WO2022145490A1 - ｉＰＳ細胞を介する再生Ｔ細胞の製造方法 - Google Patents

ｉＰＳ細胞を介する再生Ｔ細胞の製造方法 Download PDF

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WO2022145490A1
WO2022145490A1 PCT/JP2021/049028 JP2021049028W WO2022145490A1 WO 2022145490 A1 WO2022145490 A1 WO 2022145490A1 JP 2021049028 W JP2021049028 W JP 2021049028W WO 2022145490 A1 WO2022145490 A1 WO 2022145490A1
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cells
cell
ips
cancer
hematopoietic stem
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French (fr)
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新 金子
良輔 五ノ坪
光次郎 大澤
泰道 等
和男 山下
ニコラ クロード ポール サックス
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Kotai Biotechnologies Inc
Thyas Co Ltd
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Kotai Biotechnologies Inc
Thyas Co Ltd
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Priority to JP2022573131A priority Critical patent/JPWO2022145490A1/ja
Priority to EP21915348.3A priority patent/EP4273234A4/en
Priority to CN202180088550.1A priority patent/CN116744947A/zh
Priority to US18/259,687 priority patent/US20240052309A1/en
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Definitions

  • the present invention introduces a T cell receptor obtained from a CD106-positive T cell population into non-T non-B cells or iPS cells derived from monocytes or T cells to produce tumor antigen-specific regenerated T cells.
  • T cells play a central role in the immune response to foreign pathogens such as bacteria or viruses or abnormal cells such as cancer cells. Therefore, it is considered that the functional deterioration of T cells contributes to pathogen infection and the development of cancer.
  • T cell replacement therapy or regenerative therapy for a patient having a disease caused by a decrease in T cell function can be an extremely effective means for improving or treating the pathological condition of the disease in the patient.
  • T cell replacement therapy for infectious diseases or cancer specifically recognizes antigens possessed by foreign pathogens such as bacteria or viruses or abnormal cells such as cancer cells. It is known that a high therapeutic effect can be obtained by using T cells. On the other hand, it is difficult to secure a sufficient amount of T cells, and exhaustion of T cells such as a decrease in proliferative ability in T cells and a decrease in immune response to antigens such as target cells becomes obstacles in T cell replacement therapy. ing. Also, how to obtain tumor-specific T cells is a major issue.
  • iPS cells artificial pluripotent stem cells
  • Cell replacement therapy has been proposed. Since the T cell receptor (TCR) for recognizing the target antigen is formed by gene rearrangement of the genome, the TCR gene possessed by the T cell before reprogramming is conserved in the iPS cell in which the T cell is reprogrammed. Has been done. Therefore, by using T cells specific to the target antigen as a raw material for producing iPS cells, it is possible to produce regenerated T cells exhibiting the same antigen specificity as the original T cells. (Patent Document 1 and Non-Patent Document 1).
  • Non-Patent Document 2 Strict antigen specificity is required for safe and efficient T cell replacement therapy.
  • Patent Document 2 It has been reported that in regenerated T cells obtained from T cells via iPS cells as described above, the frequency of appearance of T cells having the same gene rearrangement pattern as the original T cells is not necessarily high.
  • Patent Document 2 It has also been reported that CD8 ⁇ regenerated T cells obtained from human T cells via iPS cells lose antigen specificity due to additional rearrangement of the TCR ⁇ chain gene at the CD4 / CD8 double positive stage.
  • Nishimura T et al. Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation.
  • Minagawa A et al. Enhancing T cell receptor stability in rejuvenated iPSC-derived T cells improves their use in cancer immunotherapy.
  • iPS-T cells regenerated T cells
  • iPS-T cells regenerated T cells
  • tumor cells or tumor tissues can be used. It is important to ensure the safety and efficacy of iPS-T cell replacement therapy in the treatment of cancer.
  • iPS-T cell replacement therapy when the onset or recurrence of cancer is detected, it is important to start iPS-T cell replacement therapy promptly in order to improve the treatment results.
  • An object of the present invention is to provide a method for rapidly producing TCR-introduced iPS-T cells isolated from tumor tissue infiltrating CD106-positive T cells and iPS-T cells produced by the method. Furthermore, it is an object of the present invention to provide iPS-T cells produced by the method, a pharmaceutical composition containing the iPS-T cells, and a method for preventing or treating cancer using the pharmaceutical composition. ..
  • T cell clones (5-10 clones) recognize antigens using different TCRs even for one antigen epitope.
  • the possibility that the clone differentiated to iPS-T cells is a T cell clone with optimal TCR to damage the target is also due to individual differences in the subjects who collect the cells for producing iPS-T cells or iPS. -Affected by T cell production batches.
  • T cells that specifically recognize a tumor it is necessary to administer the tumor antigen to a patient or co-culture the T cell and the tumor antigen in vitro, but the tumor antigen is used. If uncertain, it is extremely difficult to adopt such a method.
  • the CD106-positive T cell population isolated from tumor tissue is a T cell population that recognizes tumor-related antigens and has specific aggression against cancer cells.
  • iPS-T cells can be obtained by obtaining various TCR pools with antigen specificity, and by introducing TCR into non-T non-B cells or iPS cells established from monospheres or T cells.
  • the iPS-T cells can be rapidly produced, and the TCR pool and the non-T non-B cells or monospheres or T cells of the same origin or in separate bodies. We have found that it is possible to have it, and have completed the present invention.
  • T cell receptors obtained from a subject which are reactive to tumor-related antigens and which have a T cell receptor ⁇ chain and ⁇ chain for each single cell from a CD106-positive T cell population, respectively.
  • the process of preparing the cDNA to be encoded (2) Peripheral blood mononuclear cells of the subject, from which B cells and T cells have been removed, are initialized into iPS cells, and the obtained iPS cells are differentiated into T cells.
  • Steps to select high iPS cell clones (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). ) 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 obtained in step (3). A method for producing regenerated T cells via iPS cells.
  • the present invention comprises a step of selecting T cells having no allo-reaction with respect to cells derived from a subject who is a target for cancer prevention or treatment from the T cells into which the cDNA obtained in step (4) has been introduced.
  • it comprises a step of selecting a cDNA encoding a T cell receptor that does not induce an allo reaction to cells derived from a subject to be prevented or treated for cancer from the cDNA prepared in step (1).
  • step (3) 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.
  • a viral vector or a non-viral vector is used, or a genome editing technique is used.
  • step (4) the steps 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 proliferating the mature T cells are the feeder cells and PHA (phytohemagglutinin).
  • Retronectin® and anti-CD3 antibody In the presence of Retronectin® and anti-CD3 antibody, or in the presence of anti-CD3 antibody and anti-CD28 antibody, according to any one of [1] to [13].
  • the feeder cells are autologous or allogeneic peripheral blood mononuclear cells.
  • the subject in step (1) or (2) is hepatocellular carcinoma, hepatoblastoma, gastric cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, breast cancer, ovarian cancer, malignant melanoma, etc.
  • the highly immunogenic cancer is hepatocellular carcinoma, hepatoblastoma, colon cancer, lung cancer or malignant melanoma.
  • iPS-T cells regenerated T cells
  • peripheral blood mononuclear cells non-T non-B cells or monocytes
  • T cells from which T cells and B cells have been removed are initially used.
  • iPS cell clones having good differentiation efficiency into T cells are selected in advance. Therefore, it is possible to secure a sufficient amount of iPS-T cells required for treatment according to the timing required for treatment.
  • iPS cell clones derived 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 the immature T cells.
  • TCR TCR antigen
  • iPS cell clones with good differentiation efficiency into T cells are selected and used, so that the yield and degree of differentiation vary between iPS-T cell production batches, and the cells are used. It is possible to minimize the influence of individual differences of the subjects to be collected on the quality and the like of the obtained iPS-T cells. In addition, it becomes possible to efficiently produce T cells having a TCR that recognizes an antigen and efficiently kills a target.
  • the cDNA encoding the TCR is prepared for each single cell from the T cell population having genetic diversity, it is possible to prepare a cDNA group having various antigen specificities. .. Since the CD106-positive T cell population isolated from the tumor tissue is a T cell population that recognizes the tumor antigen, the tumor antigen-induced immunity or in vitro T cells are used to isolate the tumor-specific TCR in advance. There is no need for co-culture with tumor antigens. Therefore, according to the method of the present invention, the production period of iPS-T cells is shortened, and at the same time, tumor-specific iPS-T cells are produced even when the tumor antigen in a cancer patient is unknown. It is possible.
  • non-T non-B cell or monocyte-derived or T cell-derived iPS cell clones is performed prior to the preparation of the cDNA encoding the antigen-specific TCR or in parallel with the preparation of the cDNA. Therefore, iPS-T cells can be produced in a short period of time as compared with the method of sequentially performing these. Therefore, in cancer treatment to which iPS-T cell replacement therapy is applied, it is specific to the expressed antigen for tumor antigen change or development of treatment resistance or cancer recurrence in the cancer patient to be treated. It is possible to quickly prepare various iPS-T cells.
