WO2022220146A1 - Ｔ細胞受容体遺伝子を導入するためのｉＰＳ細胞により構成される細胞バンク - Google Patents

Ｔ細胞受容体遺伝子を導入するためのｉＰＳ細胞により構成される細胞バンク Download PDF

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WO2022220146A1
WO2022220146A1 PCT/JP2022/016392 JP2022016392W WO2022220146A1 WO 2022220146 A1 WO2022220146 A1 WO 2022220146A1 JP 2022016392 W JP2022016392 W JP 2022016392W WO 2022220146 A1 WO2022220146 A1 WO 2022220146A1
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cells
cell
ips
antigen
cell bank
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French (fr)
Japanese (ja)
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良輔 五ノ坪
光次郎 大澤
泰道 等
新 金子
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Thyas Co Ltd
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Thyas Co Ltd
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Priority to US18/285,023 priority Critical patent/US20250177524A1/en
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Priority to EP22788071.3A priority patent/EP4324917A4/en
Priority to CN202280027749.8A priority patent/CN117157391A/zh
Priority to AU2022259170A priority patent/AU2022259170A1/en
Priority to CA3213440A priority patent/CA3213440A1/en
Publication of WO2022220146A1 publication Critical patent/WO2022220146A1/ja
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    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K40/32—T-cell receptors [TCR]
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    • A61K40/00—Cellular immunotherapy
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    • A61K40/41—Vertebrate antigens
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    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2760/18843—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention is intended to produce induced pluripotent stem cell (iPS cell)-derived T cells (regenerative T cells) into which a T cell receptor (T cell receptor: TCR) gene that recognizes antigens derived from tumors or pathogens has been introduced.
  • iPS cell induced pluripotent stem cell
  • T cell receptor T cell receptor: TCR
  • cell banks containing iPS cell-derived hematopoietic stem cells, immature T cells and mature T cells as intermediates of
  • the present invention relates to the use of cell banks containing hematopoietic stem cells, immature T cells and mature T cells for the production of regenerative T cell preparations for cancer prevention and/or treatment.
  • 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, it is believed that decreased function of T cells contributes to the development of cancer and pathogen infection.
  • T-cell replacement therapy or regenerative therapy for patients with diseases caused by T-cell dysfunction can be extremely effective means for ameliorating and treating disease conditions in patients.
  • T cell replacement therapy In studies using humans and mice, when T cell replacement therapy is performed for cancer or infectious diseases, it specifically recognizes antigens possessed by cancer cells or abnormal cells infected with foreign pathogens such as bacteria or viruses. It is known that a high therapeutic effect can be obtained by using T cells that treat cancer. On the other hand, it is difficult to secure a sufficient amount of T cells, it takes a long time to produce T cells, and when patient-derived cells are used as materials, the proliferative ability of T cells is reduced and Exhaustion of T cells, such as decreased immune response to antigens such as target cells, is an obstacle in T cell replacement therapy.
  • iPS cells are established from antigen-specific T cells, and after proliferating the iPS cells, T cell replacement therapy using regenerated T cells differentiated into T cells. , or T cell replacement therapy using regenerated T cells differentiated from iPS cells into which TCRs have been introduced to recognize target antigens.
  • T cells specific for target antigens or TCRs for recognizing target antigens as raw materials for producing iPS cells, regenerated T cells exhibiting the same antigen specificity as the original T cells are produced. It is possible to manufacture (Patent Document 1 and Non-Patent Document 1).
  • Patent Document 2 When producing regenerated T cells from iPS cells, long-term differentiation culture is required (Patent Document 2).
  • an acquired TCR is introduced into allogeneic iPS cells, the produced regenerated T cells can be stored, so the long production period of the regenerated T cells is not a particular problem.
  • an acquired TCR is used in a patient different from the individual from which the TCR was derived, there is a risk of side reactions due to alloreactions. For this reason, the use of patient-specific antigen-specific TCRs is attracting attention for the purpose of risk avoidance.
  • each individual has different genetic mutations that make them neoantigens, which are good targets for T-cell therapy. To date, administration of vaccines against neoantigens has shown high efficacy, and therapeutic agents manufactured by methods using antigen-specific TCRs for individual patients have great benefits in cancer treatment.
  • Nishimura T et al. Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation. Cell Stem Cell. 2013; 12:114-126.
  • iPS-T cells regenerative T cells
  • the use of patient-specific TCRs is important in ensuring the safety and efficacy of regenerative T cell replacement therapy in therapy.
  • prompt initiation of iPS-T cell replacement therapy is important for improving treatment results.
  • the production of cell preparations using a patient's antigen-specific TCR needs to be performed on a patient-by-patient basis, and the problems of the labor required for production and the long production period have hindered the development of cell preparations. Particularly in allogeneic regenerated T cell preparations, the length of the manufacturing period has been a problem.
  • the present invention provides a cell group containing hematopoietic stem cells, immature T cells and mature T cells, which are cells differentiated from iPS cells, for rapid production of iPS-T cells into which a patient-specific antigen-specific TCR has been introduced,
  • the purpose is to construct a cell bank that can be provided as a manufacturing intermediate.
  • the present invention includes the use of said cell bank for the production of regenerative T-cell preparations for use in the prevention and/or treatment of cancer or infectious diseases from said production intermediates.
  • T cell clones (5 to 10 clones) recognize the antigen using different TCRs for one antigen epitope. Moreover, it has been clarified that each patient has a different TCR repertoire for one antigenic epitope.
  • cancer cells have genetic mutations that differ from one another in individual patients, making them neoantigens that are good targets for T-cell therapy. It is highly likely that regenerative T cells produced using TCRs obtained from individual patients will be T cells with TCRs that are optimal for attacking the target of T cell therapy within the corresponding patient's body. It is clear that regenerative T cells produced using TCRs obtained from individual patients do not elicit an alloreactive attack on recipient cells. Therefore, the benefits of using TCRs obtained from individual patients to produce regenerative T cells in terms of safety and efficacy in the prevention and/or treatment of cancer or infectious diseases are extremely high.
  • the present inventors obtained various TCRs with antigen specificity from T cell populations reactive to tumor-associated antigens or pathogen-specific antigens of individual patients, and induced differentiation of these TCRs from iPS cells. and can be introduced into prestored hematopoietic stem cells, immature T cells or mature T cells, or can be replaced with endogenous TCR to produce regenerated T cells; hematopoietic stem cells previously differentiated from iPS cells , the construction of a cell bank with immature T cells and mature T cells can greatly shorten the production period of regenerated T cells; and the use of the cells contained in the cell bank as an intermediate for producing regenerated T cell preparations By doing so, the inventors have found that homogenization and standardization of regenerated T cell preparations are facilitated, and have completed the present invention.
