-transduced-dTCR iPS-CL cells against target cells. HLA-A*24:02-positive B-LCLs pulsed with GPC3 peptide were used as target cells, and after coculturing TCR82-transduced-dTCR iPS-CL cells at various effector: target ratios, the percentage of target cells killed was analyzed by a non-RI cytotoxicity assay kit. dTCRba9 iPS-CL cells expressing TCR82 showed higher cytotoxic activity than dTCRba2 iPS-CL cells expressing TCR82.Analysis of Stability of TCR82 Expression in TCR82-transduced-dTCR iPS-CL cellsFIG. 16 shows that the TCR82 gene introduced into dTCR iPS-CL cells is expressed stably. The expression of TCR82 was analyzed by flow cytometry as the expression of GFP, a gene expression marker. In the TCR82-transduced-dTCR iPS-CL cells that expressed TCR82, many cells maintained GFP expression even after the second anti-CD3 antibody stimulation (2nd expansion). Particularly, GFP expression was good in TCR82-transduced-dTCRba9 iPS-CL cells.In addition, in the present application, all the disclosed contents of pamphlet of WO 2020 / 138371 (Patent Document 2) and WO 2023 / 249071 (in particular, the information on the genes and amino acid sequences of dTCRba9, dTCRba2, and TCR82) are incorporated by reference.Example 5Production of iPS-T cells using NY-ESO-1-specific 1G4 TCRT cell differentiation of 1G4 TCR-introduced iPSCs and analysis of functional stable expression of 1G4 TCRNY-ESO-1 is a cancer-testis antigen that is found to be expressed in cancers such as esophageal cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, etc. 1G4 TCR is a TCR specific to the NY-ESO-1 peptide (SLLMWITQC) presented by HLA-A*02:01 (Non-Patent Documents 12). It was verified whether iPS cells transduced with 1G4 TCR could differentiate into iPS-T cells and stably express 1G4 TCR. After introducing the 1G4 TCR gene into the human iPS cell line Ff-I01s04 using the PiggyBac system, the cells were differentiated into iPS-T cells and stained with Dextramer (NY-ESO-1-Dex) consisting of a complex of NY-ESO-1 peptide (SLLMWITQC) and HLA-A*02:01, and Dextramer (MAGE-A4-Dex) consisting of a complex of MAGE-A4 peptide (GVYDGREHTV) and HLA-A*02:01 as a negative control, and the binding of each Dextramer was analyzed by flow cytometry. At the same time, the cells were stained with TCRab antibody to detect TCR expression.The results are shown in Figure 18. In iPS-T cells transduced with the 1G4 TCR gene, NY-ESO-1-Dex and TCRab double positive cells were observed at a high rate of 88.1%, while no MAGE-A4-Dex positive cells were observed. The expression of 1G4 TCR in iPS-T cells and their specificity to the NY-ESO-1 peptide / HLA-A*02:01 antigen was confirmed. These results indicate the feasibility of immune cell therapy using iPS-T cells transfected with the 1G4 TCR gene with NY-ESO-1 as a target antigen. Because of this result, 1G4 TCR is a great candidate for transduction at iPSC-derived cytotoxic lymphocytes expressing dTCR (dTCR iPS-CL).Example 6Production of iPS-T cells using HPV E7-specific TCRCloning of HPV E7-specific TCRHPV is a virus that causes cervical cancer, and it is known that expression of the HPV-derived E7 protein is involved in carcinogenesis. In the present invention, the cloning of TCR genes specific to the HPV-E7 peptide (YMLDLQPETT) presented by HLA-A*02:01 was performed. From commercially available T cells stimulated with HPV-E7 peptide (YMLDLQPETT), Tetramer-positive single cells were sorted using a cell sorter with the tetramer of the HPV-E7 peptide / HLA-A*02:01 complex (HPV E7 Tetramer) as a probe, and two types of TCR cDNA were obtained from these cells. FIG. 19 shows the results of antigen specificity when the expression plasmids for these two types of TCRs were introduced into Jurkat cells by electroporation to express the TCRs. Jurkat cells expressing the TCRs were stained with HPV E7 Tetramer and TCRαβ antibodies and analyzed by flow cytometry. As a result, it was shown that TCR Clone1 of the two types of TCRs is specific to HPV-E7 Tetramer. This TCR Clone1 could be introduced at iPSC-derived cytotoxic lymphocytes expressing dTCR (dTCR iPS-CL).Example 7Production of cancer-specific iPS-T cells from the dTCR iPS-CL cell bankTo lose the expression of the HLA Class I and HLA Class II, the B2M and CIITA genes of iPSCs have been knocked out by gene editing, and tCD64 and SIRPα Engager have been introduced into iPSCs to avoid attack by the patient's immune system. In addition to these modifications, iPSCs introduced with IL-15 and IL-21 genes that enhance iPS-T function are being developed for the dTCR iPS-CL cell (see

