Method for producing regenerated T cells via iPS cells

Abstract

[Problem] To provide a method for producing regenerated T cells by introducing antigen-specific T cell receptors to iPS cell clones or hematopoietic stem cells differentiated from iPS cell clones, immature T cells or mature T cells. [Solution] The present invention is a method for producing regenerated T cells via iPS cells, the method comprising: (1) a step for obtaining T cells from a subject, and preparing, from individual cells in a group of T cells that is reactive to a tumor-related antigen, cDNAs that code for TCRα chain and TCRβ chain; (2) a step for initializing the subject-derived peripheral blood mononuclear cells from which B cells and T cells have been removed, or T cells, to iPS cells, and selecting, from the iPS cells thus obtained, iPS cell clones that demonstrate high differentiation efficiency into T cells; (3) a step for introducing the cDNAs from the individual cells into the iPS cell clones, into hematopoietic stem cells differentiated from the iPS cell clones, into immature T cells differentiated from the hematopoietic stem cells, or into mature T cells differentiated from the immature T cells; and (4) a step for differentiating the iPS cell clones, the hematopoietic stem cells or the immature T cells, which are obtained in step 3 and into which the cDNAs have been introduced, into mature T cells and proliferating the mature T cells.

Description

[Technical Field]

[0001] The present invention relates to a method for producing antigen-specific regenerated T cells by introducing a T cell receptor obtained from a T cell population that is reactive to tumor-associated antigens into iPS cells derived from non-T non-B cells or monocytes or T cells; regenerated T cells produced by the said method; a cancer preventive or therapeutic agent comprising the regenerated T cells as an active ingredient; a pharmaceutical composition containing the regenerated T cells; and a cancer preventive or therapeutic method using the pharmaceutical composition. [Background technology]

[0002] T cells play a central role in the immune response against foreign pathogens such as bacteria or viruses, or abnormal cells such as cancer cells. Therefore, a decline in T cell function is thought to contribute to pathogen infection and cancer development. T cell replacement therapy or regenerative therapy for patients with diseases caused by T cell dysfunction can be an extremely effective means of improving the condition and treating the disease in these patients.

[0003] Studies using humans and mice have shown that when T cell replacement therapy is performed for infectious diseases or cancer, high therapeutic efficacy can be obtained by using T cells that specifically recognize antigens present on foreign pathogens such as bacteria or viruses, or abnormal cells such as cancer cells. On the other hand, the difficulty in securing a sufficient quantity of T cells, as well as T cell exhaustion, such as a decrease in the proliferative capacity of T cells and a decrease in the immune response to antigens on target cells, are obstacles to T cell replacement therapy.

[0004] To overcome the aforementioned obstacles in T cell replacement therapy, a T cell replacement therapy has been proposed that uses regenerated T cells, which are created by establishing induced pluripotent stem cells (iPS cells) from antigen-specific T cells, proliferating them, and then differentiating them into T cells. Since T cell receptors (TCRs) for recognizing target antigens are formed by gene rearrangement of the genome, the TCR genes of the pre-reprogrammed T cells are preserved in iPS cells. Therefore, by using T cells specific to the target antigen as raw materials for producing iPS cells, it is possible to produce regenerated T cells that exhibit the same antigen specificity as the original T cells (Patent Document 1 and Non-Patent Document 1).

[0005] Strict antigen specificity is necessary for safe and efficient T cell replacement therapy. However, as mentioned above, it has been reported that the frequency of T cells with the same gene rearrangement pattern as the original T cells is not necessarily high in regenerated T cells obtained from T cells via iPS cells (Patent Document 2). Furthermore, it has been reported that CD8αβ regenerated T cells obtained from human T cells via iPS cells lose antigen specificity due to additional rearrangement of the TCRα chain gene at the CD4 / CD8 double-positive stage (Non-Patent Document 2). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2011 / 096482 pamphlet [Patent Document 2] WO2013 / 176197 pamphlet [Non-patent literature]

[0007] [Non-Patent Document 1] Nishimura T, et al. Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation. Cell Stem Cell. 2013; 12:114-126. [Non-Patent Document 2] Minagawa A, et al. Enhancing T cell receptor stability in rejuvenated iPSC-derived T cells improves their use in cancer immunotherapy. Cell Stem Cell. 2018; 23: 850-858. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] In cancer treatment using regenerated T cells (iPS-T cells) produced via iPS cells, using iPS-T cells that specifically recognize target antigens present in tumor cells or tumor tissue is crucial for ensuring the safety and efficacy of iPS-T cell replacement therapy in cancer treatment. Furthermore, promptly initiating iPS-T cell replacement therapy when cancer onset or recurrence is detected is important for improving treatment outcomes.

[0009] The present invention aims to provide a method for rapidly producing iPS-T cells into which antigen-specific TCRs have been introduced, and iPS-T cells produced by the said method. Furthermore, the present invention aims to provide iPS-T cells produced by the said method, a cancer preventive or therapeutic agent comprising the iPS-T cells as an active ingredient, a pharmaceutical composition containing the iPS-T cells, and a method for preventing or treating cancer using the pharmaceutical composition.

[0010] Recent studies have revealed that even against a single antigen epitope, multiple T cell clones (5 to 10 clones) recognize the antigen using different TCRs respectively. At present, it is extremely difficult to maintain all antigen-specific T cell clones during the process of obtaining iPS-T cells from cells isolated from a patient. The probability that a clone differentiated into iPS-T cells is a T cell clone having an optimal TCR for damaging the target is also affected by individual differences among subjects from which cells for producing iPS-T cells are collected, or by the production batch of iPS-T cells.

[0011] In addition, currently, when iPS-T cells are produced using iPS cell clones derived from cells obtained from a patient, the production process requires a long period of time. Shortening the production period is a major challenge to rapidly provide iPS-T cell replacement therapy to patients in need thereof. [Means for Solving the Problem]

[0012] The present inventors found that: various TCR pools having antigen specificity can be obtained from a T cell population reactive to tumor-associated antigens; iPS-T cells can be produced by introducing TCR into iPS cells established from non-T non-B cells or monocytes, or iPS cells established from T cells; uniform quality of regenerated T cells and shortening of the production period can be achieved by previously selecting iPS cell clones with high differentiation efficiency into T cells; the production of the iPS-T cells can be rapidly performed by adjusting the timing of obtaining the TCR pool and establishing the iPS cells at the same time; and the origin of the TCR pool and the non-T non-B cells, monocytes or T cells may be the same individual or different individuals, thereby leading to completion of the present invention.

[0013] That is, the object of the present invention is achieved by the following invention. [1] (1) a step of preparing cDNAs respectively encoding T cell receptor α chain and β chain for each single cell from a T cell population obtained from a subject, the T cell population having reactivity to a tumor-associated antigen (2) a step of reprogramming peripheral blood mononuclear cells from a subject, from which B cells and T cells have been removed, or T cells into iPS cells, and selecting an iPS cell clone having high differentiation efficiency from the obtained iPS cells into T cells (3) a step of introducing the cDNA into the iPS cell clone, a hematopoietic stem cell differentiated from the iPS cell clone, an immature T cell differentiated from the hematopoietic stem cell, or a mature T cell differentiated from the immature T cell, and (4) a step of differentiating the iPS cell clone, the hematopoietic stem cell, or the immature T cell into which said cDNA has been introduced, obtained in step (3), into mature T cells and proliferating said mature T cells A method for producing regenerated T cells via iPS cells, comprising: [2] (1) a step of contacting T cells obtained from a subject with a tumor-associated antigen, and preparing cDNAs respectively encoding T cell receptor α chain and β chain for each single cell from a T cell population having reactivity to the tumor-associated antigen (2) a step of reprogramming peripheral blood mononuclear cells from a subject, from which B cells and T cells have been removed, or T cells into iPS cells, and selecting an iPS cell clone having high differentiation efficiency from the obtained iPS cells into T cells (3) a step of introducing the cDNA into the iPS cell clone, a hematopoietic stem cell differentiated from the iPS cell clone, an immature T cell differentiated from the hematopoietic stem cell, or a mature T cell differentiated from the immature T cell, and (4) a step of differentiating the iPS cell clone, the hematopoietic stem cell, or the immature T cell into which said cDNA has been introduced, obtained in step (3), into mature T cells and proliferating said mature T cells A method for producing regenerated T cells via iPS cells, comprising: [3] (1) A step of contacting T cells obtained from a subject administered with a tumor-associated antigen with the tumor-associated antigen, and preparing cDNA encoding the α chain and β chain of the T cell receptor, respectively, from a population of T cells that are reactive to the tumor-associated antigen, for each single cell. (2) A step of reprogramming peripheral blood mononuclear cells from the subject, from which B cells and T cells have been removed, or from T cells into iPS cells, and selecting iPS cell clones from the obtained iPS cells that have a high differentiation efficiency into T cells. (3) A step of introducing the cDNA into the iPS cell clone, hematopoietic stem cells differentiated from the iPS cell clone, immature T cells differentiated from the hematopoietic stem cells, or mature T cells differentiated from the immature T cells, and (4) A step of differentiating the iPS cell clone obtained in step (3), the hematopoietic stem cell, or the immature T cell into mature T cells and proliferating the mature T cell, A method for producing regenerated T cells via iPS cells, including the above. [4] The method according to any of [1] to [3], wherein the execution of step (2) precedes the execution of step (1), or is performed in parallel with the execution of step (1). [5] The method according to any of [1] to [4], wherein the subject in steps (1) and (2) is the same individual. [6] The method according to any one of [1] to [4], wherein the subjects in step (1) and (2) are separate individuals, and the subject in step (2) is the subject of cancer prevention or treatment. [7] The method according to [6], further comprising the step of selecting T cells from the T cells into which the cDNA obtained in step (4) has been introduced that do not show an allo-reaction to cells derived from the subject in step (2). [8] The method according to [6], further comprising the step of selecting from the cDNA prepared in step (1) cDNA encoding a T cell receptor that does not induce an allochemical response to cells derived from the subject in step (2). [9] The method according to [7] or [8], wherein the cells derived from the subject are peripheral blood mononuclear cells.

[10] The method according to any one of [1] to [9], wherein the tumor-associated antigen is selected from the group consisting of GPC3, WT1, XAGE1, LMP2, NY-ESO-1, EB virus antigen and neoantigens and peptide fragments thereof.

[11] The method according to any one of [1] to [9], wherein the tumor-associated antigen is EYILSLEEL (SEQ ID NO: 1), an HLA-A24-restricted GPC3 peptide; FVGEFFTDV (SEQ ID NO: 2), an HLA-A2-restricted GPC3 peptide; or a mixture thereof.

[12] The method according to any one of [1] to

[11] , wherein in step (3), the introduction of the cDNA into the iPS cell clone, the hematopoietic stem cell differentiated from the iPS cell clone, the immature T cell differentiated from the hematopoietic stem cell, or the mature T cell differentiated from the immature T cell is performed using a viral vector, a non-viral vector, or genome editing technology.

[13] The method according to

[12] , wherein the nonviral vector is a transposon vector.

[14] The method according to

[13] , wherein the transposon vector is a piggyBac® vector.