  • the present invention can also be applied to the production of regenerated T cells into which an acquired TCR has been introduced and regenerated T cells into which a chimeric antigen receptor (CAR)
  • a cell bank composed of cells into which a T cell receptor gene is introduced, wherein the cells are iPS cells and hematopoietic stem cells differentiated from the iPS cells, immature T cells and mature T cells.
  • the cell bank which is one or more types of cells selected from.
  • the cell bank of [1] wherein the cell is a cell into which a chimeric antigen receptor gene is introduced.
  • the iPS cells are peripheral blood mononuclear cells of a subject, iPS cells obtained by reprogramming peripheral blood mononuclear cells from which B cells and T cells have been removed, [1] or The cell bank of [2].
  • [4] introduction of a T cell receptor gene into the iPS cell clone or the hematopoietic stem cells or immature T cells differentiated from the iPS cell clone uses a viral vector, transposon vector or genome editing technology; Cell bank as indicated.
  • [6] The cell bank of [5], wherein a genome editing technique is used to introduce the T cell receptor gene into the mature T cells.
  • the cell bank of [5], wherein the mature T cells express a T cell receptor that does not recognize non-tumor cells derived from a subject separate from the subject from which the mature T cells were derived.
  • the cell bank of any one of [1] to [14], wherein the immature T cells are CD8 ⁇ -chain/ ⁇ -chain double-positive.
  • the cell bank of any one of [1] to [15], wherein the mature T cells are CD8 ⁇ -chain/ ⁇ -chain double-positive and TCR ⁇ -chain/ ⁇ -chain double-positive.
  • the T cell receptor gene is prepared from a T cell population obtained from a subject, wherein each single cell is prepared from a T cell population having reactivity to a tumor-associated antigen, [1] to [ 16], the cell bank according to any one of the above.
  • the T cell receptor gene is prepared for each single cell from a T cell population reactive to the tumor-associated antigen by contacting T cells obtained from a subject with a tumor-associated antigen; [1] The cell bank according to any one of [16].
  • the T-cell receptor gene contacts T cells obtained from a subject to whom a tumor-associated antigen has been administered with the tumor-associated antigen; The cell bank according to any one of [1] to [16], which is prepared for each cell.
  • the tumor-associated antigen is selected from the group consisting of GPC3, WT1, XAGE1, LMP2, NY-ESO-1, EB virus antigens, neoantigens, and peptide fragments thereof; cell bank as described in .
  • the tumor-associated antigen is HLA-A24-restricted GPC3 peptide EYILSLEEL (SEQ ID NO: 1), HLA-A2-restricted GPC3 peptide FVGEFFTDV (SEQ ID NO: 2), or a mixture thereof;
  • the cell bank according to any one of to [19].
  • the cell into which the T-cell receptor gene or the T-cell receptor gene and the chimeric antigen receptor are introduced is an intermediate for the production of a T-cell preparation used for the prevention and/or treatment of cancer; , the cell bank according to any one of [1] to [25].
  • the cell bank of the present invention contains pre-stored hematopoietic stem cells, immature T cells and/or mature T cells that have been induced to differentiate from iPS cells, these cells can be treated with cancer or infectious diseases.
  • TCRs harvested from or acquired TCRs or CARs can be transfected to produce regenerative T cells in a short period of time. Therefore, the time required from the production of regenerated T cells into which an antigen-specific TCR has been introduced to the use of the regenerated T cells for treatment can be dramatically shortened, and the amount of regenerated T cells necessary for treatment can be reduced. can be stably secured.
  • the cell bank of the present invention can be constructed using either iPS cell clones derived from non-T non-B cells or monocytes or iPS cell clones derived from T cells.
  • Introduction of the TCR to hematopoietic stem cells, immature T cells and mature T cells differentiated from iPS cell clones derived from non-T non-B cells or monocytes allowed these cells to differentiate into regenerative T cells followed by expansion. Since rearrangement of the TCR gene does not easily occur when cultured, the antigen specificity of the introduced TCR is maintained, and as a result, the safety of the regenerated T cell preparation is enhanced.
  • regenerated T cells that are less exhausted due to expansion culture can be produced.
  • iPS cell clones with good differentiation efficiency into T cells are selected in advance and used as cells that make up the cell bank. Therefore, it is possible to minimize the influence of variations in cell yield and degree of cell differentiation between production batches of regenerated T cells, and individual differences in subjects from whom cells are collected, on the quality, etc. of the regenerated T cells obtained. Become.
  • the cell bank of the present invention it becomes possible to efficiently and rapidly produce T cells having TCRs that recognize antigens and efficiently kill targets.
  • iPS cell-derived differentiated cells iPS cell-derived differentiated cells, iPS cell-derived differentiated cells, iPS cell-differentiated hematopoietic stem cells, immature T cells, and mature T cells, which are differentiated from iPS cells in advance and constitute the cell bank of the present invention.
  • the cells that make up the cell bank are differentiated from the cloned iPS cells, so the quality of the cells is more uniform and stable.
  • the number of cells used for a single TCR introduction is 10 7 to 10 9 or more
  • the cell bank of the present invention can contain, in aliquot form, the number of cells required for 1000 or more TCR transductions. Therefore, it is expected that the cost for implementing TCR introduction once will be reduced. For this reason, in cancer therapy applying regenerative T cell replacement therapy, antigen-specific expression of tumor antigen change in cancer patients to be treated, development of treatment resistance, or cancer recurrence It is possible to quickly and easily prepare regenerative T cells.
  • the subject from whom T cells used for preparing the cDNA encoding the TCR are cancer patients to be treated by regenerative T cell replacement therapy. It may be the same individual as the subject, or it may be a separate entity from each other. Thus, safe and effective TCRs with optimal antigen specificity can be selected for individual cancer patients.
  • Fig. 2 shows the production of tumor-specific regenerative T cells from mature T cells that are induced to differentiate from iPS cells and constitute a cell bank.
  • Introduction of tumor-specific TCRs into mature T cells is done by gene replacement using genome modification techniques such as CRISPR/Cas9. It takes about two weeks from the introduction of the TCR into the cells to the administration of the TCR-introduced cells to the patient. Desirably, TCRs expressed in mature T cells do not induce alloreactions.