  • the subject from which the T cells used for preparing the cDNA encoding the TCR are collected is the same individual as the subject who is a cancer patient to be treated by iPS-T cell replacement therapy. It may be separate from each other. Therefore, it is possible to prepare a TCR pool corresponding to various antigens, and it is possible to select a TCR having optimal antigen specificity for each cancer patient.
  • the upper partial view shows a step of isolating the TCR gene from CD106-positive T cells and a step of verifying antigen-specific reactivity
  • the lower partial view shows a step of producing a TCR gene-introduced iPS cell and a step of producing the TCR gene-introduced iPS cell.
  • the step of producing the regenerated T cell from the iPS cell is shown. It is a figure which shows the step of introducing a TCR gene using a piggyBac® transposon vector. It is a figure which shows the phenotype of the regenerated T cell (tumor-related antigen-specific CD8 positive cytotoxic T cell) derived from iPS cell. It is a figure which shows the result of having analyzed the telomere as a cell senescence marker in a regenerated T cell. It is a figure which shows the result of having analyzed the expression of PD-1 and TIGHT molecules as a cell exhaustion marker in regenerated T cells.
  • IL-2 and IFN- ⁇ levels produced by stimulation with PMA and ionomycin in cytotoxic T cells (CD8 ⁇ chain / ⁇ chain double positive cells) and regenerated T cells in peripheral blood mononuclear cells of healthy subjects It is a figure. It is a figure which shows the expression of various markers in a tumor infiltrating T cell. In each panel, cells positive for the expression of the markers described within the panel are shown colored. The cells are clustered according to the expression pattern, and the circled part corresponds to the tumor-damaging T cell. It is a figure which shows the IFN- ⁇ production at the time of self-tumor stimulation in the cell subpopulation that tumor infiltrated T cells were sorted by marker expression.
  • IFN- ⁇ is produced by autotumor stimulation
  • marker-positive cell subpopulation other than CD106 IFN- ⁇ is produced by autotumor stimulation, but the marker-negative cell subpopulation. Since IFN- ⁇ is also produced in the population, it can be understood that the specificity is inferior to that of CD106.
  • the CD19 gene is a gene integrated into the same piggyBac® transposon vector in tandem with the TCR gene, and is used as a marker for gene insertion into the host chromosome and gene expression.
  • the "T cell” obtained from a subject is a cell expressing an antigen receptor called a T cell receptor (TCR) on the cell surface.
  • TCR includes a heterodimer consisting of an ⁇ chain and a ⁇ chain, and a heterodimer consisting of a ⁇ chain and a ⁇ chain.
  • T cells having a TCR consisting of an ⁇ chain and a ⁇ chain are called ⁇ type T cells, and T cells having a TCR consisting of a ⁇ chain and a ⁇ chain are called ⁇ type T cells.
  • the T cells of the present invention are preferably CD3 / ⁇ type T cells, but may be CD3 / ⁇ type T cells.
  • the TCR of T cells in the present invention is a gene-introduced one.
  • the "T cell population” refers to the above-mentioned T cell population in which the gene sequence of the T cell receptor (TCR) that recognizes an antigen is diverse as a whole. Therefore, T cells collected from a living body are a T cell population having specificity for various antigens. In the T cells existing in the living body, each T cell has a different TCR gene sequence due to the random recombination of the TCR gene when the T precursor cells develop and differentiate in the thymus, and for all antigens. It is possible to provoke an immune response.
  • CD106 is a highly specific marker for identifying T cells having aggression against cancer cells, and the TCR possessed by each CD106-positive T cell is tumor antigen-specific. Therefore, by treating CD106-positive T cells as a population, it can be understood that the T cell population is immune to various tumor antigens. Therefore, the CD106-positive T cell population collected from a living body is a T cell population having genetic diversity in that sense.
  • the "tumor-related antigen” is an antigen that is specifically or non-specifically expressed in a tumor, and is an antigen derived from a protein that is overexpressed in tumor cells and a variant thereof, an antigen derived from a tumor virus, and the like. Certain differentiation antigens and novel tumor-related antigens (neoantigens) due to gene mutations and splice abnormalities. T cells that respond to "tumor-related antigens" are recognized as CD106-positive T cells.
  • a tumor-related antigen may be referred to as a tumor antigen. When it is a protein antigen, it may be a peptide fragmented from this (peptide fragment).
  • Antigens expressed specifically or non-specifically to tumors include WT1, GPC3, XAGE1, MUC1, MUC5AC, MUC6, EGFRvIII, HER-2 / neu, MAGE A3, MAGE A1, telomerase, PRAME, SSX2 / 4, PSCA. , CTLA-4, gp100, GD2, GD3, fucosyl GM1, GM3, sLe (a), glycolipid F77, mesotelin, PD-L1, trp1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, p53.
  • Viral antigens include inactivated viruses such as inactivated HBV and HPV, and proteins derived from various viruses EBV LMP1, EBV LMP2, EBNA (EBV nuclear antigen), HPV E1, HPV E2, HPV E6, HPV E7, HBV. Examples include, but are not limited to, HBs, HTLV-1Tax, HBZ (HTLV-1bZIPFactor), and the like.
  • T cells are tumor-related presented to major histocompatibility complex (MHC) class I or class II on antigen-presenting cells. It means that T cells have a reaction caused by selectively binding / joining to an epitope peptide derived from an antigen via TCR, and in binding / joining of T cells to something other than the epitope peptide, the T cells It means that no reaction occurs.
  • MHC major histocompatibility complex
  • the T cell response resulting from binding / conjugation to an epitope peptide derived from a tumor-related antigen presented in MHC class I or class II via the TCR is cytotoxic, IFN- ⁇ and granzyme production, T. Expression of cell activation markers and activation of transcription factors such as NF-AT can be mentioned.
  • the T cells are preferably ⁇ T cells.
  • the source of T cells is preferably, but not limited to, peripheral blood because of its low invasiveness.
  • Other preferred sources are cancer or tumor tissue, lymph nodes or other tissues or organs, or blood, umbilical cord blood, lymph, tissue fluid (interstitial fluid, interstitial fluid and interstitial fluid), body cavity fluid (abdominal fluid). , Chest fluid, heart sac fluid, cerebrospinal fluid, joint fluid and atriointerstitial fluid) and all sources in the body.
  • preferred T cells are tumor tissue-derived T cells. Tumor tissue-derived T cells are usually tumor-infiltrating T cells.
  • the cancer of the cancer patient is hepatocellular carcinoma, hepatoblastoma, gastric cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, breast cancer, ovarian cancer, Skin cancer such as malignant melanoma, bladder cancer, head and neck cancer, uterine cancer, cervical cancer, glioblastoma, prostate cancer, neuroblast tumor, chronic lymphocytic leukemia, papillary thyroid cancer, colon Selected from cancer, brain tumor, sarcoma or B-cell non-Hodgkin lymphoma.
  • the cancer is preferably hepatocellular carcinoma or hepatoblastoma.
  • iPS cells are preferably produced by reprogramming non-T non-B cells or monocytes or T cells, but are not limited to non-T non-B cells or monocytes or T cells.
  • Non-T non-B cell means a mononuclear cell that is not classified as a T cell and is not classified as a B cell.
  • non-T non-B cells or monocytes can be prepared by collecting as peripheral blood mononuclear cells of a subject and then removing T cells and B cells contained in the mononuclear cells. Peripheral blood mononuclear cells can be isolated from whole human blood with a mononuclear cell isolation solution.
  • Examples of the mononuclear cell separation solution include Lymphoprep (registered trademark).
  • B cells such as CD19, CD20, CD22 or B cell receptors
  • T cells such as CD3, CD4 or CD8.
  • the antibody may be utilized to use magnetic beads such as, for example, flow cytometry or MACS® beads.
  • T cells can be isolated from whole human blood with a mononuclear cell isolation solution.
  • Examples of the mononuclear cell separation solution include Lymphoprep (registered trademark). Purification of T cells can be performed by utilizing the surface antigens of T cells, CD3, CD4 or CD8, or T cell receptors. Magnetic beads such as, for example, flow cytometry or MACS® beads may be used.
  • iPS cells can preferably be induced by introducing a cell reprogramming factor into non-T non-B cells or monocytes or T cells.
  • cell reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, klf4, klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERAs, and ECAT15-2.
  • cell reprogramming factors may be used alone or in combination.
  • these cell reprogramming factors from the viewpoint of efficiently establishing iPS cells, Oct3 / 4, Sox2, Klf4 and c-Myc (so-called Yamanaka 4 factors) are used as the non-T non-B cells or monocytes. Alternatively, it is preferably introduced into T cells.
  • the method for introducing a cell reprogramming factor into the non-T non-B cell or monocyte or T cell is not particularly limited, and a method known in the art can be adopted.
  • the gene encoding the cell reprogramming factor for example, cDNA
  • the gene encoding the cell reprogramming factor is introduced into the non-T non-B cell or monocyte. It is inserted into an expression vector containing a promoter that functions in spheres or T cells, and the expression vector is subjected to non-T by infection, lipofection method, liposome method, calcium phosphate co-precipitation method, DEAE dextran method, microinjection method or electroporation method.
  • the cell reprogramming factor can be introduced into non-B cells or monocytes or T cells.