[15] The method according to any one of [1] to

[14] , wherein in step (4), the step of differentiating the iPS cell clone into which the cDNA has been introduced, the hematopoietic stem cell, or the immature T cell into mature T cells and the proliferation of the mature T cells is carried out in the presence of feeder cells and PHA (phytohemagglutinin), in the presence of retronectin (registered trademark) and an anti-CD3 antibody, or in the presence of an anti-CD3 antibody and an anti-CD28 antibody.

[16] The method according to

[15] , wherein the feeder cells are autologous or allogeneic peripheral blood mononuclear cells.

[17] The method according to any one of [1] to

[16] , wherein the subject in step (2) is a patient with hepatocellular carcinoma or hepatoblastoma.

[18] The method according to any one of [1] to

[17] , wherein the T cell population in step (1) is CD3 / CD137 double positive.

[19] The method according to any one of [1] to

[17] , wherein the T cell population in step (1) binds to an MHC tetramer or MHC dextramer® that forms a complex with the tumor-associated antigen peptide.

[20] The method according to any one of [1] to

[19] , wherein the hematopoietic stem cells are CD34 / CD43 double-positive. 〔twenty one〕 The method according to any one of [1] to

[20] , wherein the immature T cells are CD8α / β chain double-positive. 〔twenty two〕 The method according to any one of items [1] to

[21] , wherein the iPS cell clones selected in step (2), hematopoietic stem cells differentiated from the iPS cell clones, immature T cells differentiated from the hematopoietic stem cells, or mature T cells differentiated from the immature T cells are stored to constitute a master cell bank. 〔twenty three〕 The method according to

[22] , wherein the preservation is cryopreservation. 〔twenty four〕 The method according to

[22] or

[23] , wherein steps (3) and (4) are performed on the iPS cell clones, hematopoietic stem cells, immature T cells, or mature T cells stored in the master cell bank, respectively. 〔twenty five〕 The master cell bank according to

[22] , comprising the iPS cell clone, the hematopoietic stem cell, the immature T cell, or the mature T cell.

[26] Regenerated T cells produced by any of the methods described in [1] to

[24] .

[27] A cancer preventive or therapeutic agent comprising the regenerated T cells described in

[26] as an active ingredient.

[28] A pharmaceutical composition containing the regenerated T cells described in

[26] .

[29] A method for preventing or treating cancer using the pharmaceutical composition described in

[28] . [Effects of the Invention]

[0014] In the present invention's method for producing regenerated T cells (iPS-T cells) via iPS cells, peripheral blood mononuclear cells (non-T non-B cells or monocytes) or T cells, from which T cells and B cells have been removed, collected from subjects undergoing cancer treatment, are reprogrammed to obtain iPS cells. After obtaining iPS cells, iPS cell clones with good differentiation efficiency into T cells are pre-selected. Therefore, a sufficient amount of iPS-T cells necessary for treatment can be secured in accordance with the timing required for treatment. Furthermore, in the present invention, TCRs are introduced into iPS cell clones induced from non-T non-B cells or monocytes or T cells, hematopoietic stem cells differentiated from the iPS cell clones, immature T cells differentiated from the hematopoietic stem cells, or mature T cells differentiated from the immature T cells. As a result, when these cells are differentiated into iPS-T cells and then cultured on an extended basis, TCR gene rearrangement is less likely to occur, and the antigen specificity of the introduced TCR is maintained. Moreover, it is possible to produce iPS-T cells that are less exhausted due to extended culture. Furthermore, when manufacturing iPS-T cells, iPS cell clones with good differentiation efficiency into T cells are selected and used. This minimizes the impact of variations in yield and differentiation degree between iPS-T cell manufacturing batches, as well as individual differences among subjects from whom cells are collected, on the quality of the resulting iPS-T cells. It also enables the efficient production of T cells with TCRs that recognize antigens and efficiently kill targets.

[0015] According to the method of the present invention, the preparation of cDNA encoding TCRs is performed on a single-cell basis from a T cell population with genetic diversity, making it possible to prepare a diverse group of cDNAs with different antigen specificities. Furthermore, since the creation and storage of iPS cell clones derived from non-T non-B cells, monocytes, or T cells are performed prior to or in parallel with the preparation of cDNA encoding antigen-specific TCRs, iPS-T cells can be produced in a shorter period of time compared to methods in which these steps are performed sequentially. Therefore, in cancer treatment using iPS-T cell replacement therapy, it becomes possible to rapidly prepare iPS-T cells that are specific to the expressed antigen in response to antigenic changes in tumors, the development of treatment resistance, or cancer recurrence in cancer patients being treated.

[0016] According to the method of the present invention, the subject from whom T cells used to prepare cDNA encoding TCRs are collected may be the same individual as the cancer patient being treated with iPS-T cell replacement therapy, or they may be different individuals. This makes it possible to create a TCR pool that corresponds to a variety of antigens, and to select a TCR with optimal antigen specificity for each individual cancer patient. [Brief explanation of the drawing]

[0017] [Figure 1] This diagram shows a schematic of the process for producing regenerated T cells from iPS cells that have been reprogrammed from non-T, non-B cells or monocytes in peripheral blood. [Figure 2] This figure shows the details of the process for producing regenerated T cells from iPS cells that have been reprogrammed from peripheral blood non-T non-B cells or monocytes. In Figure 2, the upper section shows the process of isolating the tumor-associated antigen X-specific TCR gene and verifying antigen-specific reactivity, while the lower section shows the process of producing TCR gene-transformed iPS cells and producing regenerated T cells from the iPS cells. [Figure 3] This diagram shows the process of introducing a TCR gene using a transposon. [Figure 4]This figure shows the phenotype of regenerated T cells (tumor-associated antigen-specific CD8-positive cytotoxic T cells) induced from iPS cells. [Figure 5] This figure shows the results of analyzing telomeres as markers of cellular senescence in regenerated T cells. [Figure 6] This figure shows the results of analyzing the expression of PD-1 and TIGHT molecules as markers of cell exhaustion in regenerated T cells. [Figure 7] This figure compares the levels of IL-2 and IFN-γ produced in cytotoxic T cells (CD8α / β double-positive cells) and regenerated T cells in peripheral blood mononuclear cells of healthy individuals, in response to stimulation with PMA and ionomycin. [Figure 8] This figure shows the antigen-specific cytotoxic activity of regenerated T cells against HLA-A24-expressing cell lines (target cells). Each point represents the mean ± standard deviation. [Figure 9] This figure shows the phenotype of mature T cells derived from iPS cells into which the GPC3 antigen-specific TCR gene has been introduced. [Figure 10] This diagram illustrates a method for selecting iPS cell clones with high differentiation efficiency into T cells in the process of producing regenerated T cells from iPS cells that have been reprogrammed into peripheral blood T cells. [Figure 11] This diagram illustrates a method for producing regenerated T cells from iPS cells that have been reprogrammed into peripheral blood T cells. Specifically, it shows how genome editing is performed on iPS cell clones selected for their high differentiation efficiency into T cells to produce regenerated T cells. [Figure 12] This diagram illustrates a method for producing regenerated T cells that recognize cancer antigens from host T cells. [Figure 13] This diagram shows a schematic of the process for producing regenerated T cells from allogeneic iPS cells that have been reprogrammed from peripheral blood T cells. [Modes for carrying out the invention]

[0018] [T cells] In this invention, "T cells" obtained from a subject are cells that express an antigen receptor called a T cell receptor (TCR) on their cell surface. TCRs exist as heterodimers consisting of α and β chains, and heterodimers consisting of γ and δ chains. T cells having a TCR consisting of α and β chains are called αβ-type T cells, and T cells having a TCR consisting of γ and δ chains are called γδ-type T cells. In one aspect of this invention, the T cells of this invention are preferably CD3 / αβ-type T cells, but may also be CD3 / γδ-type T cells. The TCR of the T cells in this invention is genetically modified.

[0019] In this invention, "T cell population" refers to a group of T cells whose T cell receptor (TCR) gene sequences, which recognize antigens, are diverse as a whole. Therefore, T cells collected from a living organism are a T cell population with specificity for diverse antigens. In living organisms, when T progenitor cells develop and differentiate in the thymus, random recombination of TCR genes occurs, resulting in individual T cells having different TCR gene sequences, making it possible to trigger an immune response to any antigen. The TCR possessed by individual T cells is determined by the antigen or peptide sequence that each cell specifically recognizes, but by considering T cells as a population, it can be understood that this T cell population targets diverse antigens for immune response. Therefore, a T cell population collected from a living organism is, in that sense, a T cell population with genetic diversity.

[0020] In the present invention, "tumor-associated antigen" refers to an antigen that is expressed specifically or nonspecifically in a tumor, and includes antigens derived from proteins overexpressed in tumor cells and their variants, antigens derived from tumor viruses, certain differentiation antigens, and novel tumor-associated antigens (neoantigens) resulting from gene mutations and splice abnormalities. In this specification, tumor-associated antigen may also be referred to as tumor antigen. If it is a protein antigen, it may also be a fragmented peptide (peptide fragment). Antigens expressed specifically or nonspecificly in tumors include WT1, GPC3, XAGE1, MUC1, MUC5AC, MUC6, EGFRvIII, HER-2 / neu, MAGE A3, MAGE Examples of such receptors include, but are not limited to, A1, telomerase, PRAME, SSX2 / 4, PSCA, CTLA-4, gp100, GD2, GD3, fucosyl GM1, GM3, sLe(a), glycolipid F77, mesothelin, PD-L1, trp1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, p53, p53 variants, NY-ESO-1, PSMA, ETV6-AML, CEA, PSA, AFP, hTERT, EpCAM, ALK, androgen receptor, EphA2, CYP1B1, OY-TES-1, MAD-CT-2, MelanA / MART1, survivorbin, Ras, Ras variants, ERG, bcr-abl, and XBP1. Examples of viral antigens include, but are not limited to, inactivated viruses such as inactivated HBV and HPV, as well as proteins derived from various viruses, such as EBV LMP1, EBV LMP2, EBNA (EBV nuclear antigen), HPV E1, HPV E2, HPV E6, HPV E7, HBV HBs, HTLV-1 Tax, and HBZ (HTLV-1 bZIP Factor).

[0021] In one embodiment of the present invention, tumor-associated antigens can be selected from the group consisting of GPC3, WT1, XAGE1, LMP2, NY-ESO-1, EB virus antigens and neoantigens, and peptide fragments thereof.

[0022] In one embodiment of the present invention, the tumor-associated antigen is EYILSLEEL, an HLA-A24-restricted GPC3 peptide; FVGEFFTDV, an HLA-A2-restricted GPC3 peptide; or a mixture thereof. In the present invention, amino acids are represented by commonly used single-letter abbreviations.

[0023] In the present invention, "reactive to tumor-associated antigens" means that T cells have a reaction that occurs when they selectively bind / conjugate via the TCR to epitope peptides derived from tumor-associated antigens presented on major histocompatibility complex (MHC) class I or class II on antigen-presenting cells, and that no T cell reaction occurs when T cells bind / conjugate to anything other than the aforementioned epitope peptides. The T cell reaction resulting from binding / conjugation to epitope peptides derived from tumor-associated antigens presented on MHC class I or class II via the TCR includes cytotoxicity, production of IFN-γ and granzymes, expression of T cell activation markers, and activation of transcription factors such as NF-AT.