  • the TCR to be introduced into the cells is a TCR obtained from a cancer patient to whom the regenerative T cell preparation is scheduled, a cancer-specific TCR that has already been obtained, or a cancer-specific CAR.
  • FIG. 2 shows the production of tumor-specific regenerative T cells from immature T cells that are induced to differentiate from iPS cells and constitute a cell bank.
  • Introduction of a tumor-specific TCR into immature T cells is performed using gene replacement using genome modification technology such as CRISPR/Cas9, lentivirus, transposon vector, or the like. It takes about 4 weeks from the introduction of the TCR into the cells to the administration of the TCR-introduced cells to the patient.
  • the TCR to be introduced into the cells is a TCR obtained from a cancer patient to whom the regenerative T cell preparation is scheduled, a cancer-specific TCR that has already been obtained, or a cancer-specific CAR.
  • FIG. 1 shows the process of introducing TCR genes into hematopoietic stem cells, immature T cells or mature T cells using transposons.
  • FIG. 2 shows phenotypes of mature T cells (CD8-positive cytotoxic T cells) induced from iPS cells. Mature T cells that make up the cell bank adopt the phenotype shown in FIG.
  • FIG. 2 shows the results of analyzing telomeres as a cellular senescence marker in mature T cells induced from iPS cells.
  • FIG. 2 shows the results of analyzing the expression of PD-1 and TIGHT molecules as cell exhaustion markers in mature T cells induced from iPS cells.
  • FIG. 2 shows phenotypes of immature T cells (CD8-positive immature T cells) induced from iPS cells.
  • the immature T cells that make up the cell bank adopt the phenotype shown in FIG.
  • FIG. 2 shows phenotypes of iPS cell-derived mature T cells into which a GPC3 antigen-specific TCR gene was introduced at the stage of mature T cells.
  • the CD19 gene is a gene integrated in the same piggyBac (registered trademark) transposon vector in tandem with the TCR gene, and is used as a marker for gene insertion into host chromosomes and gene expression.
  • FIG. 1 shows phenotypes of immature T cells (CD8-positive immature T cells) induced from iPS cells.
  • the immature T cells that make up the cell bank adopt the phenotype shown in FIG.
  • FIG. 2 shows phenotypes of iPS cell-derived mature T cells into which a GPC3 antigen-specific
  • FIG. 2 is a diagram illustrating 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.
  • iPS cell clones selected as cells with high differentiation efficiency into T cells are subjected to genome editing to produce regenerated T cells. It is a figure explaining a method.
  • This regenerated T cell is used as a master cell bank of host T cells.
  • FIG. 2 is a diagram illustrating a method for producing regenerative T cells that recognize cancer antigens from host T cells.
  • FIG. 2 is a diagram showing an outline of a process for producing regenerated T cells from allogeneic iPS cells reprogrammed peripheral blood T cells.
  • the differentiated T cells serve as a master cell bank of host T cells.
  • a "cell bank” is a collection of multiple types of cells composed of cells into which a T-cell receptor gene is to be introduced. CAR can also be introduced into the cells that make up the cell bank.
  • the cell bank of the present invention is composed of iPS cells and one or more types of cells selected from the group consisting of hematopoietic stem cells differentiated from the iPS cells, immature T cells and mature T cells. Cells contained in the cell bank of the present invention can be used to produce regenerative T cells for use in preventing and/or treating cancer or infectious diseases.
  • the “aggregate of cells” is obtained by dividing cells based on the origin of the cells, the stage of differentiation of the cells, etc., and distributing and accumulating each of the cells in independent cell containers.
  • the cell bank of the present invention can be stored in any facility or repository that allows cryopreservation of cells.
  • the "mature T cells" constituting the cell bank are differentiated cells that have been induced to differentiate from iPS cells, express CD3 and CD4 or CD8 as cell surface antigens, and express major histocompatibility complex ( cells that express functional TCRs on their cell surface that can induce cell proliferation, cytokine production and cytotoxicity in response to antigens presented on the major histocompatibility complex.
  • TCRs include heterodimers consisting of ⁇ and ⁇ chains and heterodimers consisting of ⁇ and ⁇ chains. T cells with a TCR consisting of ⁇ and ⁇ chains are called ⁇ type T cells, and T cells with a TCR consisting of ⁇ and ⁇ chains are called ⁇ type T cells.
  • the mature T cells of the present invention are preferably CD3/ ⁇ T cells, but may be CD3/ ⁇ T cells.
  • the mature T cells may be cells expressing endogenous TCR in which TCR gene rearrangement has been completed, or cells expressing exogenously introduced TCR.
  • the "immature T cells" that constitute the cell bank are differentiated cells that have been induced to differentiate from iPS cells, but are in the process of differentiating into T cells and do not express TCR on the cell surface.
  • Immature T cells include T cells at each stage from CD4/CD8 double-negative cells to CD4/CD8 double-positive cells to CD8 single-positive cells.
  • CD8 expressed on immature T cells is preferably a CD8 ⁇ heterodimer, but may be a CD8 ⁇ homodimer.
  • the "hematopoietic stem cells” that make up the cell bank are cells that can differentiate into hematopoietic cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes.
  • Hematopoietic stem cells and hematopoietic progenitor cells (HPC) are not to be distinguished from each other, and refer to the same cell unless otherwise specified.
  • Hematopoietic stem cells or hematopoietic progenitor cells are recognized, for example, by being double positive for the surface antigens CD34 and CD43.
  • the term "genetically modified cells so that the expression of endogenous TCR can be controlled” refers to a method using microRNA or the like, or replacing the endogenous TCR promoter with a tetotacycline-regulated promoter or the like. Thus, it is a cell that has been genetically modified so that the expression of endogenous TCR can be suppressed or regulated.
  • the TCRs expressed by the mature T cells that make up the cell bank recognize a single antigen and produce no or almost no alloreaction.
  • Antigens recognized by the TCR include influenza virus antigens, EB virus antigens, HPV antigens, HBV antigens, HCV antigens, HIV antigens, coronavirus antigens, HTLV antigens, and cancer antigens.
  • the antigen recognized by the TCR to be introduced into the cells constituting the cell bank is an antigen that is expressed specifically or non-specifically in the tumor, and is derived from a protein that is overexpressed in tumor cells and These include its variants, tumor virus-derived antigens, certain differentiation antigens and novel tumor-associated antigens (neoantigens) due to gene mutations and splice abnormalities. Tumor-associated antigens are sometimes referred to herein as tumor antigens. In the case of a protein antigen, it may be a peptide fragment (peptide fragment).