  • the cell reprogramming factor is in the form of a protein and the protein is introduced into the non-T non-B cells or monospheres or T cells, a method using a protein transfer reagent or a method using a protein transfer domain fusion protein. , Electroporation method and microinjection method.
  • mRNA messenger RNA
  • a method using an mRNA introduction reagent and a method of adding the mRNA to the culture medium are available. Can be mentioned.
  • the expression vector used for gene transfer by infection examples include viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpesvirus, and Sendai virus, and animal cell expression plasmids, but insertion mutations are unlikely to occur.
  • viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpesvirus, and Sendai virus
  • animal cell expression plasmids but insertion mutations are unlikely to occur.
  • the gene encoding the cell reprogramming factor is transferred to the non-T non-B cell or monosphere or T cell using Sendai virus. It is preferable to introduce it.
  • Examples of the promoter used in the expression vector used when introducing the gene encoding the cell reprogramming factor into non-T non-B cells or monospheres or T cells include SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, and the like. Examples include the RSV promoter, HSV-TK promoter, ubiquitin promoter and the like. These promoters may be capable of controlling the expression of the gene inserted downstream of the promoter depending on the presence or absence of a drug such as tetracycline.
  • the expression vector can include an enhancer, a poly A addition signal, a selectable marker gene (for example, a neomycin resistance gene), an SV40 origin of replication, and the like.
  • the culture medium used for culturing iPS cells obtained by reprogramming non-T non-B cells or monocytes or T cells is not particularly limited, but the culture medium used for culturing animal cells is used as the basal culture medium. It can be prepared by adding cytokines for maintaining the undifferentiated ability of iPS cells.
  • the basal culture medium include Isove's Modified Dulbecco's Medium (IMDM) culture medium, Medium 199 culture medium, Eagle's Minimum Essential Medium (EMEM) culture medium, ⁇ MEM culture medium, Dulbecco's modified Eagle's Medium (DMEM) culture medium, and Ham.
  • IMDM Isove's Modified Dulbecco's Medium
  • EMEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's modified Eagle's Medium
  • cytokines include bFGF, and the concentration thereof in the culture medium is, for example, 1 to 100 ⁇ g / mL (preferably 50 ⁇ g / mL).
  • the method for culturing iPS cells may be adhesive culture or suspension culture, but adhesive culture is preferable.
  • the method for isolating iPS cells include a method of physically isolating with a cell scraper or the like, a dissociation solution having protease activity, a dissociation solution having collagenase activity, or a dissociation solution having protease activity and collagenase activity (for example,).
  • An isolation method using Accutase (registered trademark), Accumax (registered trademark), etc.) can be mentioned.
  • the iPS cells are preferably 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.
  • the number of passages may be any number as long as the required amount of iPS cells is obtained, preferably 1 to 5 times or 5 to 10 times.
  • the produced iPS cells consist of a large number of iPS cell clones.
  • the colony pickup method is not particularly limited, but a method using a pipette man under a microscope, a limiting dilution method, a method using a fully automated colony picker, and the like are used.
  • the obtained iPS cell clone may form a master cell bank after expansion culture.
  • the "master cell bank" composed of the iPS cell clones is a single pool of iPS cell clones that is dispensed and accumulated in an independent cell storage container.
  • cryopreserve iPS cell clones In the master cell bank, it is preferable to cryopreserve iPS cell clones. Methods of cryopreserving cells are well known to those of skill in the art. For example, cultured iPS cell clones are collected, washed with a buffer or culture medium, the number of cells is counted, concentrated by centrifugation or the like, and suspended in a frozen medium (for example, a culture medium containing 10% DMSO). After turbidity, it can be cryopreserved at low temperature.
  • the master cell bank of the present invention can be stored in any facility or storage that can be cryopreserved. In the case of cryopreservation, the storage temperature is not particularly limited as long as it is a temperature suitable for storing cells. For example, ⁇ 20 ° C., ⁇ 80 ° C. and ⁇ 120 to -196 ° C. are mentioned, but ⁇ 150 ° C. or lower is preferable.
  • “Differentiation efficiency” refers to the hematopoietic stem cells, immature T cells, and mature T cells in all surviving cells at each stage of differentiation from iPS cells to hematopoietic stem cells, from hematopoietic stem cells to immature T cells, and from immature T cells to mature T cells, respectively. Refers to the abundance ratio. Identification of differentiated cells at each stage of differentiation can be performed by FACS analysis of surface markers. The efficiency of differentiation into hematopoietic stem cells is the proportion of CD34 / CD43 double positive cells in all viable cells, and the efficiency of differentiation into immature T cells is the proportion of CD4 / CD8 double positive cells or CD5 positive cells in all viable cells. Also, the efficiency of differentiation into mature T cells is shown as the proportion of cells in all surviving cells in which all of the CD8 ⁇ , CD8 ⁇ , TCR ⁇ and TCR ⁇ chains are positive.
  • “High differentiation efficiency” or “good / good differentiation efficiency” means that the proportion of CD34 / CD43 double positive cells, which are hematopoietic stem cells, is 5 to 15% or more in the differentiation of iPS cells into hematopoietic stem cells. It means that the ratio of CD4 / CD8 double positive cells or CD5 positive cells, which are immature T cells, is 10% or more or 50% or more, respectively, in the differentiation of hematopoietic stem cells into immature T cells; In the differentiation from immature T cells to mature T cells, it means that the proportion of cells in which all of the CD8 ⁇ chain, CD8 ⁇ chain, TCR ⁇ chain and TCR ⁇ chain are positive is 50% or more.
  • the regenerated T cells of the present invention preferably first differentiate the iPS cell clone into hematopoietic stem cells, then differentiate the hematopoietic stem cells into immature T cells, and finally the immature T cells into CD8 single positive T cells. It is produced by differentiating into mature T cells.
  • the "hematopoietic stem cell” is a cell capable of differentiating into blood cell lineage cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes and megakaryocytes. It should be noted that hematopoietic stem cells and hematopoietic progenitor cells (HPC) are not distinguished and refer to the same cells unless otherwise specified. Hematopoietic stem cells / progenitor cells are recognized, for example, by the fact that the surface antigens CD34 and CD43 are double positive.
  • the "immature T cell” is a T cell that expresses TCR ⁇ chain and ⁇ chain from the stage of T cell in which both TCR ⁇ chain and ⁇ chain are not expressed, and CD8 single via CD4 / CD8 double positive cell. Refers to T cells at each stage up to positive cells.
  • the immature T cells are preferably CD8 ⁇ chain / ⁇ chain double positive.
  • the "mature T cell” refers to a T cell that expresses a TCR ⁇ chain and a ⁇ chain and reaches a CD8 single positive cell via a CD4 / CD8 double positive cell.
  • CD8 ⁇ chain / ⁇ chain double positive is preferable.
  • Hematopoietic stem cells are preferably produced by culturing iPS cells in a culture medium supplemented with vitamin Cs.
  • vitamin Cs refers to L-ascorbic acid and its derivatives
  • L-ascorbic acid derivatives means those that are converted to vitamin C by an enzymatic reaction in the living body.
  • L-ascorbic acid includes, for example, vitamin C phosphate, ascorbic acid glucoside, ascorbic ethyl, vitamin C ester, ascorbic tetrahexyldecanoate, ascorbic stearate and ascorbic acid-2 phosphate-6 palmitic acid.
  • the derivative of L-ascorbic acid is preferably vitamin C phosphate, and examples thereof include phosphate-L ascorbate such as sodium phosphate-L ascorbate or magnesium phosphate-L-ascorbate.
  • Vitamin Cs are contained, for example, in a culture solution at a concentration of 5 to 500 ⁇ g / mL.
  • the culture medium used for producing hematopoietic stem cells is not particularly limited, but can be prepared by using the culture medium used for culturing animal cells as the basal culture medium and adding vitamin C or the like to it.
  • the basal culture medium include Isove's Modified Dulbecco's Medium (IMDM) culture medium, Medium 199 culture medium, Eagle's Minimum Essential Medium (EMEM) culture medium, ⁇ MEM culture medium, Dulbecco's modified Eagle's Medium (DMEM) culture medium, and H12 culture medium.
  • IMDM Isove's Modified Dulbecco's Medium
  • EMEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's modified Eagle's Medium
  • H12 culture medium H12 culture medium.
  • Examples include broth, RPMI 1640 broth, Fischer's broth, Neurobasal Medium (Life Technologies), StemPro34 (Life Technologies) and mixed broths thereof.
  • the culture broth may contain serum or may be serum-free.
  • the basal culture medium may be, for example, albumin, insulin, transferase, 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, cytokines and the like.
  • the culture medium used for the production of hematopoietic stem cells includes BMP4 (Bonemorphogenetic protein 4), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), SCF (stem cell factor), TPO (thrombopoietin), and FLT3L (FLT3L).
  • Cytokines selected from the group consisting of Flt3 ligand) may be further added. These concentrations are, for example, 1-100 ng / mL for BMP4, 1-100 ng / mL for VEGF, 1-100 ng / mL for bFGF, and 10-100 ng / mL for SCF.
  • TPO is 1 to 100 ng / mL
  • FLT3L is 1 to 100 ng / mL.