[0024] In the present invention, T cells are collected from subjects who are cancer patients or non-cancer patients. Subjects from whom T cells are collected may be cancer patients who have previously received, are currently receiving, or are scheduled to receive a cancer vaccine, or non-cancer patients who have not received a cancer vaccine. One or more types of cancer vaccines may be administered. A cancer vaccine is a composition containing a vaccine antigen, which is a cancer or tumor-specific protein or peptide derived from cancer or the tumor-associated antigen, for inducing a cancer or tumor-specific immune response. Typically, cancer vaccines contain an adjuvant to enhance the specific immune response induced by the vaccine antigen. Alpha-beta T cells are preferred as the T cells. Peripheral blood is preferred as the source of T cells due to its low invasiveness, but is not limited thereto. Other preferred sources of collection include cancerous tissue or tumor tissue, lymph nodes or other tissues or organs, or any source within the body such as blood, umbilical cord blood, lymph, tissue fluid (interstitial fluid, intracellular fluid, and interstitial fluid), body cavity fluid (ascites, pleural fluid, pericardial fluid, cerebrospinal fluid, synovial fluid, and aqueous humor). In one embodiment of the present invention, preferred T cells are tumor tissue-derived T cells. Tumor tissue-derived T cells are typically tumor-infiltrating T cells.

[0025] If the subject is a cancer patient, the cancer of the patient is selected from ovarian cancer, hepatoblastoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, malignant melanoma, non-small cell lung cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid carcinoma, colorectal cancer, or B-cell non-Hodgkin lymphoma. The cancer is preferably hepatocellular carcinoma or hepatoblastoma.

[0026] [iPS cell clones] In the present invention, iPS cells are preferably produced by reprogramming non-T non-B cells, monocytes, or T cells, but are not limited to non-T non-B cells, monocytes, or T cells. "Non-T non-B cells" means mononuclear cells that are not classified as T cells and are not classified as B cells. In the present invention, non-T non-B cells or monocytes can be prepared by collecting peripheral blood mononuclear cells from a subject and then removing T cells and B cells contained in the mononuclear cells. Peripheral blood mononuclear cells can be isolated from human whole blood using a mononuclear cell isolation solution. An example of a mononuclear cell isolation solution is Lymphoprep®. To remove B cells and T cells from mononuclear cells, antibodies against surface antigens of B cells, such as CD19, CD20, CD22, or B cell receptors, and surface antigens of T cells, such as CD3, CD4, or CD8, may be used, for example, by flow cytometry or by using magnetic beads such as MACS® beads. T cells can be isolated from human whole blood using a mononuclear cell isolation solution. Examples of mononuclear cell isolation solutions include Lymphoprep®. T cell purification can be performed using the surface antigens of T cells, such as CD3, CD4, or CD8, or T cell receptors. For example, flow cytometry or magnetic beads such as MACS® beads may be used.

[0027] Methods for producing iPS cells are known in the art. In the present invention, iPS cells can preferably be induced by introducing cell reprogramming factors into non-T non-B cells, monocytes, or T cells. Examples of cell reprogramming factors include genes 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. These cell reprogramming factors may be used individually or in combination. Among these cell reprogramming factors, from the viewpoint of efficiently establishing iPS cells, it is preferable to introduce Oct3 / 4, Sox2, Klf4, and c-Myc (the so-called Yamanaka 4 factors) into the aforementioned non-T non-B cells, monocytes, or T cells.

[0028] There are no particular limitations on the method for introducing the cell reprogramming factor into the non-T non-B cells, monocytes, or T cells, and methods known in the field can be employed. For example, when introducing a gene encoding the cell reprogramming factor into the non-T non-B cells or monocytes, the gene encoding the cell reprogramming factor (e.g., cDNA) can be inserted into an expression vector containing a promoter that functions in the non-T non-B cells, monocytes, or T cells, and the expression vector can be introduced into the non-T non-B cells, monocytes, or T cells by infection, lipofection, liposome method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, or electroporation method. When the cell reprogramming factor is in the form of a protein, and the protein is introduced into the non-T non-B cells, monocytes, or T cells, methods include using a protein delivery reagent, using a protein delivery domain fusion protein, electroporation, and microinjection. When the cell reprogramming factor is in the form of messenger RNA (mRNA) and the mRNA is introduced into non-T non-B cells or monocytes, methods include using an mRNA introduction reagent and adding it to a culture medium.

[0029] Examples of expression vectors used for gene transfer by infection include viral vectors such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai viruses, as well as animal cell expression plasmids. However, from the viewpoint of being less prone to insertion mutations, having high gene transfer efficiency, and producing a large number of copies of the introduced gene, it is preferable to use Sendai virus to introduce the gene encoding the cell reprogramming factor into non-T non-B cells, monocytes, or T cells.

[0030] Examples of promoters used in expression vectors for introducing the gene encoding the aforementioned cell reprogramming factor into non-T non-B cells, monocytes, or T cells include the SRα promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, and ubiquitin promoter. These promoters may also 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 promoters, expression vectors may include enhancers, poly(A) addition signals, selection marker genes (e.g., neomycin resistance genes), SV40 origin of replication, etc.

[0031] The culture medium used for culturing iPS cells obtained by reprogramming non-T non-B cells, monocytes, or T cells is not particularly limited, but a culture medium used for culturing animal cells can be used as the base culture medium, and cytokines to maintain the undifferentiated ability of iPS cells can be added to it to prepare the culture medium. Examples of base culture media include Iscove's Modified Dulbecco's Medium (IMDM) culture medium, Medium 199 culture medium, Eagle's Minimum Essential Medium (EMEM) culture medium, αMEM culture medium, Dulbecco's modified Eagle's Medium (DMEM) culture medium, Ham's F12 culture medium, RPMI 1640 culture medium, Fischer's culture medium, Neurobasal Medium (Life Technologies, Inc.), StemFit® AK03N (Ajinomoto Healthy Supply Co., Ltd.), and mixed culture media of these. The culture medium may or may not contain serum. Preferably, the cytokines include bFGF, and its concentration in the culture medium is, for example, 1 to 100 μg / mL (preferably 50 μg / mL).

[0032] In one aspect of the present invention, the method for culturing iPS cells may be adherent culture or suspension culture, but adherent culture is preferred. Examples of methods for isolating iPS cells include a method of physically isolating the cells with a cell scraper or the like, and an isolation method using a dissociation solution having protease activity, a dissociation solution having collagenase activity, or a dissociation solution having both protease activity and collagenase activity (for example, Accutase (registered trademark) and Accumax (registered trademark), etc.).

[0033] In one aspect of the present invention, iPS cells are preferably at 1×10 3 to 1×10 4 cells / cm 2 , 1×10 4 to 1×10 5 cells / cm 2 or 1×10 5 to 1×10 6 cells / cm 2 when the cell density is reached, the cells are passaged to another culture vessel. The number of passages may be any number as long as a required amount of iPS cells is obtained, and is preferably 1 to 5 times or 5 to 10 times.

[0034] The manufactured iPS cells consist of a large number of iPS cell clones. As will be described later, it is preferable to clone iPS cells by the colony pickup method in order to select iPS cell clones with high differentiation efficiency into T cells. There are no particular limitations on the colony pickup method, but methods such as using a pipettor under a microscope, the limiting dilution method, and a fully automated colony picker can be used. The obtained iPS cell clones may be cultured in an expanded culture to form a master cell bank. A "master cell bank" composed of the iPS cell clones is one in which each single pool of iPS cell clones is dispensed into an independent cell storage container and accumulated. It is preferable to cryopreserve the iPS cell clones in the master cell bank. Methods for cryopreserving cells are well known to those skilled in the art. For example, cultured iPS cell clones can be collected, washed with buffer or culture medium, the number of cells counted, concentrated by centrifugation or the like, suspended in a freezing medium (e.g., culture medium containing 10% DMSO), and then cryopreserved at a low temperature. The master cell bank of the present invention can be stored in any facility or storage facility capable of cryopreservation. When preserving cells by freezing, the storage temperature is not particularly limited as long as it is suitable for cell preservation. Examples include -20°C, -80°C, and -120 to -196°C, but -150°C or lower is preferred.

[0035] "Differentiation efficiency" refers to the proportion of hematopoietic stem cells, immature T cells, and mature T cells among all surviving cells at each differentiation stage from iPS cells to hematopoietic stem cells, from hematopoietic stem cells to immature T cells, and from immature T cells to mature T cells. Identification of differentiated cells at each differentiation stage can be performed by FACS analysis of surface markers. Differentiation efficiency to hematopoietic stem cells is expressed as the proportion of CD34 / CD43 double-positive cells among all surviving cells, differentiation efficiency to immature T cells is expressed as the proportion of CD4 / CD8 double-positive cells or CD5-positive cells among all surviving cells, and differentiation efficiency to mature T cells is expressed as the proportion of cells among all surviving cells that are positive for all CD8α, CD8β, TCRα, and TCRβ chains.

[0036] "High differentiation efficiency" or "good differentiation efficiency" means that, in the differentiation of iPS cells into hematopoietic stem cells, the proportion of CD34 / CD43 double-positive cells (hematopoietic stem cells) is 5-15% or more; in the differentiation of hematopoietic stem cells into immature T cells, the proportion of CD4 / CD8 double-positive cells or CD5-positive cells is 10% or more or 50% or more, respectively; and in the differentiation of immature T cells into mature T cells, the proportion of cells in which all of the CD8α, CD8β, TCRα, and TCRβ chains are positive is 50% or more.

[0037] The regenerated T cells of the present invention are preferably produced by first differentiating the iPS cell clone into hematopoietic stem cells, then differentiating the hematopoietic stem cells into immature T cells, and finally differentiating the immature T cells into mature T cells, which are CD8 single-positive T cells.

[0038] In this invention, "hematopoietic stem cells" refer to cells capable of differentiating into hematopoietic cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes. Hematopoietic stem cells and hematopoietic progenitor cells (HPCs) are not distinguished and refer to the same cells unless otherwise specified. Hematopoietic stem cells / progenitor cells are recognized, for example, by being double-positive for the surface antigens CD34 and CD43.

[0039] In this invention, "immature T cells" refers to T cells at each stage from the stage in which neither TCRα nor β chains are expressed, to the stage in which T cells express both TCRα and β chains, through CD4 / CD8 double-positive cells, to CD8 single-positive cells. It is preferable that immature T cells are CD8α / β chain double-positive.

[0040] In this invention, "mature T cell" refers to a T cell that expresses TCRα and β chains and has progressed through CD4 / CD8 double-positive cells to CD8 single-positive cells. Preferably, mature T cells are CD8α / β chain double-positive.