  • Antigens specifically or non-specifically expressed in 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, Mesothelin, PD-L1, trp1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, p53 , p53 mutant, NY-ESO-1, PSMA, ETV6-AML, CEA, PSA, AFP, hTERT, EpCAM, ALK, androgen receptor, EphA2, CYP1B1, OY-TES-1, MAD-CT-2, MelanA /MART1, survivin, Ras, Ras mutants, ERG, bcr-abl, XBP
  • Viral antigens include inactivated HBV and inactivated viruses such as HPV, and various virus-derived proteins 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), etc., but not limited thereto.
  • HPV 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), etc. but not limited thereto.
  • antigens recognized by TCRs introduced into cells constituting a cell bank consist of GPC3, WT1, XAGE1, LMP2, NY-ESO-1, EB virus antigens and neoantigens, and peptide fragments thereof. You can choose from the group.
  • the antigen recognized by the TCR introduced into the cells constituting the cell bank is EYILSLEEL, an HLA-A24-restricted GPC3 peptide, FVGEFFTDV, an HLA-A2-restricted GPC3 peptide, or a mixture thereof.
  • amino acids are represented by conventional one-letter abbreviations.
  • T cells are tumor-associated with 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 the TCR, and binding / joining of T cells to something other than the epitope peptide means that T cells have a reaction. It means that no reaction occurs.
  • MHC major histocompatibility complex
  • T cell responses resulting from binding/conjugation to epitope peptides derived from tumor-associated antigens presented on MHC class I or class II via the TCR include cytotoxicity, IFN- ⁇ and granzyme production, T Expression of cell activation markers, as well as activation of transcription factors such as NF-AT.
  • the TCR to be introduced into the cells that make up the cell bank is collected from subjects who are cancer patients or non-cancer patients.
  • Subjects from whom T cells are collected may be cancer patients who have been administered cancer vaccines, are currently being administered, or are scheduled to be administered in the future, and cancer vaccines are not administered. It may be a non-cancer patient.
  • One or more cancer vaccines may be administered.
  • a cancer vaccine is a cancer or tumor specific protein or peptide, a composition comprising a vaccine antigen derived from a cancer or said antigen, for inducing a cancer or tumor specific immune response. Cancer vaccines usually contain an adjuvant to enhance the specific immune response induced by the vaccine antigen.
  • T cells are preferably ⁇ T cells.
  • peripheral blood is preferable because of its low invasiveness, but it is not limited thereto.
  • Other preferred collection sources include cancer tissue or tumor tissue, lymph nodes or other tissues or organs, blood, umbilical cord blood, lymph, tissue fluid (interstitial fluid, intercellular fluid and interstitial fluid), body cavity fluid (ascites , pleural fluid, pericardial fluid, cerebrospinal fluid, synovial fluid and aqueous humor).
  • 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 ovarian cancer, hepatoblastoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, malignant melanoma cancer, non-small cell lung cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid cancer, colon cancer or B-cell non-Hodgkin's lymphoma.
  • Said cancer is preferably hepatocellular carcinoma or hepatoblastoma.
  • iPS cells may be produced by reprogramming non-T, non-B cells or monocytes. It may also be already made from non-T non-B cells or monocytes.
  • non-T non-B cells mononuclear cells that are not classified as T cells nor are they classified as B cells.
  • Non-T non-B cells can be prepared by collecting peripheral blood mononuclear cells and removing T cells and B cells contained in the mononuclear cells.
  • Peripheral blood mononuclear cells can be isolated from human whole blood with a mononuclear cell separation solution. Mononuclear cell separation solutions include, for example, Lymphoprep®.
  • the B cell surface antigens CD19, CD20, CD22 or B cell receptor and the T cell surface antigens CD3, CD4 or CD8 Antibodies may be used, for example, using flow cytometry or magnetic beads such as MACS® beads.
  • T cells can be prepared as cells expressing CD3 and CD8 after collection as peripheral blood mononuclear cells.
  • antibodies against the surface antigens of T cells, CD3, CD4 or CD8, are used, for example, by flow cytometry or magnetic beads such as MACS (registered trademark) beads. may be used.
  • iPS cells can preferably be induced by introducing cell reprogramming factors into non-T non-B cells or monocytes or T cells.
  • Cell reprogramming factors include, for example, Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, klf4, klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERAs, ECAT15-2 , Tcl1, ⁇ -catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 and Glis1.
  • These cell reprogramming factors may be used alone or in combination.
  • Oct3/4, Sox2, Klf4 and c-Myc are selected from the viewpoint that iPS cells can be efficiently established. Introduction into T cells is preferred.
  • the method of introducing the cell reprogramming factor into the non-T non-B cells or monocytes or T cells is not particularly limited, and methods known in the art can be adopted.
  • the gene encoding the cell reprogramming factor when the gene encoding the cell reprogramming factor is introduced into the non-T non-B cells or monocytes or T cells, the gene (e.g., cDNA) encoding the cell reprogramming factor can function intracellularly.
  • the expression vector can be introduced into cells by infection, lipofection, liposome, calcium phosphate coprecipitation, DEAE dextran, microinjection or electroporation.
  • the cell reprogramming factor When the cell reprogramming factor is in the form of a protein and the protein is introduced into the non-T non-B cells or monocytes or T cells, a method using a protein introduction reagent, a method using a protein transfer domain fusion protein , electroporation and microinjection methods.
  • a method using a protein introduction reagent When the cell reprogramming factor is in the form of messenger RNA (mRNA) and the mRNA is introduced into the non-T non-B cells or monocytes or T cells, a method using an mRNA introduction reagent and addition to the culture medium method.
  • mRNA messenger RNA
  • Expression vectors used for gene transfer by infection include, for example, viral vectors such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai viruses, and animal cell expression plasmids.
  • viral vectors such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai viruses, and animal cell expression plasmids.
  • Sendai virus it is preferable to use to transfer the gene encoding the cell reprogramming factor into the cell.
  • promoters used in expression vectors for introducing the cell reprogramming factor-encoding gene into cells include SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, and ubiquitin promoter. etc. These promoters may be able to control the expression of genes inserted downstream of the promoter depending on the presence or absence of drugs such as tetracycline.
  • the expression vector can contain, in addition to the promoter, an enhancer, poly A addition signal, selectable marker gene (eg, neomycin resistance gene), SV40 origin of replication, and the like.