  • TGF ⁇ inhibitors are small molecule inhibitors that interfere with TGF ⁇ family signal transduction, such as SB431542 and SB202190 (R.K.Lindemann et al., Mol. Cancer 2: 20 (2003)), SB505124 (GlaxoSmithKline). , NPC30345, SD093, SD908 and SD208 (Scios), and LY2109761, LY364947 and LY580276 (Lilly Research Laboratories), and the concentration added to the culture solution is preferably 0.5 to 100 ⁇ M.
  • iPS cells are 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): It may be co-cultured with feeder cells such as 367-77).
  • the method for culturing iPS cells at the time of producing hematopoietic stem cells may be adhesive culture or suspension culture, but suspension culture is preferable.
  • iPS cells can be subjected to suspension culture after releasing colonies cultured to 80% confluence with respect to the dish used, dissociating them into single cells.
  • a method for isolating iPS cells for example, a method of physically isolating with a cell scraper or the like, a dissociation solution having protease activity and collagenase activity (for example, Accutase® and Accumax®), or An isolation method using a dissociation solution having collagenase activity can be mentioned.
  • Floating culture is to culture cells in a non-adherent state to the culture vessel.
  • the suspension culture is not particularly limited, but is a culture vessel that has not been artificially treated (for example, a coating treatment with an extracellular matrix or the like) for the purpose of improving adhesion to cells, or artificially suppresses adhesion. It can be carried out using a culture vessel which has been treated (for example, coated with polyhydroxyethyl methacrylate (po1y-HEMA) or a nonionic surfactant (Pluronic F-127, etc.)).
  • po1y-HEMA polyhydroxyethyl methacrylate
  • Pluronic F-127 nonionic surfactant
  • Hematopoietic stem cells can also be prepared from cyst-like structures (also referred to as iPS-sac) obtained by culturing iPS cells.
  • cyst-like structure also referred to as iPS-sac
  • the "cyst-like structure” is a three-dimensional sac-like (with space inside) structure derived from iPS cells, which is formed by an endothelial cell population or the like and contains hematopoietic stem cells inside. be.
  • the temperature conditions for culturing to produce hematopoietic stem cells from iPS cells are not particularly limited, but are, for example, about 37 ° C to about 42 ° C, preferably about 37 ° C to about 39 ° C. Further, the culture period can be appropriately determined by those skilled in the art while monitoring the number of hematopoietic stem cells and the like.
  • the number of culture days is not particularly limited as long as hematopoietic stem cells can be obtained, but for example, at least 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, Or 14 days or more, preferably 14 days.
  • the long culture period is not a problem in the production of hematopoietic stem cells.
  • the cells may be cultured under low oxygen conditions, and in one embodiment of the present invention, the low oxygen conditions include, for example, 15%, 10%, 9%, 8%, 7%, 6%, 5% or less. Oxygen concentration is mentioned.
  • CD4 / CD8 double positive T cells are T cells that are both positive for surface antigens CD4 and CD8 (CD8 + CD4 + ), and T cells are positive for surface antigens CD3 and CD45.
  • CD4 / CD8 double positive T cells can be identified as cells positive for CD4, CD8, CD3 and CD45.
  • CD4 / CD8 double positive T cells can be induced to differentiate into CD4 single positive cells or CD8 single positive cells.
  • CD4 / CD8 double positive T cells can be produced by a method comprising culturing hematopoietic stem cells in a culture medium supplemented with a p38 inhibitor and / or SDF-1.
  • P38 inhibitor is defined as a substance that inhibits the function of p38 protein (p38MAP kinase).
  • p38 inhibitors include, but are not limited to, chemical inhibitors of p38, dominant negative variants of p38, or nucleic acids encoding them.
  • Chemical inhibitors of p38 include SB203580 (4- (4-fluorophenyl) -2- (4-methylsulfonylphenyl) -5- (4-pyridyl) -1H-imidazole) and its derivatives, SB202190 (4-).
  • the p38 inhibitor is added to the culture medium, for example, in the range of about 1 ⁇ M to about 50 ⁇ M.
  • Dominant negative variants of p38 include p38T180A, which is a point mutation of threonine at position 180 located in the DNA binding region of p38 to alanine, and p38Y182F, which is a point mutation of tyrosine at position 182 of p38 to phenylalanine in humans and mice. Can be mentioned.
  • SDF-1 (Stromal cell-derived factor 1) is not only SDF-1 ⁇ or its mature form, but also isoforms such as SDF-1 ⁇ , SDF-1 ⁇ , SDF-1 ⁇ , SDF-1 ⁇ or SDF-1 ⁇ , or them. It may be a mature form of the above, or it may be a mixture of any proportions thereof and the like. Preferably SDF-1 ⁇ is used. SDF-1 may also be referred to as CXCL-12 or PBSF.
  • SDF-1 may be substituted, deleted and / or added with one or several amino acids in its amino acid sequence as long as it has activity as a chemokine, and similarly, sugar chains may be substituted or deleted. And / or may be added.
  • SDF-1 may be of a mammal, eg, a non-human mammal such as a monkey, sheep, cow, horse, pig, dog, cat, rabbit, rat, or mouse.
  • GenBank registration number: NP_954637 can be used as human SDF-1 ⁇
  • a protein registered with GenBank registration number: NP_000600 can be used as SDF-1 ⁇ .
  • SDF-1 a commercially available product may be used, a product purified from nature may be used, or a product manufactured by peptide synthesis or a genetic engineering method may be used. SDF-1 is added to the culture medium, for example, in the range of about 10 ng / mL to about 100 ng / mL.
  • the culture medium used for producing the CD4 / CD8 double positive T cells is not particularly limited, but the culture medium used for culturing animal cells is used as the basal culture medium, and p38 inhibitor and / or SDF-1 is more preferable. It can be prepared by adding vitamin Cs.
  • the types of vitamin C used in the production of CD4 / CD8 double positive T cells are as described above, for example, and the concentration of vitamin C is, for example, 5 to 200 ⁇ g / mL.
  • basal culture medium examples include Isove's Modified Dulbecco's Medium (IMDM) culture medium, Medium 199 culture medium, Eagle's Minimum Essential Medium (EMEM) culture medium, ⁇ MEM culture medium, Dulbecco's modified Eagle's Medium (DMEM) culture medium, and Ham. , RPMI 1640 culture medium, Fischer's Neurobasal Medium culture solution (Life Technologies), and a mixed culture solution thereof. Serum may be added to the culture broth, or serum-free.
  • IMDM Isove's Modified Dulbecco's Medium
  • EMEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's modified Eagle's Medium
  • Ham Ham.
  • RPMI 1640 culture medium Fischer's Neurobasal Medium culture solution (Life Technologies), and a mixed culture solution thereof. Serum may be added to the culture broth, or serum-free.
  • basal culture solutions include, for example, albumin, insulin, transferase, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerols, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors. , Low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and one or more substances selected from cytokines and the like.
  • Cytokines selected from the group consisting of SCF, TPO (thrombopoietin), FLT3L and IL-7 may be further added to the culture medium used for producing CD4 / CD8 double positive T cells. These concentrations are, for example, 10-100 ng / mL for SCF, 1O-200 ng / mL for TPO, 1-100 ng / mL for FLT3L, and 1-100 ng / mL for IL-7. Is.
  • Hematopoietic stem cells may be adherently cultured or suspension-cultured, but adherent culture is preferable.
  • the culture vessel may be coated and used.
  • Matrigel Niwa A, et al. PLoS One. 6 (7): e22261, 2011
  • collagen gelatin
  • laminin heparan sulfate proteoglycan
  • retronectin Fc-DLL4 or entactin
  • the culture temperature conditions for culturing hematopoietic stem cells for producing CD4 / CD8 double positive T cells are not particularly limited, but are preferably about 37 ° C to about 42 ° C, preferably about 37 ° C to about 39 ° C, for example. Is more preferable. Further, the culture period can be appropriately determined by those skilled in the art while monitoring the number of CD4 / CD8 double positive T cells and the like. As long as CD4 / CD8 double positive T cells can be obtained, the number of culture days is not particularly limited, but for example, at least 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, 22 days or more. , Or 23 days or more, preferably 23 days.
  • the obtained CD4 / CD8 double positive T cells may be isolated and used, or may be used as a cell population containing other cell types.
  • isolation methods well known to those of skill in the art can be used. For example, a method of labeling with an antibody against CD4, CD8, CD3 and / or CD45 and isolation using a flow cytometer, or a method of purification using an affinity column on which a desired antigen is immobilized can be mentioned.
  • CD8 single positive T cells that is, mature T cells, are cells (CD8 + CD4- ) positive for the surface antigen CD8 among T cells, and 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 positive for CD8, CD3 and CD45 and negative for CD4. ..
  • CD8 single positive T cells can be produced by a method including a step of culturing CD4 / CD8 double positive T cells in a culture medium supplemented with a corticohormonal agent.
  • the corticosteroid agent is preferably a glucocorticoid or a derivative thereof, and examples thereof include cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, or beclomethasone propionate.
  • the corticosteroid agent is dexamethasone. Its concentration in the culture medium is, for example, 1 to 100 nM.