[0041] [Culture of hematopoietic stem cells and immature T cells] Hematopoietic stem cells are preferably produced by culturing iPS cells in a culture medium supplemented with vitamin C derivatives. Here, "vitamin C derivatives" refers to L-ascorbic acid and its derivatives, and "L-ascorbic acid derivatives" refers to those that are converted to vitamin C by enzymatic reactions in the body. Examples of L-ascorbic acid derivatives include vitamin C phosphate, ascorbyl 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, such as sodium L-ascorbate phosphate or magnesium L-ascorbate phosphate. Vitamin C derivatives are included in the culture medium at a concentration of 5 to 500 μg / mL, for example.

[0042] The culture medium used for the production of hematopoietic stem cells is not particularly limited, but it can be prepared by using a culture medium used for culturing animal cells as a base culture medium and adding vitamin C and other substances to it. Examples of base culture media include Iscove's Modified Dulbecco's Medium (IMDM) culture medium, Medium 199 culture medium, Eagle's Minimum Essential Medium (EMEM) culture medium, αMEM culture medium, Dulbecco's modified Eagle's Medium (DMEM) culture medium, Ham's F12 culture medium, RPMI 1640 culture medium, Fischer's culture medium, Neurobasal Medium (Life Technologies Inc.), StemPro34 (Life Technologies Inc.), and mixed culture media of these. The culture medium may contain serum or may be serum-free. If necessary, the basal culture medium may also contain one or more substances selected from, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines.

[0043] The culture medium used for the production of hematopoietic stem cells may also contain cytokines 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). For example, the concentrations of these cytokines are 1-100 ng / mL for BMP4, 1-100 ng / mL for VEGF, 1-100 ng / mL for bFGF, 10-100 ng / mL for SCF, 1-100 ng / mL for TPO, and 1-100 ng / mL for FLT3L.

[0044] A TGFβ inhibitor may be added to the culture medium of hematopoietic stem cells. A "TGFβ inhibitor" is a small molecule inhibitor that interferes with the signaling of the TGFβ family, and examples include SB431542 and SB202190 (RKLindemann et al., Mol. Cancer 2: 20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908 and SD208 (Scios), and LY2109761, LY364947 and LY580276 (Lilly Research Laboratories). The concentration added to the culture medium is preferably 0.5 to 100 μM.

[0045] iPS cells may be co-cultured with feeder cells such as C3H10T1 / 2 (Takayama N., et al. J Exp Med. 2817-2830, 2010) or heterologous stromal cells (Niwa A et al. J Ce11 Physiol. 2009 Nov; 221 (2):367-77).

[0046] The method for culturing iPS cells during hematopoietic stem cell production may be adherent culture or suspension culture, but suspension culture is preferred. For example, iPS cells can be cultured in a dish until 80% confluence, then the colonies are released, dissociated into single cells, and then subjected to suspension culture. Methods for isolating iPS cells include, for example, physical isolation using a cell scraper, isolation using a dissociation solution having protease activity and collagenase activity (e.g., Accutase® and Accumax®), or isolation using a dissociation solution having collagenase activity.

[0047] Suspension culture is a method of culturing cells in a non-adherent state to a culture vessel. Suspension culture is not particularly limited, but it can be performed using culture vessels that have not been artificially treated to improve cell adhesion (e.g., coated with extracellular matrix, etc.), or culture vessels that have been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (po1y-HEMA) or nonionic surfactant polyols (Pluronic F-127, etc.)). When performing suspension culture, it is preferable to form embryoid bodies (EBs) and then culture them. If hematopoietic stem cells are obtained by suspension culture of embryoid bodies, it is preferable to dissociate them into single cells and then perform adherent culture.

[0048] Hematopoietic stem cells can also be prepared from cyst-like structures (also called iPS-sacs) obtained by culturing iPS cells. Here, a "cyst-like structure" is a three-dimensional sac-like (with an internal space) structure derived from iPS cells, formed from endothelial cell populations, etc., and containing hematopoietic stem cells.

[0049] The temperature conditions for culturing iPS cells to produce hematopoietic stem cells are not particularly limited, but for example, they are about 37°C to about 42°C, preferably about 37°C to about 39°C. The culture period can be appropriately determined by those skilled in the art while monitoring the number of hematopoietic stem cells, etc. The number of culture days is not particularly limited as long as hematopoietic stem cells are obtained, but for example, it is at least 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, preferably 14 days. A long culture period is not a problem in the production of hematopoietic stem cells. Furthermore, culture may be performed under low-oxygen conditions, and in one embodiment of the present invention, examples of low-oxygen conditions include oxygen concentrations of 15%, 10%, 9%, 8%, 7%, 6%, 5%, or less than these.

[0050] "CD4 / CD8 double-positive T cells" are T cells in which both the surface antigens CD4 and CD8 are positive (CD8 + CD4+ Since T cells can be recognized by being positive for the surface antigens CD3 and CD45, CD4 / CD8 double-positive T cells can be identified as cells that are positive for CD4, CD8, CD3, and CD45. CD4 / CD8 double-positive T cells can be differentiated into CD4 single-positive cells or CD8 single-positive cells by induction.

[0051] CD4 / CD8 double-positive T cells can be produced by a method that includes the step of culturing hematopoietic stem cells in a culture medium supplemented with a p38 inhibitor and / or SDF-1.

[0052] A "p38 inhibitor" is defined as a substance that inhibits the function of the p38 protein (p38MAP kinase). Examples of p38 inhibitors include, but are not limited to, chemical inhibitors of p38, dominant-negative variants of p38, or nucleic acids that encode them.

[0053] Chemical inhibitors of p38 include, but are not limited to, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole) and its derivatives, SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole) and its derivatives, SB239063 (trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazole-1-yl]cyclohexanol) and its derivatives, SB220025 and its derivatives, PD169316, RPR200765A, AMG-548, BIRB-796, SCl0-469, SCIO-323, VX-702, or FR167653. These compounds are commercially available; for example, SB203580, SB202190, SC239063, SB220025, and PD169316 are available from Calbiochem, and SCl0-469 and SCIO-323 are available from Scios, among others. The p38 inhibitors are added to the culture medium in concentrations ranging from approximately 1 μM to approximately 50 μM.

[0054] Examples of dominant-negative mutants of p38 include p38T180A, which has a point mutation in which threonine at position 180 in the DNA-binding region of p38 is replaced with alanine, and p38Y182F, which has a point mutation in which tyrosine at position 182 of p38 in humans and mice is replaced with phenylalanine.

[0055] SDF-1 (Stromal cell-derived factor 1) may be SDF-1α or its mature form, as well as isoforms such as SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε, or SDF-1φ, or their mature forms, or a mixture of any proportion thereof. Preferably, SDF-1α is used. SDF-1 is also sometimes referred to as CXCL-12 or PBSF.

[0056] SDF-1 may have one or more amino acids substituted, deleted, and / or added in its amino acid sequence, as long as it retains its chemokine activity, and similarly, its glycans may be substituted, deleted, and / or added. Amino acid mutations are permissible as long as at least four cysteine ​​residues (Cys30, Cys32, Cys55, and Cys71 in the case of human SDF-1α) are retained and the mutations maintain at least 90% identity with the amino acid sequence of the native organism. SDF-1 may be from mammals, such as humans, monkeys, sheep, cattle, horses, pigs, dogs, cats, rabbits, rats, or non-human mammals such as mice. For example, the protein registered with GenBank registry number: NP_954637 can be used as human SDF-1α, and the protein registered with GenBank registry number: NP_000600 can be used as SDF-1β.

[0057] SDF-1 may be commercially available, purified from natural sources, or produced by peptide synthesis or genetic engineering techniques. SDF-1 is added to the culture medium in a range of approximately 10 ng / mL to 100 ng / mL, for example.

[0058] The culture medium used for the production of CD4 / CD8 double-positive T cells is not particularly limited, but it can be prepared by using a culture medium used for culturing animal cells as the base culture medium and adding a p38 inhibitor and / or SDF-1, and more preferably vitamin C derivatives. The type of vitamin C derivatives used in the production of CD4 / CD8 double-positive T cells is as described above, for example, and the concentration of vitamin C derivatives is, for example, 5 to 200 μg / mL. Examples of base culture media include Iscove's Modified Dulbecco's Medium (IMDM) culture medium, Medium 199 culture medium, Eagle's Minimum Essential Medium (EMEM) culture medium, αMEM culture medium, Dulbecco's modified Eagle's Medium (DMEM) culture medium, Ham's F12 culture medium, RPMI 1640 culture medium, Fischer's Neurobasal Medium culture medium (Life Technologies Inc.), and mixed culture media of these. The culture medium may or may not contain serum. If necessary, the basal culture medium may also contain one or more substances selected from, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines.

[0059] The culture medium used for producing CD4 / CD8 double-positive T cells may also contain cytokines selected from the group consisting of SCF, TPO (thrombopoietin), FLT3L, and IL-7. For example, the concentrations of these cytokines are 10-100 ng / mL for SCF, 10-200 ng / mL for TPO, 1-100 ng / mL for FLT3L, and 1-100 ng / mL for IL-7.

[0060] Hematopoietic stem cells may be cultured in adherent culture or suspension culture, but adherent culture is preferred. In the case of adherent culture, the culture vessel may be coated. Examples of coating agents include Matrigel (Niwa A, et al. PLoS One. 6(7):e22261, 2011), collagen, gelatin, laminin, heparan sulfate proteoglycan, retronectin, Fc-DLL4 or entactin, and combinations thereof.

[0061] The culture temperature conditions when culturing hematopoietic stem cells to produce CD4 / CD8 double-positive T cells are not particularly limited, but are preferably around 37°C to 42°C, and more preferably around 37°C to 39°C. As for the culture period, those skilled in the art can determine it appropriately while monitoring the number of CD4 / CD8 double-positive T cells. The number of culture days is not particularly limited as long as CD4 / CD8 double-positive T cells are obtained, but is preferably at least 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, or 23 days.

[0062] The obtained CD4 / CD8 double-positive T cells may be used isolated or as a cell population containing other cell types. When isolating, methods well known to those skilled in the art can be used. For example, this may involve labeling with antibodies against CD4, CD8, CD3, and / or CD45 and isolating them using a flow cytometer, or purifying them using an affinity column immobilized with the desired antigen.

[0063] "CD8 single-positive T cells," or mature T cells, are T cells in which the surface antigen CD8 is positive (CD8 + CD4 -These are also called cytotoxic T cells. Since T cells can be recognized by being positive for the surface antigens CD3 and CD45, CD8 single-positive T cells can be identified as cells that are positive for CD8, CD3, and CD45, and negative for CD4.

[0064] CD8 single-positive T cells can be produced by a method that includes culturing CD4 / CD8 double-positive T cells in a culture medium supplemented with adrenocortical hormones.

[0065] The corticosteroid is preferably a glucocorticoid or a derivative thereof, such as cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, or beclomethasone propionate. Preferably, the corticosteroid is dexamethasone. Its concentration in the culture medium is, for example, 1 to 100 nM.

[0066] The culture medium used for producing CD8 single-positive T cells is not particularly limited, but it can be prepared by using a culture medium used for culturing animal cells as the base culture medium and adding adrenocortical hormones to it. Examples of base culture media include Iscove's Modified Dulbecco's Medium (IMDM) medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's Neurobasal Medium medium (Life Technologies Inc.), and mixed culture media of these. The culture medium may or may not contain serum. If necessary, the basal culture medium may also contain one or more substances selected from, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines.