  • the culture medium used for culturing iPS cells obtained by reprogramming is not particularly limited. can be prepared by adding cytokines of Examples of the basal culture medium include Iscove's Modified Dulbecco's Medium (IMDM) medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, and Ham's F12 medium. , RPMI 1640 culture medium, Fischer' culture medium, Neurobasal Medium (Life Technologies), StemFit (registered trademark) AK03N (Ajinomoto Healthy Supply Co.) and mixed cultures thereof.
  • the culture medium may be serum-supplemented or serum-free.
  • Cytokines preferably include bFGF, and its concentration in the culture medium is, for example, 1 to 100 ⁇ g/mL (preferably 50 ⁇ g/mL).
  • the iPS cell culture method may be adherent culture or suspension culture, but adherent culture is preferred.
  • Methods for isolating iPS cells include, for example, 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 (e.g., Examples include isolation methods using Accutase (registered trademark) and Accumax (registered trademark).
  • 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 cells/cm 2 . Passage to another incubator when a cell density of 6 cells/cm 2 is reached. 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.
  • iPS cells reprogrammed by introducing Yamanaka factors consist of a large number of iPS cell clones.
  • the method of colony picking is not particularly limited, but a method using a Pipetman under a microscope, a limiting dilution method, a method using a fully automatic colony picker, and the like are used.
  • One or more, preferably 3 or more, more preferably 6 or more iPS cell clones obtained are preferably preserved to constitute a cell bank.
  • iPS cell clones are preferably cryopreserved.
  • iPS cell clones are collected, washed with a buffer or culture medium, counted, concentrated by centrifugation or the like, and suspended in a freezing medium (e.g., culture medium containing 10% DMSO). After turbidity, it can be cryopreserved.
  • a freezing medium e.g., culture medium containing 10% DMSO. After turbidity, it can be cryopreserved.
  • iPS cell clones that have already been established and that have good differentiation efficiency into T cells may be used.
  • “Differentiation efficiency” refers to hematopoietic stem cells, immature T cells, and mature T cells in all viable 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. It refers to the existence 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 expressed as the percentage of CD34/CD43 double-positive cells in total viable cells, and the efficiency of differentiation into immature T cells is expressed as the percentage of CD4/CD8 double-positive cells or the percentage of CD5-positive cells in total viable cells. , and the efficiency of differentiation into mature T cells is shown as the percentage of cells positive for all of the CD8 ⁇ , CD8 ⁇ , TCR ⁇ and TCR ⁇ chains among all viable cells.
  • “Good differentiation efficiency” means that the percentage of CD34/CD43 double-positive cells, which are hematopoietic stem cells, is 5 to 15% or more in the differentiation from iPS cells to hematopoietic stem cells; In differentiation into cells, the percentage of CD4 / CD8 double positive cells or CD5 positive cells that are immature T cells refers to 10% or more or 50% or more, respectively; from immature T cells to mature T cells In differentiation, it refers to 50% or more of cells in which all of the CD8 ⁇ chain, CD8 ⁇ chain, TCR ⁇ chain and TCR ⁇ chain are positive.
  • the mature T cells in the present invention preferably differentiate the iPS cell clone first into hematopoietic stem cells, then differentiate the hematopoietic stem cells into immature T cells, and finally convert the immature T cells into CD8 single-positive T cells. It is produced by differentiating into The immature T cells in the present invention are preferably produced by first differentiating the iPS cell clone into hematopoietic stem cells and then differentiating the hematopoietic stem cells into immature T cells. Hematopoietic stem cells in the present invention are preferably produced by differentiating the iPS cell clone into hematopoietic stem cells.
  • Hematopoietic stem cells are preferably produced by culturing iPS cells in a culture medium supplemented with vitamin Cs.
  • vitamin C refers to L-ascorbic acid and its derivatives
  • L-ascorbic acid derivative means one that is converted to vitamin C in vivo by an enzymatic reaction.
  • L-ascorbic acid includes, for example, vitamin C phosphate, glucoside ascorbate, ethyl ascorbyl, vitamin C esters, ascorbyl tetrahexyldecanoate, ascorbyl stearate and ascorbate-diphosphate-6-palmitate.
  • the derivative of L-ascorbic acid is preferably vitamin C phosphate, for example phosphate-L ascorbate salts such as sodium phosphate-L-ascorbate or magnesium phosphate-L-ascorbate.
  • Vitamin C is contained, for example, at a concentration of 5-500 ⁇ g/mL in the culture solution.
  • the culture solution used for the production of hematopoietic stem cells is not particularly limited, but it can be prepared by using the culture solution used for culturing animal cells as the basal culture solution and adding vitamin C or the like to this.
  • basic culture media include Iscove'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's F12 culture medium.
  • liquid RPMI 1640 culture medium, Fischer's culture medium, Neurobasal Medium (Life Technologies), StemPro34 (Life Technologies), and mixed cultures thereof.
  • the culture medium may contain serum or may be serum-free.
  • the basal medium contains e.g. 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, pyruvic acid, buffers, inorganic salts, cytokines, and the like.
  • albumin e.g. 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, pyruvic acid, buffers, inorganic salts, cytokines, and the like.
  • BMP4 (Bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), SCF (stem cell factor), TPO (thrombopoietin), and FLT3L ( A cytokine selected from the group consisting of Flt3 ligand) may be further added.
  • 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 1–100 ng/mL
  • FLT3L is 1–100 ng/mL.
  • a TGF ⁇ inhibitor may be added to the hematopoietic stem cell culture medium.
  • a "TGF ⁇ inhibitor” is a small molecule inhibitor that interferes with the signaling of the TGF ⁇ family, e.g. , NPC30345, SD093, SD908 and SD208 (Scios), and LY2109761, LY364947 and LY580276 (Lilly Research Laboratories), and the concentration added to the culture medium is preferably 0.5-100 ⁇ 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): 367-77) and other feeder cells.
  • the method for culturing iPS cells during production of hematopoietic stem cells may be adherent culture or suspension culture, but suspension culture is preferred.
  • iPS cells can be cultured to 80% confluency in the dish used, released, dissociated into single cells, and subjected to suspension culture.
  • Methods for isolating iPS cells include, for example, a method of physically isolating with a cell scraper or the like, a dissociation solution having protease activity and collagenase activity (e.g., Accutase (registered trademark) and Accumax (registered trademark), etc.), or Isolation methods using a dissociation solution with collagenase activity are included.