  • the culture medium used for producing CD8 single positive T cells is not particularly limited, but can be prepared by using the culture medium used for culturing animal cells as the basal culture medium and adding an corticohormonal agent to it.
  • the basal culture medium include Isove's Modified Dulbecco's Medium (IMDM) culture medium, Medium 199 culture medium, Eagle's Minimum Essential Medium (EMEM) culture medium, ⁇ MEM culture medium, Dulbecco's modified Eagle's Medium (DMEM) culture medium, and H12 culture medium.
  • IMDM Isove's Modified Dulbecco's Medium
  • EMEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's modified Eagle's Medium
  • H12 culture medium examples thereof include a solution, RPMI 1640 culture solution, Fischer's Neurobasal Medium culture solution (Life Technologies), and a mixed culture solution thereof.
  • Serum may be added to the culture broth, or serum-free.
  • the basal culture medium may be, for example, albumin, insulin, transferase, selenium, fatty acid, trace elements, 2-mercaptoethanol, thiolglycerol, monothiolglycerol, lipid, amino acid, L-glutamine, non-essential amino acids, It may also contain one or more substances selected from vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like.
  • the culture medium used for producing CD8 single positive T cells preferably further contains anti-CD3 antibodies, vitamin Cs, or cytokines.
  • cytokine examples include IL-2, IL-7, IL-15, IL-21 and the like.
  • the anti-CD3 antibody is not particularly limited as long as it is an antibody that specifically recognizes CD3, and examples thereof include an antibody produced from an OKT3 clone.
  • the concentration of the anti-CD3 antibody in the culture medium is, for example, 10 to 1000 ng / mL.
  • the vitamin Cs used for producing CD8 single positive T cells are, for example, those described above, and can be used under the same conditions as described above.
  • the concentration of cytokines used for the production of CD8 single positive T cells in the culture medium is, for example, 10 to 1000 U / mL for IL-2 and 1 to 100 ng / mL for IL-7.
  • the temperature conditions for culturing CD4 / CD8 double positive T cells for producing CD8 single positive T cells are not particularly limited, but are preferably about 37 ° C to about 42 ° C, preferably about 37 ° C to about 38 ° C, for example. About ° C is more preferable.
  • the culture period can be appropriately determined by those skilled in the art while monitoring the number of CD8 single positive T cells and the like. As long as CD8 single positive T cells can be obtained, the number of culture days is not particularly limited, but is, for example, at least 1 day or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more, preferably 3 days. ..
  • the cDNA encoding the TCR ⁇ chain and the ⁇ chain for each single cell.
  • the T cells collected from the subjects are a population of T cells with genetic diversity as a whole, but were identified as CD106-positive T cells without determining the antigen or peptide sequence recognized by the TCR of each individual T cell.
  • the antigen specificity of individual T cells is different. It is preferable to prepare the cDNA for each single cell in order to select the TCR that is optimal for each tumor-related antigen, that is, highly responsive to each tumor-related antigen.
  • a T cell population that responds to a tumor-related antigen may be isolated as a single cell by a cell sorter or the like using CD106 as a marker.
  • cell surface CD137 may be used as a marker for isolating cells.
  • Known techniques for isolating human T cells include, for example, flow cytometry using antibodies against T cell surface markers such as CD106, CD3 and CD137 and cell sorters.
  • Gene cloning can be performed from the obtained single T cells using the PCR method to amplify the cDNAs encoding the TCR ⁇ chain and ⁇ chain, respectively.
  • PCR fragments amplified using the isolated TCR cDNA as a template can be incorporated into viral or non-viral vectors using, for example, the Gibson assembly system.
  • a gene in which an isolated TCR ⁇ chain gene and a TCR ⁇ chain gene are linked via a T2A sequence is bound to the downstream of the ubiquitin promoter, and further downstream to the IRES (internal ribosome entry site) sequence.
  • a marker gene such as EGFR (EGFRt, truncated EGFR) excluding the ligand binding site and the intracellular domain or CD19 lacking the intracellular domain is ligated, and this construct is integrated into a viral vector or a non-viral vector.
  • a viral vector and a non-viral vector can be used, but a non-viral vector is preferable.
  • a transposon vector is preferable, and a piggyBac® transposon vector is further preferable.
  • the transposon method is a next-generation gene transfer method that is cheaper and safer than the conventional viral vector method.
  • a method for introducing a cDNA pair encoding a TCR ⁇ chain and a ⁇ chain into the mature T cell differentiated from a T cell a method using a viral vector, a method using a non-viral vector, or replacement of TCR using a genome editing technique is used. Any of these can be adopted.
  • virus vector examples include viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus and Sendai virus, and animal cell expression plasmids, with retrovirus or lentivirus being preferred.
  • viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus and Sendai virus, and animal cell expression plasmids, with retrovirus or lentivirus being preferred.
  • a spin infection method or the like When a non-viral vector is used, the transposon method is preferable.
  • the CRISPR / Cas9 method, the CRISPR / MAD method and the CRISPR / CAS3 method may be used to replace the TCR using the genome editing technique.
  • Examples of the method for introducing a gene of a non-viral vector or the method for introducing a guide RNA and donor DNA for genome editing include a lipofection method, a liposome method, a calcium phosphate co-precipitation method, a DEAE dextran method, a microinjection method and an electroporation method. .. PCR products can also be introduced directly into cells without the use of vectors. It is preferable to use an electroporation method for cell transfer of a transposon vector or PCR product. As the electroporation device, the gene transfer device ExPERT® system (MaxCyte) is preferable.
  • Genome editing techniques such as CRISPR / Cas9 and TALEN may be used as a method for introducing cDNA pairs encoding the TCR ⁇ chain and ⁇ chain, respectively.
  • the introduction of the TCR gene by CRISPR / Cas9 is, for example, homologous to an endogenous TCR ⁇ chain and a guide RNA designed in the gene for the ⁇ chain (guide RNA to the sense strand and guide RNA to the antisense strand), as well as the endogenous TCR ⁇ strand.
  • the promoter used in the expression vector used when introducing the cDNA pair into the cell examples include EF1 ⁇ promoter, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, ubiquitin promoter and the like. Can be mentioned. These promoters may be capable of controlling the expression of the gene inserted downstream of the promoter depending on the presence or absence of a drug such as tetracycline.
  • the expression vector can include an enhancer, a poly A addition signal, a selectable marker gene (for example, a neomycin resistance gene), an SV40 origin of replication, and the like.
  • the iPS cell clone into which the cDNA pair has been introduced, 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 the above-mentioned culture medium.
  • culture conditions such as culture solution composition and culture temperature, regenerated T cells expressing TCR consisting of ⁇ -chain and ⁇ -chain encoded by the cDNA pair can be obtained.
  • 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 are derived is the same, and “allogeneic” means that the peripheral blood mononuclear cells and the iPS cell clone are derived from. It means that the subjects who do this are different.
  • the iPS cell into which the cDNA pair has been introduced, the hematopoietic stem cell, the immature T cell, or the mature T cell may be cloned.
  • a colony pickup method for cloning it is preferable to perform a colony pickup method for cloning.
  • the colony pickup method is not particularly limited, and examples thereof include a method using a pipette man under a microscope, a limiting dilution method, and a method using a fully automated colony picker.
  • TCRV ⁇ analysis kit BECKMAN COULTER: Catalog No. IM-3497. Since marker genes such as EGFRt (truncated EGFR) and CD19t (truncated CD19) are incorporated into the vector, the expression of TCR ⁇ chain and ⁇ chain can be inferred by analyzing the expression of the marker gene.
  • the iPS cell clone, the hematopoietic stem cell, the immature T cell or the mature T cell may form a master cell bank after expansion culture.
  • master cell bank means that the iPS cell clone, the hematopoietic stem cell, the immature T cell or the mature T cell is dispensed into a separate cell storage container for each single pool of a single clone. It is an accumulation. Usually, it is preferable to proliferate the cells within a range that does not change the properties of the cells and dispense them into a plurality of cell storage containers.
  • the cells stored in the master cell bank are cells into which a TCR gene or CAR (chimeric antigen receptor) gene is introduced and used as a starting material for producing regenerated T cells, and are mastered for each production of the regenerated T cells.
  • a cell storage container containing the required number of the cells can be taken out from the cell bank. Therefore, the regenerated T cells of the present invention can be repeatedly supplied with the same quality.
  • a master cell bank it is preferable to cryopreserve the cells dispensed into the cell storage container.
  • Methods of cryopreserving cells are well known to those of skill in the art. For example, a single clone of expanded-cultured cells is collected, washed with a buffer or culture medium, the number of cells is counted, concentrated by centrifugation or the like, and frozen medium (for example, a culture medium containing 10% DMSO). After suspending in, it can be cryopreserved at low temperature.
  • a master cell bank can be constructed by accumulating 200 to 1000 cell storage containers containing 1 ⁇ 10 6 to 10 7 cells per cell in a cell storage container stocker or the like.
  • the master cell bank of the present invention can be stored in any facility or storage that can be cryopreserved.
  • the storage temperature is not particularly limited as long as it is a temperature suitable for storing cells.