[0067] The culture medium used for producing CD8 single-positive T cells preferably further contains anti-CD3 antibodies, vitamin C derivatives, or cytokines. Examples of such cytokines include IL-2, IL-7, IL-15, and IL-21.

[0068] The anti-CD3 antibody is not particularly limited as long as it is an antibody that specifically recognizes CD3, but examples include antibodies produced from OKT3 clones. The concentration of the anti-CD3 antibody in the culture medium is, for example, 10 to 1000 ng / mL.

[0069] Vitamin C derivatives used in the production of CD8 single-positive T cells include, for example, those mentioned above, and can be used under the same conditions as described above.

[0070] The concentrations of cytokines used in the culture medium for producing CD8 single-positive T cells are, for example, 10-1000 U / mL for IL-2 and 1-100 ng / mL for IL-7.

[0071] The temperature conditions for culturing CD4 / CD8 double-positive T cells to produce CD8 single-positive T cells are not particularly limited, but are preferably around 37°C to 42°C, and more preferably around 37°C to 38°C. The culture period can be appropriately determined by those skilled in the art while monitoring the number of CD8 single-positive T cells. The number of culture days is not particularly limited as long as CD8 single-positive T cells are obtained, but is preferably at least 1 day, 2 days, 3 days, 4 days, or 5 days, and is preferably 3 days.

[0072] [Preparation of cDNA encoding the TCR] In the present invention, it is preferable to prepare the cDNA encoding the TCRα chain and β chain, respectively, for each single cell. Although T cells collected from a subject are a T cell population with overall genetic diversity, the antigen or peptide sequence recognized by the TCR of each individual T cell has been determined, and the antigen specificity of each T cell differs. It is preferable to prepare the cDNA for each single cell in order to select the TCR that is optimal for each tumor-associated antigen, that is, the TCR that has high reactivity to each tumor-associated antigen.

[0073] It is preferable to culture and proliferate T cells obtained from subjects together with the target tumor-associated antigen. Single cells may be isolated from the T cell population responding to the tumor-associated antigen using an activation marker, such as a cell sorter. Cell surface CD137 is preferred as the activation marker. Known methods for isolating human T cells include, for example, flow cytometry using antibodies against T cell surface markers such as CD3 and CD137 and a cell sorter. Gene cloning can be performed from the obtained single T cells using PCR, and the cDNA encoding the TCRα and β chains can be amplified, respectively.

[0074] [Isolation of TCR gene by single-cell PCR] To obtain single cells, CD8-positive T cells specific to tumor antigens obtained from peripheral blood or other sources can be bound to MHC dextramers (registered trademark) that form a complex with the antigen peptide, and then sorted into single cells using a cell sorter. MHC dextramers are compounds composed of a dextran polymer skeleton in which MHC and a fluorescent dye molecule are bound. Instead of MHC dextramers, MHC tetramers may be used. MHC tetramers are tetramers formed by tetramerizing a complex of an antigen peptide and an MHC molecule with biotin and avidin. In another embodiment, CD8-positive T cells specific to tumor antigens obtained from peripheral blood or other sources can be cultured and proliferated in the presence of the antigen, and then sorted into single cells using a cell sorter to form CD3 / CD137 double-positive cells. In another embodiment, a population of cells that bind to MHC dextramers that form a complex with the antigen peptide can be sorted into single cells using a cell sorter from CD3 / CD137 double-positive cells. RNA can be extracted from the obtained single cells, and the TCR gene pair (TCRα chain gene and TCRβ chain gene) can be isolated by PCR using cDNA obtained by reverse transcription. Sequence analysis can be performed on the isolated TCR gene pair to analyze the types of tumor antigen-reactive T cells (TCR repertoire) and their frequency of occurrence.

[0075] [Construction of vectors] PCR fragments amplified using isolated TCR cDNA as a template can be incorporated into viral or non-viral vectors (transposon vectors) using, for example, the Gibson Assembly System. Specifically, a gene consisting of isolated TCRα and TCRβ chain genes linked via a T2A sequence is attached downstream of the ubiquitin promoter, and further downstream, a marker gene such as EGFR (EGFRt, truncated EGFR) or CD19 lacking the intracellular domain is attached to the IRES (internal ribosome entry site) sequence. This construct is then incorporated into a viral or non-viral vector. While both viral and non-viral vectors can be used, non-viral vectors are preferred. Among non-viral vectors, the piggyBac® vector is preferred among transposon vectors. The transposon method is a next-generation gene transfer method that is inexpensive and safe compared to conventional viral vector methods.

[0076] [Cell introduction of TCR cDNA] As a method for introducing cDNA pairs encoding the TCRα and β chains, respectively, into non-T non-B cells or iPS cell clones derived from monocytes or T cells that are well differentiated into T cells, hematopoietic stem cells differentiated from the iPS cell clones, immature T cells differentiated from the hematopoietic stem cells, or mature T cells differentiated from the immature T cells, any of the following can be employed: a method using a viral vector, a method using a non-viral vector, or TCR replacement using genome editing technology. Examples of viral vectors include lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai viruses, as well as animal cell expression plasmids, but retroviruses or lentiviruses are preferred. When performing retrovirus or lentivirus infection, it is preferable to use a method such as spin infection. When using a non-viral vector, the transposon method is preferred. For TCR replacement using genome editing technology, the CRISPR / Cas9 method, CRISPR / MAD method, and CRISPR / CAS3 method may be used. Methods for introducing non-viral vectors into cells or for introducing guide RNA and donor DNA for genome editing include lipofection, liposome therapy, calcium phosphate coprecipitation, DEAE dextran therapy, microinjection, and electroporation. PCR products can also be directly introduced into cells without the use of vectors. Electroporation is preferred for introducing transposon vectors or PCR products into cells. The ExPERT® system (MaxCyte) is preferred as the electroporation equipment.

[0077] Examples of promoters used in expression vectors for introducing the cDNA pair into cells include the EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, and ubiquitin promoter. These promoters may also 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 promoters, expression vectors may include enhancers, poly(A) addition signals, selection marker genes (e.g., neomycin resistance genes), SV40 origin of replication, etc.

[0078] The iPS cell clone into which the cDNA pair has been introduced, the hematopoietic stem cells differentiated from the iPS cell clone, the immature T cells differentiated from the hematopoietic stem cells, or the mature T cells differentiated from the immature T cells can be cultured under the culture conditions described above, such as the culture medium, culture medium composition, and culture temperature, to obtain regenerated T cells expressing a TCR consisting of the α and β chains encoded by the cDNA pair, respectively. When these cells are proliferated in this culture, it is preferable to do so in the presence of feeder cells and PHA (phytohemagglutinin), in the presence of retronectin® and anti-CD3 antibody, or in the presence of anti-CD3 antibody and anti-CD28 antibody. The feeder cells are preferably autologous or allogeneic peripheral blood mononuclear cells. "Autologous" means that the subject from which the peripheral blood mononuclear cells and the iPS cell clone originated is the same, and "allogeneic" means that the subject from which the peripheral blood mononuclear cells and the iPS cell clone originated is different.

[0079] Whether or not the target TCR is expressed can be confirmed, for example, by repertoire analysis of the TCRβ gene using a TCRVβ analysis kit (BECKMAN COULTER: catalog number IM-3497). Since the vector incorporates marker genes such as EGFRt (truncated EGFR) and CD19t (truncated CD19), the expression of TCRα and β chains can be inferred by analyzing the expression of these marker genes.

[0080] The iPS cell clones, hematopoietic stem cells, immature T cells, or mature T cells may constitute a master cell bank after expansion culture. A "master cell bank" as used herein refers to a collection of iPS cell clones, hematopoietic stem cells, immature T cells, or mature T cells, each collected in separate cell storage containers for a single pool of single clones. Typically, it is preferable to proliferate the cells to a degree that does not alter their properties and then dispense them into multiple cell storage containers. The cells stored in the master cell bank are used as starting materials for producing regenerated T cells by introducing a TCR gene or CAR gene. Each time regenerated T cells are produced, the required number of cell storage containers containing these cells can be taken from the master cell bank. Therefore, the regenerated T cells of the present invention can be repeatedly supplied with the same quality.

[0081] In the master cell bank, it is preferable to cryopreserve the cells dispensed into cell storage containers. Methods for cryopreserving cells are well known to those skilled in the art. For example, a single clone of a cell cultured on a large scale can be collected, washed with a buffer or culture medium, the number of cells counted, concentrated by centrifugation or the like, suspended in a freezing medium (e.g., a culture medium containing 10% DMSO), and then cryopreserved at a low temperature. For example, 1 × 10⁶ cells per container. 6 ~10 7A master cell bank can be constructed by accumulating 200 to 1000 cell storage containers, each containing individual cells, in a cell storage container stocker or similar device. The master cell bank of the present invention can be stored in any facility or storage facility capable of cryopreservation. When cryopreserving, the storage temperature is not particularly limited as long as it is suitable for cell preservation. Examples include -20°C, -80°C, and -120 to -196°C, but -150°C or lower is preferred.

[0082] [Production of regenerated T cells from iPS cells] It is said that there are 5 to 10 TCR pairs corresponding to tumor-associated antigens per epitope, and each TCR pair is predicted to have different sensitivities to point mutation variants of tumor-associated antigens. Therefore, even if a point mutation occurs in a tumor-associated antigen, for example, there is a high probability that a TCR capable of recognizing the epitope exists, and antigen-specific regenerated T cells can be rapidly prepared even for point mutation variants. Furthermore, even if the tumor-associated antigen changes completely as a result of the mutation, and resistance to cancer treatment with regenerated T cell replacement therapy occurs, it is possible to analyze the patient's responsiveness to multiple tumor antigens and start producing TCRs that respond to those tumor-associated antigens from already obtained iPS cells with high differentiation potential into T cells or differentiated cells derived from said iPS cells, thereby enabling the rapid production of regenerated T cells.

[0083] [T cells from which TCR is obtained] It is preferable to collect T cells from peripheral blood that serve as the source for obtaining TCR pairs corresponding to tumor-associated antigens. If it is necessary to administer tumor-associated antigens to subjects in advance in order to make tumor-associated antigen-specific T cells appear in the peripheral blood, it is preferable to collect T cells from peripheral blood after administering the tumor-associated antigens to the subjects. Examples of such tumor-associated antigens include GPC3. On the other hand, if tumor-specific T cells are present in peripheral blood even without administration of tumor-associated antigens, tumor-associated antigen-specific T cells may be isolated from peripheral blood using, for example, an MHC dextramer, without administering tumor-associated antigens. Examples of such tumor-associated antigens include WT1, NYESO-1, and EBV antigen.

[0084] As described above, T cells collected from the peripheral blood of subjects either after administration of tumor-associated antigens or without administration of tumor-associated antigens can, in either case, preferably be stimulated with tumor-associated antigens in vitro to amplify tumor-responsive cell populations, and then isolated using activation markers. If the frequency of tumor-associated antigen-specific T cells in peripheral blood is extremely high, in vitro stimulation with tumor-associated antigens may be omitted.