  • Suspension culture is to culture cells in a non-adherent state to the culture vessel.
  • Suspension culture is not particularly limited, but culture vessels that have not undergone artificial treatment (for example, coating treatment with extracellular matrix, etc.) for the purpose of improving adhesion to cells, or artificially suppress adhesion (For example, coating treatment with polyhydroxyethyl methacrylate (poly-HEMA) or nonionic surfactant polyol (Pluronic F-127, etc.)).
  • poly-HEMA polyhydroxyethyl methacrylate
  • Pluronic F-127 nonionic surfactant polyol
  • Hematopoietic stem cells can also be prepared from cyst-like structures (also called iPS-sac) obtained by culturing iPS cells.
  • cyst-like structure also called iPS-sac
  • cyst-like structure refers to a three-dimensional sac-like structure (having a space inside) derived from iPS cells, formed of an endothelial cell population or the like, and containing 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.
  • a person skilled in the art can appropriately determine the culture period while monitoring the number of hematopoietic stem cells and the like.
  • the number of days of culture is not particularly limited as long as hematopoietic stem cells can be obtained. Or 14 days or more, preferably 14 days. A long culture period is not a problem in the production of hematopoietic stem cells.
  • hypoxic conditions include, for example, 15%, 10%, 9%, 8%, 7%, 6%, 5% or less. of oxygen concentration.
  • CD4/CD8 double-positive T cells are T cells that are positive for both the surface antigens CD4 and CD8 (CD8 + CD4 + ), and the T cells are 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 since they can be recognized by being .
  • 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, for example, chemical inhibitors of p38, dominant-negative mutants of p38, or nucleic acids encoding same.
  • Chemical inhibitors of p38 include 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-imidazol-1-yl]cyclohexanol) and its derivatives, SB220025 and its derivatives, PD169316, RPR200765A, AMG-548, BIRB-796, SCl0-469, SCIO-323, Examples include but are not limited to VX-702, or FR167653.
  • a p38 inhibitor is added to the culture medium, for example, in the range of about 1 ⁇ M to about 50 ⁇ M.
  • the dominant-negative mutants of p38 include p38T180A, in which threonine at position 180 located in the p38 DNA-binding region is mutated to alanine, and p38Y182F, in which tyrosine at position 182 of p38 is mutated to phenylalanine in humans and mice. is mentioned.
  • SDF-1 stromal cell-derived factor 1
  • SDF-1 ⁇ includes 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 their or a mixture thereof at any ratio.
  • SDF-1 ⁇ is used.
  • SDF-1 is sometimes referred to as CXCL-12 or PBSF.
  • SDF-1 may have one or several amino acid substitutions, deletions and/or additions 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. A range in which at least four cysteine residues (Cys30, Cys32, Cys55 and Cys71 in the case of human SDF-1 ⁇ ) are retained in SDF-1 and have 90% or more identity to the natural amino acid sequence Amino acid mutations are allowed.
  • the SDF-1 may be of a mammal, eg, a non-human mammal such as a human, eg, monkey, sheep, cow, horse, pig, dog, cat, rabbit, rat, or mouse.
  • GenBank accession number: NP_954637 can be used as human SDF-1 ⁇
  • NP_000600 can be used as SDF-1 ⁇ .
  • SDF-1 may be commercially available, purified from nature, or produced by peptide synthesis or genetic engineering techniques. 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 the production of CD4/CD8 double-positive T cells is not particularly limited. It can be prepared by adding vitamin C.
  • the types of vitamin C used in the production of CD4/CD8 double-positive T cells are, for example, as described above, and the concentration of vitamin C is, for example, 5-200 ⁇ g/mL.
  • Examples of the basal culture medium include Iscove's Modified Dulbecco's Medium (IMDM) medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, and Ham's F12 medium. , RPMI 1640 culture medium, Fischer's Neurobasal Medium culture medium (Life Technologies), and their mixed culture medium.
  • the culture medium may be serum-supplemented or serum-free.
  • the basal medium contains e.g. 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, pyruvic acid, buffers, inorganic salts, cytokines, and the like.
  • a cytokine selected from the group consisting of SCF, TPO (thrombopoietin), FLT3L and IL-7 may be further added to the culture medium used to produce CD4/CD8 double-positive T cells.
  • concentrations are, for example, 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. is.
  • Hematopoietic stem cells may be adherently cultured or suspended cultured, but adherently cultured is preferred.
  • the culture vessel may be coated.
  • 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. is mentioned.
  • the culture temperature conditions for culturing hematopoietic stem cells to produce CD4/CD8 double-positive T cells are not particularly limited. is more preferred.
  • 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.
  • the number of days of culture is not particularly limited as long as CD4/CD8 double-positive T cells can be obtained. , 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.
  • methods well known to those skilled in the art can be used. Examples thereof include 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 or the like on which a desired antigen is immobilized.
  • CD8 single-positive T cells are T cells whose surface antigen CD8 is positive (CD8 + CD4 ⁇ ), 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 that are CD8, CD3 and CD45 positive and CD4 negative.
  • CD8 single-positive T cells can be produced by a method that includes the step of culturing CD4/CD8 double-positive T cells in a culture medium supplemented with an adrenocortical hormone agent.
  • 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.
  • the corticosteroid is dexamethasone. Its concentration in the culture medium is, for example, 1-100 nM.
  • the culture medium used for the production of 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 adrenocortical hormone agent thereto.
  • Examples of basic culture media include Iscove'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's F12 culture medium.
  • IMDM Iscove's Modified Dulbecco's Medium
  • EMEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's modified Eagle's Medium
  • Ham's F12 culture medium Ham's F12 culture medium.
  • liquid RPMI 1640 culture medium, Fischer's Neurobasal Medium culture medium (Life Technologies), and mixed culture medium thereof.
  • the culture medium may be serum-supplemented or serum-free
  • the basal medium contains, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothiolglycerol, lipids, 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, pyruvic acid, 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.
  • cytokines include, for example, IL-2, IL-7, IL-15 and IL-21.
  • the anti-CD3 antibody is not particularly limited as long as it 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, 10-1000 ng/mL.
  • the vitamin Cs used for the production of CD8 single-positive T cells are, for example, those mentioned above, and can be used under the same conditions as above.
  • the concentrations of cytokines used in the production of CD8 single-positive T cells in the culture medium are, for example, 10-1000 U/mL for IL-2 and 1-100 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. °C is more preferable.