  • ⁇ 20 ° C., ⁇ 80 ° C. and ⁇ 120 to -196 ° C. are mentioned, but ⁇ 150 ° C. or lower is preferable.
  • the number of TCR pairs corresponding to tumor-related antigens is said to be 5 to 10 per epitope, and each TCR pair is predicted to have different susceptibility to point mutant variants of tumor-related antigens. There is. Therefore, for example, even when a point mutation occurs in a tumor-related antigen, there is a high possibility that there is a TCR capable of recognizing the epitope, and preparation of antigen-specific regenerated T cells for the point mutation variant is also possible. Can be done quickly.
  • TCRs that respond to those tumor-related antigens can be started from already acquired iPS cells having high differentiation potential into T cells or differentiated cells derived from iPS cells, it is possible to rapidly produce regenerated T cells. Is possible.
  • T cells from the TCR acquisition source The T cells that are the source of the TCR pair corresponding to the tumor-related antigen are preferably collected from the tumor tissue.
  • the collected T cells may be isolated using CD106 as a marker after amplifying a tumor-responsive cell population by co-culture with tumor tissue in vitro.
  • T cells for obtaining a TCR pair can be collected in advance at any time independently of the treatment time of the regenerated T cell replacement therapy, the regenerated T cell replacement therapy can be started promptly. ..
  • the obtained TCR pair is the subject to be treated. It is necessary to confirm that it does not show an allo reaction to cells.
  • the method of confirmation is a known method. For example, it can be confirmed by mixed lymphocyte culture (MLR) or the like that the obtained T cells having the TCR pair do not respond to the cells of the subject to be treated. Alternatively, it is confirmed that the T cells having the obtained TCR pair do not respond to the antigen in which one or more residues of the constituent amino acids of the antigen are replaced with other amino acids.
  • MLR mixed lymphocyte culture
  • the pharmaceutical composition containing the regenerated T cells of the present invention can be used for treating a cancer treatment target.
  • the pharmaceutical composition of the present invention can be produced by a method commonly used in the field of pharmaceutical technology, for example, the method described in the Japanese Pharmacopoeia.
  • the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable additive. Examples of the additive include a cell culture solution, a physiological saline solution, an appropriate buffer solution (for example, a phosphoric acid-based buffer solution), and the like.
  • the pharmaceutical composition of the present invention can be produced by suspending regenerated T cells in physiological saline, an appropriate buffer solution (for example, a phosphate-based buffer solution), or the like. It is preferable to contain, for example, 1 ⁇ 10 7 or more cells as a single dose so that the desired therapeutic effect is exhibited. A more preferable cell content is 1 ⁇ 10 8 or more, and even more preferably 1 ⁇ 10 9 or more. The cell content can be appropriately adjusted in consideration of the sex, age, body weight, condition of the affected area, cell condition, etc. of the administration subject.
  • the pharmaceutical composition of the present invention may contain dimethyl sulfoxide (DMSO), serum albumin and the like for the purpose of protecting the cells.
  • DMSO dimethyl sulfoxide
  • antibiotics and the like may be contained, and vitamins and cytokines may be contained for the purpose of promoting cell activation and differentiation.
  • the pharmaceutical composition of the present invention contains other components (eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives) that are acceptable for the formulation. , Physiological saline, etc.) may be contained.
  • the pharmaceutical composition containing the regenerated T cells of the present invention as an active ingredient can be cryopreserved.
  • the storage temperature is not particularly limited as long as it is a temperature suitable for storing cells.
  • ⁇ 20 ° C., ⁇ 80 ° C. and ⁇ 120 to -196 ° C. are mentioned, but ⁇ 150 ° C. or lower is preferable.
  • cells are preferably stored in suitable containers such as freezing vials and freezing bags. Procedures for minimizing the risk of cell damage during freezing and thawing of regenerated T cells are well known to those of skill in the art.
  • the T cells are recovered from the culture medium, washed with a buffer solution or a culture solution, the number of cells is counted, and then concentrated by centrifugation or the like to be concentrated in a frozen medium (for example, for example). After suspending in a culture medium containing 10% DMSO), it is cryopreserved at low temperature.
  • Regenerated T cells may be stored as individual clones or as a mixture of each clone.
  • the pharmaceutical composition containing the regenerated T cells of the present invention contains, for example, 5 ⁇ 10 4 to 9 ⁇ 10 10 regenerated T cells per container such as a freezing vial and a freezing bag, but for the cancer of interest. It can be changed according to the type, administration target, administration route, and the like.
  • Examples of the route of administration of the pharmaceutical composition containing the regenerated T cells of the present invention include infusion, intratumoral injection, intraarterial injection, portal vein injection, intraperitoneal administration and the like.
  • the route of administration is not limited to these as long as the regenerated T cells, which are the active ingredients in the pharmaceutical composition of the present invention, are delivered to the affected area.
  • the administration schedule may be a single dose or multiple doses. As the period of the multiple administration, for example, a method of repeating the administration once every 2 to 4 weeks, a method of repeating the administration once every 6 months to 1 year, and the like can be adopted.
  • the gender, age, body weight, pathological condition, etc. of the target patient can be taken into consideration.
  • the pharmaceutical composition containing the regenerated T cells of the present invention is used for the prevention or treatment of cancer in a cancer patient, if the TCR on the regenerated T cells is allogeneic, the allo in the body of the cancer patient.
  • a pharmaceutical composition containing regenerated T cells introduced with TCR that does not show an allo reaction to normal cells of a cancer patient is used.
  • the pharmaceutical composition of the present invention is used for the prevention or treatment of cancer.
  • Cancers include hepatocellular carcinoma, hepatoblastoma, gastric cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, breast cancer, ovarian cancer, skin cancer such as malignant melanoma, bladder cancer, Head and neck cancer, uterine cancer, cervical cancer, glioblastoma, prostate cancer, neuroblast tumor, chronic lymphocytic leukemia, papillary thyroid cancer, colon cancer, brain tumor, sarcoma and B-cell non-hodgkin lymphoma
  • cancers include hepatocellular carcinoma, hepatoblastoma, gastric cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, breast cancer, ovarian cancer, skin cancer such as malignant melanoma, bladder cancer, Head and neck cancer, uterine cancer, cervical cancer, glioblastoma, prostate cancer, neuroblast tumor, chronic lympho
  • the pharmaceutical composition of the present invention can be suitably used for highly immunogenic cancers such as hepatocellular carcinoma, hepatoblastoma, colon cancer, lung cancer and malignant melanoma.
  • Highly immunogenic cancer is a cancer that has a high ability to induce an immune response, and many gene mutations are found in cancer cells (1000 or more per cell or 5000 or more), including T cells. It refers to cancer that is recognized by the immune cells, the immune checkpoint inhibitor is effective, and the tumor tissue is heavily infiltrated with many immune cells (many immune cells are found by histopathological staining).
  • FIG. 1 shows a manufacturing process of regenerated T cells of the present invention.
  • Mononuclear cells in the tumor tissue of a cancer patient (subject) being treated are collected, T cells that specifically react with the tumor antigen are isolated as CD106-positive T cells, and the TCR gene is isolated from the CD106-positive T cells.
  • Fig. 1- (2') A TCR gene that is expected to have a higher therapeutic effect than the TCR binding ability encoded by the TCR gene to a tumor was selected (FIG. 1- (3')).
  • TCR repertoire TCR repertoire
  • iPS cells were produced from non-T non-B cells or monocytes in the peripheral blood of the patient (FIGS. 1- (1) and (2)).
  • iPS cell clones having good differentiation efficiency from the obtained iPS cells to T cells were selected (Fig. 1- (3)). By this selection, it was confirmed that the variation in the induction efficiency to T cells due to the difference in iPS cell clones was remarkably reduced, and the robustness and efficiency of the production of regenerated T cells were improved.
  • the above-mentioned selected TCR gene group that reacts specifically with a tumor antigen is expressed in the iPS cell clone having good differentiation efficiency into T cells (Fig. 1- (4)), and each selected TCR gene is expressed. Differentiated and proliferated into mature T cells (Fig. 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 (Fig. 1- (6)).
  • a step of producing iPS cells (FIGS. 1- (1) to (3)) and a step of acquiring a TCR gene that reacts with a tumor antigen (FIGS. 1- (2') and (3')). Since these steps can be performed in parallel, these steps can be completed in a shorter period (about 55 days) as compared with the case where these steps are sequentially performed.
  • Tumor antigen-specific TCR genes were isolated and antigen-specific reactivity was verified as follows (see the upper partial diagram of FIG. 2). After co-culturing a tumor (specimen) isolated from a cancer patient (subject) with CD8-positive cytotoxic T cells, CD106-positive cytotoxic T cells activated by a cell sorter (CD8-positive) And CD106-positive cells) were single-cell sorted (Fig. 2- (2)). TCR gene pairs (TCR ⁇ chain gene and TCR ⁇ chain gene) are isolated from isolated single cells by single cell PCR, sequence analysis is performed on the isolated TCR gene pairs, and tumor-reactive T cell types (TCR). Repatoa) and its frequency of appearance were analyzed (Fig. 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 (Fig. 2- (4)). Subsequently, the reactivity (antigen-specific reactivity) between the reconstructed TCR gene complex and the tumor was verified (Fig. 2- (5)). That is, the T cell line expressing the TCR gene pair and the target tumor cell are co-cultured, but when the expressed TCR and the tumor are antigen-specifically bound, the TCR gene signal is activated and targeted. A reporter gene integrated downstream of the gene promoter is expressed. Then, the reporter gene activity was measured, and the antigen-specific reactivity was verified by evaluating the binding of the tumor antigen-specific TCR.