[0085] If the subjects from whom T cells are collected to obtain TCR pairs and the subjects receiving cancer treatment with regenerative T cell replacement therapy are different individuals, it is not necessary to collect antigen-specific T cells from the subjects receiving treatment. Therefore, T cells for obtaining TCR pairs can be collected at any time, independently of the timing of regenerative T cell replacement therapy, allowing for rapid initiation of regenerative T cell replacement therapy.

[0086] [Pharmaceutical Compositions] The pharmaceutical composition containing regenerated T cells of the present invention can be used to treat cancer patients. The pharmaceutical composition of the present invention can be manufactured by methods commonly used in the pharmaceutical technology, such as those described in the Japanese Pharmacopoeia. The pharmaceutical composition of the present invention may contain pharmaceutically acceptable additives. Examples of such additives include cell culture medium, physiological saline, and suitable buffers (e.g., phosphate-based buffers).

[0087] The pharmaceutical composition of the present invention can be prepared by suspending regenerated T cells in physiological saline or a suitable buffer (e.g., a phosphate-based buffer). To achieve the desired therapeutic effect, the amount for a single dose should be, for example, 1 × 10⁶ 7 It is preferable to contain more than 10 cells. More preferably, the cell content is 1 × 10⁶. 8 One or more, more preferably 1 × 10 9 The number of cells is one or more. The cell content can be appropriately adjusted considering the gender, age, weight, condition of the affected area, and condition of the cells of the recipient. In addition to regenerated T cells, the pharmaceutical composition of the present invention may contain dimethyl sulfoxide (DMSO) and serum albumin, etc., for the purpose of protecting the cells. Furthermore, antibiotics, etc., may be included to prevent bacterial contamination, and vitamins and cytokines, etc., may be included to promote cell activation and differentiation. Moreover, the pharmaceutical composition of the present invention may contain other pharmaceutically acceptable components (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, preservatives, antiseptics, physiological saline, etc.).

[0088] The pharmaceutical composition containing regenerated T cells as an active ingredient of the present invention can be cryopreserved. When cryopreserved, the storage temperature is not particularly limited as long as it is suitable for cell preservation. For example, -20°C, -80°C, and -120 to -196°C are examples, but -150°C or lower is preferred. When cryopreserved, it is preferable to store the cells in appropriate containers such as cryopreservation vials and cryopreservation bags. Procedures to minimize the risk of cell damage during freezing and thawing of regenerated T cells are well known to those skilled in the art.

[0089] In the cryopreservation of regenerated T cells according to the present invention, the T cells are recovered from the culture medium, washed with buffer or culture medium, the number of cells is counted, concentrated by centrifugation or the like, suspended in a freezing medium (for example, a culture medium containing 10% DMSO), and then cryopreserved at a low temperature. The regenerated T cells may be stored individually or as a mixture of each clone. The pharmaceutical composition containing the regenerated T cells of the present invention is available in quantities of, for example, 5 × 10⁶ per container such as a freezing vial or freezing bag. 4 pieces~9×10 10 It contains individual regenerated T cells, but this can be changed depending on the type of cancer being treated, the target patient, the route of administration, etc.

[0090] The administration routes for the pharmaceutical composition containing regenerated T cells of the present invention include, for example, infusion, intratumor injection, intra-arterial injection, portal vein injection, and intraperitoneal administration. However, the administration routes are not limited to these, as long as the regenerated T cells, which are the active ingredient in the pharmaceutical composition of the present invention, are delivered to the affected area. The administration schedule can be a single dose or multiple doses. For multiple doses, for example, a method of repeating administration once every 2 to 4 weeks or a method of repeating administration once every six months to one year can be adopted. When creating the administration schedule, the gender, age, weight, and disease state of the target patient may be taken into consideration.

[0091] When using a pharmaceutical composition containing regenerated T cells of the present invention for the prevention or treatment of cancer in cancer patients, if the TCR on the regenerated T cells is allogeneic, a pharmaceutical composition containing regenerated T cells into which a TCR that does not show an allogeneic reaction to normal cells of the cancer patient is used in order to avoid allogeneic reactions in the cancer patient's body.

[0092] The pharmaceutical compositions of the present invention are used for the prevention or treatment of cancer. Examples of cancers include, but are not limited to, ovarian cancer, hepatoblastoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, malignant melanoma, non-small cell lung cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid carcinoma, colorectal cancer, and B-cell non-Hodgkin lymphoma.

[0093] When using a pharmaceutical composition containing regenerated T cells of the present invention in combination with a cancer vaccine for the prevention or treatment of cancer, the timing of administration of the pharmaceutical composition and the cancer vaccine is not limited. The pharmaceutical composition and the cancer vaccine may be administered to the target patient simultaneously or with a time difference. The pharmaceutical composition and the cancer vaccine may be formulated separately or as a combination preparation. The dosage of the pharmaceutical composition and the cancer vaccine may be in accordance with clinically used dosages and can be appropriately selected depending on the disease, target patient, route of administration, and combination with other drugs.

[0094] The administration method of the pharmaceutical composition and cancer vaccine of the present invention is not particularly limited, and ultimately, both the pharmaceutical composition and the cancer vaccine of the present invention are administered. Examples of such administration methods include (1) individual administration of the pharmaceutical composition and cancer vaccine of the present invention via the same administration route with a time difference, (2) simultaneous administration of the pharmaceutical composition and cancer vaccine of the present invention via different administration routes, and (3) individual administration of the pharmaceutical composition and cancer vaccine of the present invention via different administration routes with a time difference.

[0095] By combining the pharmaceutical composition of the present invention with a cancer vaccine, the following excellent effects can be obtained. (1) Compared to administering the pharmaceutical composition or cancer vaccine of the present invention alone, the dosage can be reduced. (2) By administering the pharmaceutical composition and cancer vaccine of the present invention, a long treatment period can be established. (3) By administering the pharmaceutical composition and cancer vaccine of the present invention, the therapeutic effect can be sustained, (4) When the pharmaceutical composition of the present invention is used in combination with a cancer vaccine, a synergistic effect in anticancer or antitumor effects can be obtained.

[0096] In one embodiment of the present invention, cytotoxic T cells against GPC3 can be isolated from the peripheral blood of hepatoblastoma patients administered a cancer peptide vaccine (e.g., a cancer vaccine containing GPC3), and a TCR can be prepared prior to T cell replacement therapy. Furthermore, a TCR prepared in advance from another individual, distinct from the patient receiving T cell replacement therapy, can be introduced into the production of autologous regenerated T cells derived from non-T non-B cells or monocytes of multiple such patients. The resulting regenerated T cells maintain antigen specificity against GPC3 and exhibit antigen-specific cytotoxicity against cancer cells (tumors) expressing GPC3 in multiple such patients. Thus, the method of the present invention and the regenerated T cells produced by the method of the present invention can be used to obtain a pharmaceutical composition containing regenerated T cells that is effective against cancer.

[0097] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. [Examples]

[0098] [Production of regenerated T cells from iPS cells that have been reprogrammed from non-T, non-B cells or monocytes in peripheral blood] Figure 1 shows the manufacturing process of regenerated T cells according to the present invention. Peripheral blood mononuclear cells were collected from cancer patients (subjects) receiving treatment with tumor antigen X peptide (Figure 1-(1')), and TCR genes were isolated from T cells that specifically react to tumor antigen X (Figure 1-(2')). Based on the frequency of appearance of the TCR genes and their tumor antigen-specific binding ability, TCR genes with a higher expected therapeutic effect were selected (Figure 1-(3')). If tumor recurrence or tumor cell mutation occurs in the treated patient, it is possible to select TCR genes that are effective for treatment again. To obtain sufficient therapeutic effect, it is important to obtain a large variety of TCRs (TCR repertoire).

[0099] Separately, iPS cells were produced from non-T non-B cells or monocytes in the peripheral blood of the aforementioned patients (Figure 1-(1) and (2)). Next, iPS cell clones with good differentiation efficiency into T cells were selected from the obtained iPS cells (Figure 1-(3)). This selection significantly reduced the variability in T cell induction efficiency due to differences in iPS cell clones, and it was confirmed that the robustness and efficiency of regenerated T cell production were improved. The selected TCR gene group that reacts specifically to tumor antigens was expressed in the iPS cell clones with good T cell differentiation efficiency (Figure 1-(4)), and differentiation and proliferation into mature T cells were performed for each selected TCR gene (Figure 1-(5)). The mature T cells are tumor antigen-specific cytotoxic T cells and are regenerated T cells. These regenerated T cells are administered to cancer patients (Figure 1-(6)).

[0100] In the method of the present invention, the iPS cell manufacturing process (Figure 1-(1)~(3)) and the process of obtaining the TCR gene that reacts to tumor antigen X (Figure 1-(1')~(3')) can be carried out in parallel. Therefore, these processes can be completed in a shorter period (approximately 55 days) compared to when they are carried out sequentially.

[0101] The above will be explained in more detail. As described below, we isolated tumor antigen X-specific TCR genes and verified their antigen-specific reactivity (see upper section of Figure 2). Peripheral blood mononuclear cells were collected from the peripheral blood of cancer patients (subjects) in whom tumor antigen X-reactive T cells were amplified after peptide administration therapy against the tumor antigen (Figure 2-(1)). Tumor antigen X peptide was added to the obtained peripheral blood mononuclear cells to amplify and culture CD8-positive cytotoxic T cells that react to tumor antigen X. Then, single-cell sorting of activated cytotoxic T cells (CD8-positive and CD137-positive cells) that react to tumor antigen X was performed using a cell sorter (Figure 2-(2)). From the isolated single cells, TCR gene pairs (TCRα chain gene and TCRβ chain gene) were isolated by single-cell PCR, and sequence analysis was performed on the isolated TCR gene pairs to analyze the types of tumor antigen X-reactive T cells (TCR repertoire) and their frequency of appearance (Figure 2-(3)). The isolated TCR gene pair was expressed in a TCR gene-deficient T cell line incorporating a reporter gene to reconstruct the TCR gene complex (Figure 2-(4)). Subsequently, the reactivity (antigen-specific reactivity) between the reconstructed TCR gene complex and cells expressing HLA presenting the tumor antigen X peptide was verified (Figure 2-(5)). Specifically, a T cell line expressing the TCR gene pair and cells expressing HLA presenting the target tumor antigen X peptide were co-cultured. When the expressed TCR and the HLA presenting the tumor antigen X peptide bound in an antigen-specific manner, the TCR gene signaling pathway was activated, and the reporter gene incorporated downstream of the target gene promoter was expressed. After that, the reporter gene activity was measured, and antigen-specific reactivity was verified by evaluating the binding of the tumor antigen-specific TCR. Through the above steps, several TCR gene pairs that showed specific reactivity to tumor antigen X were selected with high frequency of appearance and used as transgenes for non-T non-B cells or monocyte-derived iPS cells (M-iPS cells) as described later.