  • a person skilled in the art can appropriately determine the culture period while monitoring the number of CD8 single-positive T cells and the like.
  • the number of culture days is not particularly limited as long as CD8 single-positive T cells can be obtained. For example, it is 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 cDNAs encoding the TCR ⁇ chain and ⁇ chain to be introduced into the cells constituting the cell bank are preferably prepared for each single cell.
  • T cells collected from a subject are a T cell population with genetic diversity as a whole. Specificity is different.
  • the CAR-encoding cDNA to be introduced into the cells constituting the cell bank comprises a target binding site of an antibody or phage that binds to a target cell surface antigen or a ligand or receptor, a linker and a cell transmembrane site (e.g., CD8).
  • the CD3 ⁇ gene and the CD28 or CD137 intracellular signaling site linked via the transmembrane site of the molecule are prepared by constructing a plasmid vector or a viral vector.
  • T cells obtained from the subject with the target tumor-associated antigen may be isolated from the T cell population that responds to the tumor-associated antigen used using an activation marker, such as by a cell sorter.
  • a preferred activation marker is cell surface CD137.
  • Known techniques 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 cell using the PCR method to amplify cDNAs encoding the TCR ⁇ chain and ⁇ chain, respectively.
  • TCR gene by single cell PCR tumor antigen-specific CD8-positive T cells obtained from peripheral blood or the like are combined with an MHC dextramer (registered trademark) that forms a complex with the antigen peptide.
  • MHC dextramer registered trademark
  • single cell sorting can be performed by a cell sorter.
  • An MHC dextramer is a compound composed of a dextran polymer backbone in which MHC and a fluorescent dye molecule are bound. MHC tetramer may be used instead of MHC dextramer.
  • An MHC tetramer is a tetramer of a complex of an antigen peptide and an MHC molecule with biotin and avidin.
  • tumor antigen-specific CD8-positive T cells obtained from peripheral blood or the like are cultured in the presence of the antigen, and then CD3/CD137 double-positive cells are isolated by a cell sorter. Single cell sorting is possible.
  • the cell population that binds to the MHC dextramer complexed with the antigenic peptide can be single cell sorted by a cell sorter from the CD3/CD137 double positive cells.
  • a TCR gene pair (TCR ⁇ chain gene and TCR ⁇ chain gene) can be isolated by PCR method using cDNA obtained by extracting RNA from the obtained single cell and reverse transcription reaction. 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.
  • a PCR fragment amplified using the isolated TCR cDNA as a template can be incorporated into a viral vector or non-viral vector (transposon vector) using, for example, the Gibson assembly system.
  • a gene in which the isolated TCR ⁇ chain gene and TCR ⁇ chain gene are linked via the T2A sequence is bound, and further downstream thereof, an IRES (internal ribosome entry site) sequence is attached.
  • IRES internal ribosome entry site
  • viral vectors and non-viral vectors can be used, but non-viral vectors are preferred.
  • PiggyBac vectors are preferred among transposon vectors as non-viral vectors.
  • the transposon method is a cheap and safe next-generation gene transfer method compared to the conventional viral vector method.
  • iPS cell clones derived from non-T non-B cells or monocytes in a good state of differentiation into T cells the hematopoietic stem cells differentiated from the iPS cell clones, or the TCR ⁇ chain and ⁇ to the immature T cells differentiated from the hematopoietic stem cells
  • Either a method using a viral vector or a method using a non-viral vector can be adopted as a method for introducing a cDNA pair encoding each strand.
  • Viral vectors include viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus and Sendai virus, and animal cell expression plasmids, preferably retroviruses or lentiviruses. When retrovirus or lentivirus infection is performed, it is preferable to use a spin infection method or the like. When using non-viral vectors, the transposon method is preferred. Non-viral vector gene transfer methods include the lipofection method, the liposome method, the calcium phosphate co-precipitation method, the DEAE dextran method, the microinjection method and the electroporation method. PCR products can also be introduced directly into cells without using vectors. Electroporation is preferably used for cell introduction of transposon vectors or PCR products. As an electroporation device, the gene transfer device ExPERT (registered trademark) system (MaxCyte) is preferred.
  • ExPERT registered trademark
  • MaxCyte MaxCyte
  • promoters used in expression vectors for introducing the cDNA pair into the cells include EF-1 ⁇ promoter, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, and ubiquitin promoter. etc. These promoters may be able to control the expression of genes inserted downstream of the promoter depending on the presence or absence of drugs such as tetracycline.
  • the expression vector can contain, in addition to the promoter, an enhancer, poly A addition signal, selectable marker gene (eg, neomycin resistance gene), SV40 origin of replication, and the like.
  • TCR into mature T cells using genome editing technology Introduction of TCR into mature T cells can be performed using genome editing technology.
  • the endogenous TCR ⁇ chain and ⁇ chain gene sequences to be targeted confirming the sequences including the upstream thereof, having high cleavage activity for the endogenous TCR ⁇ chain and ⁇ chain genes, and genome other than the target gene Create guide RNAs (guide RNA for the sense strand and guide RNA for the antisense strand) that do not cut the above sequence.
  • donor DNA is generated that contains homologous recombination sites upstream and downstream of the insertion sites for the TCR ⁇ and ⁇ chains to be introduced.
  • the target TCR By introducing the guide RNA, Cas9 and the donor DNA into mature T cells, the target TCR can be introduced.
  • Introduction of the guide RNA, Cas9 and the donor DNA can be performed by using a viral vector, a non-viral vector, an electroporation method, or the like.
  • Mature T cells in which the endogenous TCR has been replaced with the desired TCR can be selected using binding to the desired tumor-associated antigen as an index.
  • marker genes such as EGFRt (truncated EGFR) and CD19t (truncated CD19
  • Regenerative T cell production can be initiated by introducing a TCR pair reactive to a tumor-associated antigen into the mature T cells, immature T cells or hematopoietic stem cells that make up the cell bank of the present invention. Therefore, it is possible to rapidly produce regenerative T cells used for prevention and/or treatment of cancer or infectious disease.
  • T cells from which TCR pairs corresponding to tumor-associated antigens are obtained are preferably collected from the patient's tumor tissue or patient's peripheral blood.
  • the tumor-associated antigen is administered from the tumor tissue or peripheral blood after administration of the tumor-associated antigen to the subject.
  • T cells are harvested.
  • tumor-associated antigens include, for example, GPC3.
  • tumor-specific T cells are present in peripheral blood without administration of tumor-associated antigens
  • MHC dextramer can be used to extract tumor cells from peripheral blood without administration of tumor-associated antigens.