  • TCR gene pairs showing tumor-specific reactivity are selected with high frequency of appearance, and non-T non-B cells or monocyte-derived iPS cells (M-iPS) described later are selected. It was used as a transgene to cells).
  • TCR gene-introduced iPS cells and regenerated T cells was performed as follows (see the lower partial view of FIG. 2).
  • IPS cells were prepared from peripheral blood mononuclear cells of cancer patients (Fig. 2- (2')).
  • the M-iPS cells described in the figure are iPS cells that are not derived from T cells, and are cells that have not undergone gene rearrangement of the TCR gene.
  • M-iPS cell lines having good differentiation efficiency into T cells were selected (FIG. 2- (3')).
  • a tumor-specific TCR gene (both TCR ⁇ chain gene and TCR ⁇ chain gene, see FIG. 2- (3)) was introduced into the selected M-iPS cell line using a transposon vector (FIG. 2).
  • TCR-iPS cell line was selected.
  • hematopoietic stem progenitor cells were induced using the embryoid body method, and differentiation was induced into mature T cells via immature T cells by a stepwise differentiation induction method that mimics T cell development in vivo. ..
  • FIG. 4 shows the analysis results of the regenerated T cells using the cell surface antigen marker by the flow cytometer.
  • the expression of CD45 + TCR ⁇ + CD3 + CD4 - CD8 ⁇ + which is observed in mature cytotoxic T cells in vivo, was confirmed in the regenerated T cells derived from iPS cells.
  • FIG. 5 shows the results of analysis of telomeres (index of rejuvenation), which is a cell senescence marker, for regenerated T cells mediated by iPS cells of the present invention.
  • telomeres index of rejuvenation
  • A peripheral blood mononuclear cells
  • B tumor antigen-specific T cells
  • C iPS cells
  • D tumor antigen-specific regenerated T cells
  • regenerated T cells mediated by iPS cells of the present invention For regenerated T cells mediated by iPS cells of the present invention, the expression of PD-1 and TIGIT molecules, which are one of the cell exhaustion markers related to immune checkpoints, was stained with anti-TIGIT antibody and anti-PD-1 antibody, and flow cytometer was used. was analyzed by. For comparison, pre-regeneration tumor antigen-specific T cells were also analyzed. The results are shown in FIG. It was confirmed that the regenerated T cells of the present invention had significantly reduced expression of PD-1 and TIGIT molecules as compared with 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.
  • Cytotoxic T cells CD8 ⁇ chain / ⁇ chain double positive cells
  • regenerated T cells of the present invention in peripheral blood mononuclear cells collected from healthy subjects were used as PMA (Phorbol 12-Myristate 13-Acetate) and ionomycin (Ionomycin).
  • PMA Phorbol 12-Myristate 13-Acetate
  • Ionomycin ionomycin Stimulated and produced IL-2 and IFN- ⁇ amounts were compared. The results are shown in FIG. It was confirmed that the number of cells producing IL-2 and IFN ⁇ was significantly increased in the regenerated T cells of the present invention as compared with the cytotoxic T cells obtained from healthy subjects.
  • Mononuclear cells were isolated from peripheral blood collected from patients with hepatocellular carcinoma or hepatoblastoma using a mononuclear cell separation 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.
  • Sendai virus (CytoTune® 2.0) and SV40Tag-encoded Sendai virus (CytoTune® 2.0) carrying Yamanaka 4 factors (Oct3 / 4, Sox2, Klf4 and c-Myc) in the obtained non-T non-B cell or monocyte cell population.
  • the virus was infected with a MOI (multiplicity of infection) of 5 to 20.
  • the SV40 may be excluded.
  • the obtained iPS cells consist of a large number of iPS cell clones. Therefore, a colony pick was performed and cloned. All cloned iPS cells were cryopreserved. The cloned iPS cells were cultured in a differentiation medium for about 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 about 21 days on a plate coated with FcDLL4, which is a fusion protein of the DLL4 protein and the Fc region of immunoglobulin, to induce differentiation into T cells.
  • FcDLL4 is a fusion protein of the DLL4 protein and the Fc region of immunoglobulin
  • the frequency of immature cytotoxic T cells obtained after culturing for the above 21 days was verified by the CD8 ⁇ chain / ⁇ chain double positive rate, and the clone with the highest frequency of appearance of CD8 ⁇ chain / ⁇ chain double positive cells was selected.
  • iPS cell clones with good differentiation efficiency into T cells are expanded and cultured in iPS cell maintenance medium for 2 weeks, then dispensed into a cell storage container and cryopreserved to construct a master cell bank. did.
  • T cell receptor ⁇ -chain and ⁇ -chain with confirmed tumor antigen specificity for iPS cell clones derived from non-T non-B cells or monocytes with good differentiation efficiency into T cells obtained in Example 2.
  • a piggyBac® transposon vector having the gene encoding the above was introduced using the electroporation method.
  • iPS cells expressing the target T cell receptor ⁇ chain and ⁇ chain were isolated by a cell sorter using the expression of the marker molecule CD19 as an index.
  • the isolated iPS cells were cultured in a differentiation medium for about 10 days to induce CD34 / CD43 double-positive hematopoietic stem cells, and isolated by a cell sorter.
  • the isolated blood stem cells were cultured on a plate coated with FcDLL4 for about 21 days to induce differentiation into T cells.
  • Immature T cells with double positive CD8 ⁇ chain / ⁇ chain obtained after the above 21-day culture were isolated and purified using a cell sorter. Immature T cells were then co-located as PHA (phytohemagglutinin) and feeder cells in the presence of peripheral blood mononuclear cells, in the presence of Retronectin® and anti-CD3 antibody, or in the presence of anti-CD3 and anti-CD28 antibodies. It was cultured and induced into mature cytotoxic T cells. These stimuli were given more than once. The performance of the obtained T cells was confirmed by the cytotoxic activity, IFN- ⁇ production and antigen-binding ability of GPC3-specific target cells.
  • PHA phytohemagglutinin
  • Tumor tissues were obtained from surgical specimens before the start of treatment with anti-PD-1 antibody in melanoma patients for whom anti-PD-1 antibody was significantly effective. Collagenase and DNase were added to the obtained tumor tissue, and the cells were separated by enzymatic treatment by stirring at 37 ° C. for 30 minutes. From a cell mixture of tumor tissue-derived cells and tumor-infiltrating immune cells, CD3-positive cells were sorted using a BD FACSAria III® cell sorter (BD Biosciences) to obtain cells expressing TCR. rice field.
  • BD FACSAria III® cell sorter BD Biosciences
  • T cells For each of the obtained T cells, a single sequence was used for both the gene expression of the marker candidate and the TCR sequence using the Chromium system (10X Genomics) 5-prime kit and V (D) J enrichment kit. Cell gene expression analysis was performed. HiSeq3000 (Illumina) was used as the sequencer.
  • Fig. 8 The results are shown in Fig. 8.
  • cells positive for the expression of the marker described in the panel are shown colored.
  • the cells are clustered according to the expression pattern, and the circled part corresponds to the tumor-damaging T cell.
  • CD106 VCAM1 positive cells are highly specific to the tumor-damaging T cell population. It is shown that CD106 can be used as a selectable marker for tumor-damaging T cells with higher specificity than surface markers other than CD106.
  • the TCR sequence was obtained in cells having a ⁇ chain of 90% or more and cells having an ⁇ chain of 60 to 90%.
  • T cells of tumor tissue were separated, CD3-positive cells were sorted from the obtained cell mixture using BD FACSAria III, and T cells expressing TCR were designated as tumor-infiltrating T cells. Obtained.
  • the tumor-infiltrating T cells and a cell line established from an autologous tumor as a stimulant were co-cultured, and IFN- ⁇ production was compared with the case of no stimulation. IFN- ⁇ production was measured by intracellular staining with an anti-IFN- ⁇ antibody.
  • Cell staining was performed using anti-PD-1 antibody, anti-TIGIT antibody, anti-LAG3 antibody, and anti-CD106 antibody. As a negative control, an isotype control antibody against the anti-CD106 antibody was used.
  • IFN- ⁇ is produced by stimulation with the autotumor cell line, and in the marker-positive cell subpopulation other than CD106, IFN- ⁇ is produced by autotumor stimulation, but the marker. Since IFN- ⁇ is also produced in the negative cell subpopulation, it can be understood that the specificity is inferior to that of CD106.
  • TCR gene into mature T cells differentiated from iPS cell clones From the non-T non-B cell or monocyte-derived iPS cell clones obtained in Example 2 having good differentiation efficiency into T cells, to mature T cells differentiated via hematopoietic stem cells and immature T cells.