[0102] The production of TCR gene-transformed iPS cells and regenerated T cells was carried out as follows (see the lower part of Figure 2). In parallel with the administration of tumor antigen X peptide to cancer patients (subjects) (Figure 2-(1)), iPS cells were generated from peripheral blood mononuclear cells of the said cancer patients (Figure 2-(2')). The M-iPS cells shown in the figure are iPS cells that do not originate from T cells and are cells in which the TCR gene has not undergone gene rearrangement. Next, M-iPS cell lines with good differentiation efficiency into T cells were selected from the generated group of M-iPS cell lines (Figure 2-(3')). Selected M-iPS cell lines were transpoised with tumor antigen X-specific TCR genes (both TCRα chain gene and TCRβ chain gene, see Figure 2-(3)) using transposons (see Figures 2-(6) and 3). Using the expression of the marker molecule CD19 as an indicator, M-iPS cell lines expressing the TCR gene (TCR-iPS cell lines) were selected using a cell sorter (MACSQuant Tyto® cell sorter, Miltenyi Biotec). Next, hematopoietic stem progenitor cells were induced using the embryoid body method, and differentiation was induced from immature T cells to mature T cells using a stepwise differentiation induction method that mimics T cell development in vivo.

[0103] The phenotype of regenerated T cells (tumor antigen-specific CD8-positive cytotoxic T cells) induced from iPS cells was analyzed as described above. Figure 4 shows the results of flow cytometry analysis of regenerated T cells using cell surface antigen markers. Regenerated T cells induced from iPS cells expressed CD45, which is expressed in mature cytotoxic T cells in vivo. + TCRαβ + CD3 + CD4 - CD8αβ + The expression of [the specified substance] was confirmed. Furthermore, it was found that the aforementioned CD8-positive cells specifically bind to the HLA fragment displaying the tumor antigen X peptide (Antigen-X binding).

[0104] Figure 5 shows the results of analyzing telomeres (an indicator of rejuvenation), a marker of cellular senescence, in regenerated T cells via iPS cells according to the present invention. Compared to the telomere length of patient peripheral blood mononuclear cells (A) and tumor antigen-specific T cells before regeneration (B), the telomere length of iPS cells produced from the aforementioned tumor antigen-specific T cells (C) was approximately three times longer, and the telomere length of tumor antigen-specific regenerated T cells according to the present invention (D) was approximately twice as long, confirming the recovery of telomere length in C and D. In other words, improvement of cellular senescence is suggested in the regenerated T cells of the present invention.

[0105] The expression of PD-1 and TIGIT molecules, which are cell exhaustion markers related to immune checkpoints, in regenerated T cells via iPS cells according to the present invention was stained with anti-TIGIT antibody and anti-PD-1 antibody and analyzed by flow cytometry. For comparison, tumor antigen-specific T cells before regeneration were also analyzed. The results are shown in Figure 6. It was confirmed that the expression of PD-1 and TIGIT molecules was significantly reduced in the regenerated T cells of the present invention compared to the tumor antigen-specific T cells before regeneration. Therefore, it is suggested that the regenerated T cells of the present invention have high cytotoxic activity.

[0106] We analyzed cytokine production capacity, an important indicator of cytotoxicity. Cytotoxic T cells (CD8α-chain / β-chain double-positive cells) from peripheral blood mononuclear cells collected from healthy individuals and regenerated T cells of the present invention were stimulated with PMA (Phorbol 12-Myristate 13-Acetate) and ionomycin, and the amounts of IL-2 and IFN-γ produced were compared. The results are shown in Figure 7. Compared to cytotoxic T cells obtained from healthy individuals, the regenerated T cells of the present invention showed a significant increase in cells producing IL-2 and IFNγ.

[0107] The antigen-specific cytotoxic activity of the regenerated T cells of the present invention was evaluated. HLA-A24-expressing cell lines (target cells) were cultured in the presence or absence of the antigen X peptide to produce antigen X peptide-expressing cells or antigen X peptide-non-expressing cells, respectively. The regenerated T cells of the present invention were co-cultured with these target cells, and the cytotoxic activity of the regenerated T cells against the target cells was measured. The cell number ratios of regenerated T cells to target cells during co-culture were 20:1, 10:1, and 5:1. The results are shown in Figure 8. The regenerated T cells of the present invention did not show cytotoxic activity against antigen X peptide-non-expressing cells (open circles), but showed cytotoxic activity against antigen X peptide-expressing cells (closed circles), and this activity was enhanced in proportion to the increase in the number of regenerated T cells relative to the target cells. [Examples]

[0108] [Preparation of cDNA encoding TCR] HLA-A24-restricted GPC3 peptide (EYILSLEEL, SEQ ID NO: 1) and HLA-A2-restricted GPC3 peptide (FVGEFFTDV, SEQ ID NO: 2) were lysed and mixed in a 1:1 (v / v) ratio with the immunoadjuvant incomplete Freund's adjuvant (Montanide ISA-51, Seppic, France). These mixtures were administered subcutaneously to patients with HLA-matched hepatocellular carcinoma or hepatoblastoma. The GPC3 peptides were administered at least five times, once or less than once a week. Peripheral blood mononuclear cells were isolated from peripheral blood collected from patients who had completed immunization with GPC3 peptides, using the mononuclear cell isolation solution Lymphoprep®. The resulting peripheral blood mononuclear cells were cultured for approximately 12 days in a culture medium containing HLA-matched HLA-restricted GPC3 peptides. Cells activated with the GPC3 peptide were fractionated as a CD3 / CD137 double-positive cell population using a cell sorter (MACSQuant Tyto® cell sorter, Miltenyi Biotec).

[0109] As described above, GPC3-responsive T cells activated and proliferated with the GPC3 peptide were stained with antibodies against the T cell marker CD3 and the T cell activation marker CD137, and fractionated using a cell sorter to obtain single cells. RNA was extracted from the obtained single cells, and the cDNA of the T cell receptor α and β chains was isolated and amplified using RT-PCR. CDNA pairs of the T cell receptor α and β chains were obtained from single cells of multiple GPC3-responsive T cells. The gene sequences of the obtained T cell receptor α and β chains were determined, and the TCR repertoire was analyzed. [Examples]

[0110] [Creation of iPS cell clones] Mononuclear cells were isolated from peripheral blood collected from hepatocellular carcinoma or hepatoblastoma patients before or after GPC3 peptide administration using the mononuclear cell isolation solution Lymphoprep®. From the obtained mononuclear cells, CD19 / CD20-positive B cells and CD3 / CD4 / CD8-positive T cells were removed using FACS or MACS beads to obtain non-T non-B cells or monocytes. The obtained non-T non-B cell population or monocyte cell population was infected with Sendai virus (CytoTune® 2.0) carrying the Yamanaka 4 factors (Oct3 / 4, Sox2, Klf4, and c-Myc) and Sendai virus encoding SV40Tag at MOI (multiplicity of infection) of 5-20. Note that SV40 may be omitted.

[0111] The obtained iPS cells consisted of numerous iPS cell clones. Therefore, colony picking was performed and cloned. All cloned iPS cells were cryopreserved. The cloned iPS cells were cultured in differentiation medium for approximately 10 days to induce hematopoietic stem cells, and CD34 / CD43 double-positive hematopoietic stem cells were isolated. The isolated hematopoietic stem cells were cultured for approximately 21 days on plates coated with FcDLL4, a fusion protein of the DLL4 protein and the Fc region of an immunoglobulin, to induce differentiation into T cells.

[0112] The frequency of immature cytotoxic T cells obtained after 21 days of culture was verified by the CD8α / β chain double-positive rate, and the clone with the highest frequency of CD8α / β chain double-positive cells was selected. It is preferable to complete the creation of iPS cell clones before immunization of cancer patients (subjects) with GPC3 peptide is complete, in order to rapidly administer T cell replacement therapy.

[0113] Several iPS cell clones with good differentiation efficiency into T cells were cultured in iPS cell maintenance medium for two weeks, then dispensed into cell storage containers and cryopreserved to establish a master cell bank. [Examples]

[0114] [Introduction of TCR gene into iPS cell clones] In Example 3, non-T non-B cell or monocyte-derived iPS cell clones with good T cell differentiation efficiency were introduced using a piggyBac vector containing genes encoding T cell receptor α and β chains with confirmed GPC3 antigen specificity, via electroporation. Next, iPS cells expressing the target T cell receptor α and β chains were isolated using a cell sorter, with CD19 expression as the marker molecule. The isolated iPS cells were cultured in differentiation medium for approximately 10 days to induce CD34 / CD43 double-positive hematopoietic stem cells, which were then isolated using a cell sorter. The isolated hematopoietic stem cells were cultured on FcDLL4-coated plates for approximately 21 days to induce differentiation into T cells.

[0115] Immature CD8 α / β chain double-positive T cells obtained after 21 days of culture were isolated and purified using a cell sorter. Next, the immature T cells were co-cultured in the presence of PHA (phytohemagglutinin) and peripheral blood mononuclear cells as feeder cells, in the presence of retronectin® and anti-CD3 antibody, or in the presence of anti-CD3 antibody and anti-CD28 antibody to induce mature cytotoxic T cells. These stimuli were performed at least once. The performance of the resulting T cells was confirmed by GPC3-specific target cell cytotoxicity, IFN-γ production, and antigen binding ability. [Examples]

[0116] [Introduction of TCR genes into mature T cells differentiated from iPS cell clones] In Example 3, mature T cells differentiated from non-T non-B cell or monocyte-derived iPS cell clones with good T cell differentiation efficiency were introduced into the piggyBac system using the same method as in Example 4, using the genes (cDNA) encoding the α and β chains of the GPC3 antigen-specific T cell receptor. These were obtained via hematopoietic stem cells and immature T cells.

[0117] Figure 9 shows the results of flow cytometry analysis of the phenotype of mature T cells derived from gene-transfected iPS cells. In Figure 9, "No EP" refers to the analysis results of the mature T cells used for gene transfer that express the WT1 antigen-specific T cell receptor α-chain and β-chain; "EGFP" refers to the analysis results of the mature T cells into which an expression vector incorporating the tracer gene (EGFP (enhanced green fluorescent protein) gene) used as an indicator of gene transfer operations was introduced; "Empty-CD19" refers to the analysis results of the mature T cells into which a piggyBac vector incorporating only the intracellularly deficient human CD19 gene and a transposon vector were introduced as tracer genes; and "TCR-CD19" refers to the analysis results of the mature T cells into which a piggyBac vector incorporating the GPC3 antigen-specific T cell receptor α-chain and β-chain gene, an intracellularly deficient human CD19 gene and a transposon vector were introduced.

[0118] In the mature T cells derived from iPS cells, indicated as "No EP," only the T cell marker CD3 was detected. This confirmed that the cells used for gene transfer were T cells.

[0119] In mature T cells derived from iPS cells, indicated by "EGFP," expression of both the CD3 gene and the EGFP gene was detected. This indicates that the gene transfer procedure was performed correctly.

[0120] In mature T cells derived from iPS cells, labeled "Empty-CD19," expression of the CD3 gene and the intracellular knockout human CD19 gene, a tracer gene incorporated into the piggyBac vector, was detected. This indicates that the gene transfer procedure was performed correctly.