[0025] and FIG. 2).By creating the dTCR iPS-CL cell bank and introducing any cancer-specific TCR gene into the iPS-T cells derived from the dTCR iPS-CL cell bank, it is possible to produce cancer-specific iPS-T cells that can be used to treat the desired cancer. As shown in FIG. 20, by introducing the TCR82 gene, GPC3-specific iPS-T cells can be produced, and used to treat cancers expressing GPC3, such as hepatocellular carcinoma, ovarian cancer, and the like. Similarly, the 1G4 TCR gene can be transduced to produce NY-ESO-1-specific iPS-T cells to treat cancers expressing NY-ESO-1, and the HPV E7 TCR gene can be transduced to produce HPV E7-specific iPS-T cells to treat cancers expressing HPV E7. Similarly, any TCRs could be introduced to dTCR iPS-CL. It is possible to produce iPS-T cells for cancer therapy for various cancers by changing the TCR to be transduced. In the future, it will be possible to produce iPS-T cells for personalized cancer therapy by introducing genes such as TCRs specific to the cancer of individual patients, e.g., neoantigen-specific TCRs.SEQUENCE LISTING<110> Shinobi Therapeutics Co. Ltd.<120>　 A cytotoxic lymphocyte expressing a variable region-deleted T cell receptor<130>　SHINOBI24001<160> 4<170><210> 1<211> 9<212> PRT<213> Artificial Sequence<220> Tumor-Associated Antigen<223> HLA-A24-restricted GPC3 epitope peptide<400> 1Glu Tyr Ile Leu Ser Leu Glu Glu Leu1 5<210> 2<211> 9<212> PRT<213> Artificial Sequence<220> Tumor-Associated Antigen<223> HLA-A2-restricted GPC3 epitope peptide<400> 2Phe Val Gly Glu Phe Phe Thr Asp Val1 5<210> 3<211> 379<212> PRT<213> Artificial Sequence<220> Variable region-deleted T cell receptor<223> Amino acid sequence of ba2<400> 3Met Leu Ser Leu Leu Leu Leu Leu Leu Gly Leu Gly Ser Val Phe Ser1 5 10 15Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro20 25 30Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu35 40 45Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn50 55 60Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys65 70 75 80Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu85 90 95Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys100 105 110Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp115 120 125Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg130 135 140Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser145 150 155 160Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala165 170 175Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp180 185 190Ser Arg Gly Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys195 200 205Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Cys Pro Gly Phe Leu210 215 220Trp Ala Leu Val Ile Ser Thr Cys Leu Glu Phe Ser Met Ala Asn Ile225 230 235 240Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser245 250 255Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val260 265 270Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu275 280 285Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser290 295 300Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile305 310 315 320Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys325 330 335Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn340 345 350Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe355 360 365Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser370 375<210> 4<211> 322<212> PRT<213> Artificial Sequence<220> Variable region-deleted T cell receptor<223> Amino acid sequence of ba9<400> 4Met Leu Ser Leu Leu Leu Leu Leu Leu Gly Leu Gly Ser Val Phe Ser1 5 10 15Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro20 25 30Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu35 40 45Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn50 55 60Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys65 70 75 80Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu85 90 95Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys100 105 110Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp115 120 125Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg130 135 140Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser145 150 155 160Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala165 170 175Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp180 185 190Ser Arg Gly Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys195 200 205Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Cys Pro Gly Phe Leu210 215 220Trp Ala Leu Val Ile Ser Thr Cys Leu Glu Phe Ser Met Ala Lys Ser225 230 235 240Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn245 250 255Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro260 265 270Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp275 280 285Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu290 295 300Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp305 310 315 320Ser Ser