  • a gene (cDNA) encoding a GPC3 antigen-specific T cell receptor ⁇ -chain ⁇ -chain was introduced using the piggyBac® system in the same manner as in Example 3.
  • FIG. 10 shows the results of analysis of the phenotype of mature T cells derived from gene-introduced iPS cells by a flow cytometer.
  • “No EP” is the analysis result of the mature T cell used for gene transfer and expressing the WT1 antigen-specific T cell receptor ⁇ chain ⁇ chain; “EGFP”.
  • EGFP enhanced green fluorescent protein
  • TCR-CD19 is a GPC3 antigen-specific T cell receptor ⁇ -chain ⁇ -chain gene and intracellular.
  • the analysis result of the mature T cell which introduced the piggyBac® transposon vector in which the defective human CD19 gene was integrated into the tandem is shown.
  • GPC3 antigen specificity In mature T cells derived from iPS cells represented by "TCR-CD19", in addition to the expression of the CD3 gene and the intracellular defective human CD19 gene, which is a tracer gene integrated into the piggyBac® transposon vector, GPC3 antigen specificity. Binding to the GPC3 peptide / HLA complex (GPC3-Dex) recognized by the target T cell receptor ⁇ chain ⁇ chain was detected. That is, GPC3 antigen-specific T expressed on the cells by introducing the GPC3 antigen-specific T cell receptor ⁇ -chain ⁇ -chain gene into mature T cells derived from iPS cells using the piggyBac® system.
  • the cell receptor ⁇ -chain ⁇ -chain functions as a molecule that recognizes the GPC3 peptide / HLA complex (GPC3-Dex). Therefore, a novel tumor-related antigen (neoantigen) and other tumor-related antigen-specific T cell receptor ⁇ -chain ⁇ -chain genes should be used as the T-cell receptor ⁇ -chain ⁇ -chain gene to be introduced into mature T cells derived from iPS cells. It was shown that mature T cells derived from iPS cells that recognize these antigens can be produced.
  • FIG. 11 shows a method for selecting iPS cell clones having high efficiency of differentiation into T cells in the step of producing regenerated T cells from iPS cells reprogrammed with peripheral blood T cells.
  • Mononuclear cells are isolated from the peripheral blood of subjects infected with EBV (Epstein-Barr virus), and the isolated mononuclear cells are stimulated with the EBV antigen in vitro to obtain a CD8-positive T cell population that recognizes the EBV antigen. Isolated.
  • EBV Epstein-Barr virus
  • Yamanaka 4 factors (Oct3 / 4, Sox2, Klf4 and c-Myc) and SV40T antigen were introduced into the isolated CD8-positive T cell population using a Sendai virus vector to obtain an iPS cell population.
  • the iPS cell clones were separated from the obtained iPS cell population, the ability to differentiate into T cells was examined for each clone, and the iPS cell clones having high differentiation efficiency into T cells were selected.
  • T cells that recognize the EBV antigen are cells that are unlikely to cause an allo reaction even when allogeneic transplantation is performed.
  • iPS cell clones selected as cells having high differentiation efficiency into T cells, and regenerated T cells were performed.
  • the method for producing cells is shown.
  • the iPS cell clones derived from CD8-positive T cells that recognize EBV antigens and have high differentiation efficiency into T cells are ⁇ 2M genes and CIITA genes involved in the expression of MHC class I and MHC class II, and natural killer (NK) cells.
  • the PVR gene involved in the activation of NK cells and the Rag2 gene involved in the rearrangement of T cell receptors were deleted using CRISPR / Cas9, while ⁇ 2 ⁇ / binding peptide / HLA, which is an inhibitory ligand for NK cells.
  • the -E fusion gene (HLA-E * ) was expressed to obtain iPS cells that were not attacked by host T cells and NK cells.
  • cells are expressed after in vivo administration by expressing a suicide gene such as drug-induced caspase-9 and / or a specific marker gene (EGFR (epidermal growth factor receptor), CD19, CD20, etc.). It can also be removed.
  • the 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 from the host T cells.
  • the host T cells can be used as a material for producing regenerated T cells into which a TCR gene or a CAR (chimeric antigen receptor) gene has been introduced. Since the host T cell is a T cell that recognizes an EBV antigen, it is unlikely to cause an allo reaction even if it is transferred into a living body.
  • "Host T cells” are used as starting materials for the production of prophylactic or therapeutic agents for cancers containing regenerated T cells as an active ingredient, although the cells themselves are not used to treat patients. Means T cells.
  • FIG. 13 shows a method for producing regenerated T cells that recognize cancer antigens from the host T cells.
  • T cell receptor ⁇ chains that recognize reconstituted EBV antigens by gene replacement by genome editing using CRISPR / Cas9 and T cells that recognize cancer antigens, respectively. It was replaced with a T2a-mediated conjugate of the receptor ⁇ chain and the T cell receptor ⁇ chain.
  • the T cell receptor ⁇ chain that recognizes the EBV antigen was removed by genome editing using CRISPR / Cas9.
  • FIG. 13 it was possible to produce regenerated T cells that recognize cancer antigens.
  • FIG. 14 collectively describes the methods shown in FIGS. 11 to 13.
  • Regenerated T cells iPS-T cells
  • iPS-T cells Regenerated T cells produced from the same type of universal iPS cells in which peripheral blood T cells have been reprogrammed can be used as a starting material for producing T cells into which a TCR gene or CAR gene has been introduced.
  • Universal iPS cells have very low immunogenicity, so they do not cause rejection in patients with any type of MHC (Major Histocompatibility Complex). Means iPS cells that can be used. That is, 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 a ligand that suppresses NK cells.
  • the viral vector used for the production of T cells used in conventional T cell replacement therapy or regeneration therapy may be used.
  • iPS-T cells as a starting material, it becomes possible to produce desired T cells as an active ingredient of a preventive or therapeutic agent for cancer in a short period of time.

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013176197A1 (ja) 2012-05-22 2013-11-28 国立大学法人 東京大学 抗原特異的ｔ細胞の製造方法
WO2016010148A1 (ja) * 2014-07-18 2016-01-21 国立大学法人京都大学 多能性幹細胞から免疫細胞療法用ｔ細胞を誘導する方法
WO2017179720A1 (ja) * 2016-04-15 2017-10-19 国立大学法人京都大学 Ｃｄ８陽性ｔ細胞を誘導する方法
WO2018182817A1 (en) * 2017-03-29 2018-10-04 Iovance Biotherapeutics, Inc. Processes for production of tumor infiltrating lymphocytes and uses of same in immunotherapy
WO2021006316A1 (ja) * 2019-07-10 2021-01-14 国立研究開発法人国立がん研究センター がん細胞を特異的に攻撃しているｔ細胞を同定するための特異的マーカー

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013176197A1 (ja) 2012-05-22 2013-11-28 国立大学法人 東京大学 抗原特異的ｔ細胞の製造方法
WO2016010148A1 (ja) * 2014-07-18 2016-01-21 国立大学法人京都大学 多能性幹細胞から免疫細胞療法用ｔ細胞を誘導する方法
WO2017179720A1 (ja) * 2016-04-15 2017-10-19 国立大学法人京都大学 Ｃｄ８陽性ｔ細胞を誘導する方法
WO2018182817A1 (en) * 2017-03-29 2018-10-04 Iovance Biotherapeutics, Inc. Processes for production of tumor infiltrating lymphocytes and uses of same in immunotherapy
WO2021006316A1 (ja) * 2019-07-10 2021-01-14 国立研究開発法人国立がん研究センター がん細胞を特異的に攻撃しているｔ細胞を同定するための特異的マーカー

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
MINAGAWA A ET AL.: "Enhancing T cell receptor stability in rejuvenated iPSC-derived T cells improves their use in cancer immunotherapy", CELL STEM CELL, vol. 23, 2018, pages 850 - 858
MINAGAWA ATSUTAKA; YOSHIKAWA TOSHIAKI; YASUKAWA MASAKI; HOTTA AKITSU; KUNITOMO MIHOKO; IRIGUCHI SHOICHI; TAKIGUCHI MAIKO; KASSAI Y: "Enhancing T Cell Receptor Stability in Rejuvenated iPSC-Derived T Cells Improves Their Use in Cancer Immunotherapy", CELL STEM CELL, ELSEVIER, CELL PRESS, AMSTERDAM, NL, vol. 23, no. 6, 6 December 2018 (2018-12-06), AMSTERDAM, NL , pages 850, XP085555973, ISSN: 1934-5909, DOI: 10.1016/j.stem.2018.10.005 *
NISHIMURA T ET AL.: "Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation", CELL STEM CELL, vol. 12, 2013, pages 114 - 126, XP055567898, DOI: 10.1016/j.stem.2012.11.002
NIWA A ET AL., J CELL PHYSIOL., vol. 221, no. 2, November 2009 (2009-11-01), pages 367 - 77
NIWA A ET AL., PLOS ONE, vol. 6, no. 7, 2011, pages e22261
R. K. LINDEMANN ET AL., MOL. CANCER, vol. 2, 2003, pages 20
See also references of EP4273234A4
TAKAYAMA N. ET AL., J EXP MED., 2010, pages 2817 - 2830

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* Cited by examiner, † Cited by third party
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