[0121] In iPS cell-derived mature T cells, indicated by "TCR-CD19," in addition to the expression of the CD3 gene and the intracellular knockout human CD19 gene, a tracer gene incorporated into the piggyBac vector, binding to the GPC3 peptide / HLA complex (GPC3-Dex), recognized by the GPC3 antigen-specific T cell receptor α-chain and β-chain, was detected. In other words, by introducing the GPC3 antigen-specific T cell receptor α-chain and β-chain gene into iPS cell-derived mature T cells using the piggyBac system, it was shown that the GPC3 antigen-specific T cell receptor α-chain and β-chain expressed on the cells function as molecules that recognize the GPC3 peptide / HLA complex (GPC3-Dex). Therefore, it was shown that by using novel neoantigens and other tumor-related antigen-specific T cell receptor α-chain and β-chain genes as T cell receptor α-chain and β-chain genes introduced into iPS cell-derived mature T cells, it is possible to create iPS cell-derived mature T cells that recognize these antigens. [Examples]

[0122] [Production of regenerated T cells from iPS cells that have been reprogrammed from peripheral blood T cells] Figure 10 shows a method for selecting iPS cell clones with high differentiation efficiency into T cells in the process of producing regenerated T cells from iPS cells reprogrammed from peripheral blood T cells. Mononuclear cells were isolated from the peripheral blood of subjects infected with EBV (Epstein-Barr virus), and the isolated mononuclear cells were stimulated with EBV antigen in vitro to isolate a population of CD8-positive T cells that recognize the EBV antigen. The isolated CD8-positive T cell population was introduced with the Yamanaka 4 factors (Oct3 / 4, Sox2, Klf4, and c-Myc) and SV40 T antigen using a Sendai virus vector to obtain an iPS cell population. iPS cell clones were separated from the obtained iPS cell population, and the differentiation potential into T cells was examined for each clone to select iPS cell clones with high differentiation efficiency into T cells. T cells that recognize the EBV antigen are cells that do not easily produce allochemical reactions even when allogeneic transplantation is performed.

[0123] Figure 11 shows a method for producing regenerated T cells from iPS cells reprogrammed from peripheral blood T cells, by performing genome editing on iPS cell clones selected as cells with high differentiation efficiency into T cells. iPS cell clones derived from CD8-positive T cells that recognize the EBV antigen and have high differentiation efficiency into T cells were edited using CRISPR / Cas9 to delete the β2M and CIITA genes involved in the expression of MHC class I and MHC class II, the PVR gene involved in the activation of natural killer (NK) cells, and the Rag2 gene involved in the re-reconstruction of the T cell receptor. On the other hand, the β2M / binding peptide / HLA-E fusion gene (HLA-E), which is an inhibitory ligand for NK cells, was edited. *iPS cells were created that are not attacked by host T cells and NK cells by expressing a drug-induced caspase 9 and / or specific marker genes (such as EGFR (epidermal growth factor receptor), CD19, and CD20) in this genome editing, making it possible to remove the cells after administration to the body. After genome editing, recloning may be performed to confirm high differentiation efficiency into T cells, and the regenerated T cells derived from iPS cells may be stored as clones to construct a master cell bank. Regenerated T cells differentiated and proliferated from iPS cells are host T cells for producing regenerated T cells for cancer treatment, and a master cell bank may be constructed using the said host T cells. The said host T cells can be used as material to produce regenerated T cells into which the TCR gene or CAR (chimeric antigen receptor) gene has been introduced. Since these host T cells are T cells that recognize the EBV antigen, they are less likely to cause allochemical reactions even when introduced into the body. "Host T cells" refer to T cells that are not used to treat patients themselves, but are used as starting materials for manufacturing cancer preventive or therapeutic agents that use regenerated T cells as the active ingredient.

[0124] Figure 12 shows a method for producing regenerated T cells that recognize cancer antigens from the host T cells. In host T cells that recognize EBV antigens, the T cell receptor β chain that recognizes EBV antigens, which had undergone rearrangement by gene substitution using genome editing with CRISPR / Cas9, was replaced with a T2a-mediated conjugate of a T cell receptor β chain that recognizes cancer antigens and a T cell receptor α chain. On the other hand, the T cell receptor α chain that recognizes EBV antigens was removed by genome editing using CRISPR / Cas9. It was possible to produce regenerated T cells that recognize cancer antigens using the method described in Figure 12.

[0125] Figure 13 summarizes the methods shown in Figures 10-12. Regenerated T cells (iPS-T cells) produced from allogeneic universal iPS cells reprogrammed from peripheral blood T cells can be used as a starting material for producing T cells into which the TCR gene or CAR gene has been introduced. "Universal iPS cells" refer to iPS cells that have very low immunogenicity and can be used in patients with any type of MHC (Major Histocompatibility Complex) without causing rejection. In other words, they are iPS cells that can be administered to patients without considering MHC matching. Universal iPS cells can be produced by knocking out MHC class I or MHC class II molecules and expressing ligands that suppress NK cells.

[0126] For the introduction of TCR genes or CAR genes, viral vectors used in the production of T cells for conventional T cell replacement therapy or regenerative therapy may be used. By using iPS-T cells as a starting material, it becomes possible to produce desired T cells that will be the active ingredient of cancer prevention or treatment agents in a short period of time. Furthermore, the introduction of TCRs that recognize neoantigens is also easy, and by introducing multiple TCR genes that recognize different neoantigens, it is possible to produce iPS-T cells while maintaining polyclonality.

Claims

1. (1) A step of preparing cDNA encoding the α chain and β chain of the T cell receptor, respectively, from a population of T cells obtained from a subject that are reactive to tumor-associated antigens, for each single cell. (2) A step of reprogramming peripheral blood mononuclear cells from the subject, from which B cells and T cells have been removed, or from peripheral blood mononuclear cells or T cells into iPS cells, and selecting iPS cell clones from the obtained iPS cells that have a high differentiation efficiency into T cells. (3) A step of introducing the cDNA into hematopoietic stem cells differentiated from the iPS cell clone, immature T cells differentiated from the hematopoietic stem cells, or mature T cells differentiated from the immature T cells, and (4) A step of differentiating the hematopoietic stem cells or immature T cells obtained in step (3) into mature T cells and proliferating the mature T cells, A method for producing regenerated T cells via iPS cells, comprising the steps (2) being performed prior to or in parallel with the performance of step (1).

2. (1) A step of contacting T cells obtained from a subject with a tumor-associated antigen, and preparing cDNA encoding the α chain and β chain of the T cell receptor, respectively, from a population of T cells that are reactive to the tumor-associated antigen, for each single cell. (2) A step of reprogramming peripheral blood mononuclear cells from the subject, from which B cells and T cells have been removed, or from peripheral blood mononuclear cells or T cells into iPS cells, and selecting iPS cell clones from the obtained iPS cells that have a high differentiation efficiency into T cells. (3) A step of introducing the cDNA into hematopoietic stem cells differentiated from the iPS cell clone, immature T cells differentiated from the hematopoietic stem cells, or mature T cells differentiated from the immature T cells, and (4) A step of differentiating the hematopoietic stem cells or immature T cells obtained in step (3) into mature T cells and proliferating the mature T cells, A method for producing regenerated T cells via iPS cells, comprising the steps (2) being performed prior to or in parallel with the performance of step (1).

3. (1) A step of contacting T cells obtained from a subject administered with a tumor-associated antigen with the tumor-associated antigen, and preparing cDNA encoding the α chain and β chain of the T cell receptor, respectively, from a population of T cells that are reactive to the tumor-associated antigen, for each single cell. (2) A step of reprogramming peripheral blood mononuclear cells from the subject, from which B cells and T cells have been removed, or from peripheral blood mononuclear cells or T cells into iPS cells, and selecting iPS cell clones from the obtained iPS cells that have a high differentiation efficiency into T cells. (3) A step of introducing the cDNA into hematopoietic stem cells differentiated from the iPS cell clone, immature T cells differentiated from the hematopoietic stem cells, or mature T cells differentiated from the immature T cells, and (4) A step of differentiating the hematopoietic stem cells or immature T cells obtained in step (3) into mature T cells and proliferating the mature T cells, A method for producing regenerated T cells via iPS cells, comprising the steps (2) being performed prior to or in parallel with the performance of step (1).

4. The method according to any one of claims 1 to 3, wherein the subject in steps (1) and (2) is the same individual.

5. The method according to any one of claims 1 to 3, wherein the subjects in step (1) and (2) are separate individuals, and the subject in step (2) is a target for cancer prevention or treatment.

6. The method according to claim 5, further comprising the step of selecting T cells from the T cells into which the cDNA obtained in step (4) has been introduced that do not show an allochemical reaction to the cells derived from the subject in step (2).

7. The method according to claim 5, further comprising the step of selecting from the cDNA prepared in step (1) cDNA encoding a T cell receptor that does not induce an allochemical response to cells derived from the subject in step (2).

8. The method according to claim 6 or 7, wherein the cells derived from the subject are peripheral blood mononuclear cells.

9. The method according to any one of claims 1 to 8, wherein the tumor-associated antigen is selected from the group consisting of GPC3, WT1, XAGE1, LMP2, NY-ESO-1, EB virus antigen, neoantigens, and peptide fragments thereof.

10. The method according to any one of claims 1 to 8, wherein the tumor-associated antigen is EYILSLEEL (SEQ ID NO: 1), which is an HLA-A24-restricted GPC3 peptide; FVGEFFTDV (SEQ ID NO: 2), which is an HLA-A2-restricted GPC3 peptide; or a mixture thereof.

11. The method according to any one of claims 1 to 10, wherein in step (3), the introduction of the cDNA into the hematopoietic stem cells differentiated from the iPS cell clone, the immature T cells differentiated from the hematopoietic stem cells, or the mature T cells differentiated from the immature T cells is performed using a viral vector, a non-viral vector, or genome editing technology.

12. The method according to claim 11, wherein the non-viral vector is a transposon vector.

13. The method according to claim 12, wherein the transposon vector is a piggyBac® vector.

14. The method according to any one of claims 1 to 13, wherein in step (4), the step of differentiating the hematopoietic stem cells or immature T cells into mature T cells and proliferating the mature T cells is carried out in the presence of feeder cells and PHA (phytohemagglutinin), in the presence of retronectin (registered trademark) and anti-CD3 antibody, or in the presence of anti-CD3 antibody and anti-CD28 antibody.

15. The method according to claim 14, wherein the feeder cells are autologous or allogeneic peripheral blood mononuclear cells.

16. The method according to any one of claims 1 to 15, wherein the subject in step (2) is a patient with hepatocellular carcinoma or hepatoblastoma.

17. The method according to any one of claims 1 to 16, wherein the T cell population in step (1) is CD3 / CD137 double positive.

18. The method according to any one of claims 1 to 16, wherein the T cell population in step (1) binds to an MHC tetramer or MHC dextramer (registered trademark) that forms a complex with the tumor-associated antigen.

19. The method according to any one of claims 1 to 18, wherein the hematopoietic stem cells are CD34 / CD43 double-positive.

20. The method according to any one of claims 1 to 19, wherein the immature T cells are CD8α / β double-positive.

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