Claims

1. A cytotoxic lymphocyte expressing a variable region-deleted T cell receptor (TCR) comprising a first polypeptide and a second polypeptide, the first polypeptide including a constant region of a human TCRα chain or a fragment thereof, and not including a variable region of the human TCRα chain, the second polypeptide including a constant region of a human TCRβ chain or a fragment thereof, and not including a variable region of the human TCRβ chain, wherein: the cytotoxic lymphocyte lacks antigen specificity due to the expression of the variable region-deleted TCR; the cytotoxic lymphocyte is differentially induced from a pluripotent stem cell into which a nucleic acid encoding the variable region-deleted TCR is introduced from outside the cell; and the cytotoxic lymphocyte is an intermediate for producing an antigen-specific killer T cell by expressing one or more receptors that may recognize an identical or different antigen.

2. The cytotoxic lymphocyte according to claim 1, wherein the cytotoxic lymphocyte comprises the nucleic acid encoding the variable region-deleted TCR introduced from outside the cell.

3. The cytotoxic lymphocyte according to claim 1, wherein the cytotoxic lymphocyte comprises an expression vector containing the nucleic acid encoding the variable region-deleted TCR.

4. The cytotoxic lymphocyte according to claim 1, wherein the pluripotent stem cell is a human induced pluripotent stem cell.

5. The cytotoxic lymphocyte according to claim 1, wherein one of the one or more receptors that may recognize an identical or different antigen is a TCR containing a variable region recognizing an antigen (an antigen-recognizing TCR) and the cytotoxic lymphocyte expresses CD8α and CD8β chains double positively functioning as co-receptors for the antigen-recognizing TCR.

6. A cell bank comprising the cytotoxic lymphocyte according to any one of claims 1 to 5.

7. The cell bank according to claim 6, wherein the cytotoxic lymphocyte is a cell to be introduced with one or more nucleic acids, each encoding a different receptor that may recognize an identical or different antigen.

8. The cell bank according to claim 6, wherein the cytotoxic lymphocyte is cryopreserved.

9. The cell bank according to claim 7, wherein one or more nucleic acids, each encoding a different receptor that may recognize an identical or different antigen, are introduced into the cytotoxic lymphocyte, wherein the cytotoxic lymphocyte serves as an intermediate for producing the antigen-specific killer T cell used for prophylaxis and / or treatment of cancer.

10. Use of the cell bank according to claim 6 for producing the antigen-specific killer T cell used for prophylaxis and / or treatment of cancer.

11. An antigen-specific killer T cell produced from the cytotoxic lymphocyte stored in the cell bank according to claim 6.

12. A pharmaceutical composition comprising the antigen-specific killer T cell according to claim 11.

13. A method for prophylaxis and / or treatment of cancer, using the pharmaceutical composition according to claim 12.

14. The cytotoxic lymphocyte according to any one of claims 1 to 5, wherein the cytotoxic lymphocyte is capable of expressing one or more receptors that may recognize an identical or different antigen.

15. The cytotoxic lymphocyte according to claim 14, wherein the cytotoxic lymphocyte comprises one or more nucleic acids, each encoding a different receptor that may recognize an identical or different antigen.

16. The cytotoxic lymphocyte according to claim 14, wherein the cytotoxic lymphocyte comprises one or more expression vectors, each including a different nucleic acid encoding a different receptor that may recognize an identical or different antigen.

17. The antigen-specific killer T cell induced by expressing one or more receptors that may recognize an identical or different antigen in the cytotoxic lymphocyte according to claim 14.

18. The antigen-specific killer T cell according to claim 17, wherein one of the one or more receptors that may recognize an identical or different antigen is a TCR containing variable region recognizing antigen (an antigen-recognizing TCR).

19. The antigen-specific killer T cell according to claim 18, wherein the antigen-recognizing TCR recognizes a single antigen.

20. The antigen-specific killer T cell according to claim 19, wherein the single antigen is a viral antigen or a tumor-associated antigen.

21. The antigen-specific killer T cell according to claim 20, wherein the viral antigen is selected from the group consisting of viral proteins and fragments thereof produced in cells infected with influenza virus, EBV, HPV, HBV, HCV, HIV, coronavirus, CMV, dengue virus, West Nile virus, hantavirus, Ebola virus, and HTLV-1.

22. The antigen-specific killer T cell according to claim 20, wherein the tumor-associated antigen is selected from the group consisting of GPC3, WT1, XAGE1, LMP2, NY-ESO-1, KRAS, PI3K, MUC1, EGFR, HER-2 / neu, MAGEA-3, PSMA, CEA, MART1, gp100, bcr-abl, hTERT, p53, BCMA, MUC5A1, MUC6, MAGE-A1, PRAME, SSX2 / 4, PSCA, CTLA-4, GD2, GD3, fucosyl GM1, GM3, sLe(a), glycolipid F77, mesothelin, PD-L1, trp1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, ETV6-AML, PSA, AFP, EpCAM, ALK, androgen receptor, EphA2, CYP1B1, OY-TES-1, MAD-CT-2, survivin, Ras, EGR, XBP-1, neoantigens due to genetic mutations, neoantigens due to splicing abnormalities, and fragments thereof.

23. The antigen-specific killer T cell according to claim 20, wherein the tumor-associated antigen is HLA-A24-restricted GPC3 peptide EYILSLEEL (Seq. No. 1) or HLA-A2-restricted GPC3 peptide FVGEFFTDV (Seq. No. 2).

24. A pharmaceutical comprising the antigen-specific killer T cell according to claim 18.

25. The pharmaceutical according to claim 24, for use in prophylaxis and / or treatment of cancer.

26. A cytotoxic agent for cells expressing a viral antigen, the cytotoxic agent comprising the antigen-specific killer T cell according to claim 18.

27. The cytotoxic agent according to claim 26, wherein the viral antigen is selected from the group consisting of viral proteins and fragments thereof produced in cells infected with influenza virus, EBV, HPV, HBV, HCV, HIV, coronavirus, CMV, dengue virus, West Nile virus, hantavirus, Ebola virus, and HTLV-1.

28. A cytotoxic agent for cells expressing a tumor-associated antigen, the cytotoxic agent comprising the antigen-specific killer T cell according to claim 18.

29. The cytotoxic agent according to claim 28, wherein the tumor-associated antigen is selected from the group consisting of GPC3, WT1, XAGE1, LMP2, NY-ESO-1, KRAS, PI3K, MUC1, EGFR, HER-2 / neu, MAGEA-3, PSMA, CEA, MART1, gp100, bcr-abl, hTERT, p53, BCMA, MUC5A1, MUC6, MAGE-A1, PRAME, SSX2 / 4, PSCA, CTLA-4, GD2, GD3, fucosyl GM1, GM3, sLe(a), glycolipid F77, mesothelin, PD-L1, trp1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, ETV6-AML, PSA, AFP, EpCAM, ALK, androgen receptor, EphA2, CYP1B1, OY-TES-1, MAD-CT-2, survivin, Ras, EGR, XBP-1, neoantigens due to genetic mutations, neoantigens due to splicing abnormalities, and fragments thereof.

30. A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the antigen-specific killer T cell according to claim 18 to the mammal.

31. A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the pharmaceutical according to claim 24 to the mammal.

32. A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the cytotoxic agent according to claim 26 to the mammal.

33. A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the cytotoxic agent according to claim 28 to the mammal.

34. The antigen-specific killer T cell according to claim 18, for use in prophylaxis and / or treatment of cancer.

35. The antigen-specific killer T cell according to claim 18, for producing an agent for prophylaxis and / or treatment of cancer.

36. The antigen-specific killer T cell according to claim 17, wherein the receptors comprise both an antigen-recognizing TCR and a chimeric antigen receptor (CAR), each of which may recognize an identical or different antigen.

37. A pharmaceutical comprising the antigen-specific killer T cell according to claim 36.

38. The pharmaceutical according to claim 37, for use in prophylaxis and / or treatment of cancer.

39. A cytotoxic agent for cells expressing a viral antigen, the cytotoxic agent comprising the antigen-specific killer T cell according to claim 36.

40. A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the antigen-specific killer T cell according to claim 36 to the mammal.

41. A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the pharmaceutical according to claim 37 to the mammal.

42. A method for prophylaxis and / or treatment of cancer in a mammal, comprising administering an effective amount of the cytotoxic agent according to claim 39 to the mammal.

43. The antigen-specific killer T cell according to claim 36, for use in prophylaxis and / or treatment of cancer.

44. The antigen-specific killer T cell according to claim 36, for producing an agent for prophylaxis and / or treatment of cancer.

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