  • Relevant antigen-specific T cells may be isolated.
  • tumor-associated antigens include, for example, WT1, NYESO-1 and EBV antigens.
  • T cells harvested from tumor tissue or peripheral blood of a subject after administration of a tumor-associated antigen to the subject, or without administration of the tumor-associated antigen are in any case preferred.
  • In vitro stimulation with a tumor-associated antigen may not be performed when the appearance frequency of tumor-associated antigen-specific T cells is extremely high in peripheral blood.
  • the TCR It is preferable to confirm that the pair does not elicit an allo-response against the cells of the subject to be treated for cancer.
  • a pharmaceutical composition containing regenerated T cells produced by introducing an antigen-specific TCR into the iPS cell-derived differentiated cells of the present invention can be used for treating cancer and infectious diseases.
  • the pharmaceutical composition of the present invention can be produced by a method commonly used in the field of formulation technology, such as the method described in the Japanese Pharmacopoeia.
  • the pharmaceutical composition of the present invention may contain pharmaceutically acceptable additives. Examples of such additives include cell culture media, physiological saline, and suitable buffers (eg, phosphate buffers).
  • the pharmaceutical composition can be produced by suspending regenerated T cells in physiological saline or an appropriate buffer (eg, phosphate buffer). It is preferable to contain, for example, 1 ⁇ 10 7 cells or more as a single dosage amount so as to exert the desired therapeutic effect. More preferably, the cell content is 1 ⁇ 10 8 or more, more preferably 1 ⁇ 10 9 or more. The content of cells can be appropriately adjusted in consideration of the sex, age, weight, condition of the affected area, condition of cells, etc. of the subject to be administered.
  • the pharmaceutical composition may contain dimethylsulfoxide (DMSO), serum albumin, etc. for the purpose of protecting the cells, in addition to the regenerated T cells as active ingredients.
  • DMSO dimethylsulfoxide
  • the pharmaceutical composition may contain antibiotics, etc., and vitamins, cytokines, etc., for the purpose of promoting activation and differentiation of cells.
  • the pharmaceutical composition may contain other pharmaceutically acceptable components (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline solution, etc.).
  • the hematopoietic stem cells, immature T cells, or mature T cells that constitute the cell bank of the present invention are divided according to the amount (1 ⁇ 10 7 to 1 ⁇ 10 9 ) to be used for one TCR introduction, and cryopreserved. can be done.
  • the preservation temperature is not particularly limited as long as it is suitable for preserving cells. For example, it may be -150 to -196°C, preferably -150°C or lower.
  • cryopreserved cells are preferably stored in suitable containers such as cryovials and cryobags. Manipulations to minimize the risk of cell damage during freezing and thawing of regenerated T cells are well known to those skilled in the art.
  • the amount of cells that constitute the cell bank of the present invention is preferably an amount that allows TCR transduction to be performed 1000 times or more.
  • the pharmaceutical composition is used for prevention and/or treatment of cancer or infectious disease.
  • Cancers include 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, and glioblastoma. including, but not limited to, blastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid cancer, colorectal cancer, and B-cell non-Hodgkin's lymphoma.
  • FIG. 1 shows the process of producing antigen-specific regenerative T cells from mature T cells that constitute the cell bank of the present invention.
  • TCR genes isolated from patient-derived T cells that specifically react to tumor antigen X, TCR genes specific to tumor antigen X, or chimeric receptor genes specific to tumor antigen X constitute a cell bank. Introduce into mature T cells. It takes about two weeks to produce regenerated T cells from the mature T cells that make up the cell bank.
  • FIG. 3 shows the steps of introducing a TCR gene using a transposon.
  • the cell bank of the present invention which is an aggregate of frozen cells, was constructed.
  • the mature T cells are T cells that recognize a single antigen and do not initiate an alloreaction, and after thawing the frozen cells that constitute the cell bank, tumor-specific TCRs are introduced.
  • regenerative T cells can be produced by introducing a TCR gene that reacts with tumor antigen X into mature T cells that have been induced to differentiate from iPS cells and cryopreserved in advance. can. For this reason, compared to the case where the step of producing iPS cells, the step of inducing the differentiation of iPS cells into mature T cells, and the step of producing regenerated T cells by introducing a TCR gene into mature T cells are sequentially performed, the time period is shorter. (approximately 2 weeks), tumor-specific regenerative T cells can be produced. Therefore, administration of tumor-specific regenerative T cells to patients can be performed rapidly.
  • FIG. 4 shows the results of flow cytometric analysis of regenerated T cells using cell surface antigen markers. Mature T cells induced from iPS cells were confirmed to express CD45 + TCR ⁇ + CD3 + CD4 ⁇ CD8 ⁇ + , the expression of which is observed in mature cytotoxic T cells in vivo.
  • Fig. 5 shows the results of analyzing telomeres (indicators of rejuvenation), which are cell senescence markers, for mature T cells that are induced to differentiate from iPS cells and constitute the cell bank of the present invention.
  • telomeres indicator of rejuvenation
  • iPS cells prepared from the tumor antigen-specific T cells C
  • C tumor antigen-specific T cells
  • D tumor antigen-specific regenerative T cells
  • the expression of PD-1 which is one of the cell exhaustion markers related to immune checkpoints, and the TIGIT molecule, is measured with anti-TIGIT antibody and anti-PD-1. They were stained with antibodies and analyzed by a flow cytometer. 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 have greatly reduced expressions of PD-1 and TIGIT molecules compared to tumor antigen-specific T cells before regeneration. Therefore, it is suggested that the regenerated T cells of the present invention have high cytotoxic activity.
  • FIG. 2 shows the process of producing antigen-specific regenerative T cells from immature T cells that constitute the cell bank of the present invention.
  • TCR genes isolated from patient-derived T cells that specifically react to tumor antigen X, TCR genes specific to tumor antigen X, or chimeric receptor genes specific to tumor antigen X constitute a cell bank. Introduce into immature T cells. In patients undergoing cancer therapy, it is possible to reselect a therapeutically effective TCR gene even in the event of tumor recurrence or tumor cell mutations. It takes about 4 weeks to produce regenerated T cells from the immature T cells that make up the cell bank. Genome editing using CRISPR/Cas9 or the like, lentivirus, or transposon vector can be used to introduce the tumor antigen X-specific TCR gene or CAR gene.
  • the cell bank of the present invention which is an aggregate of frozen cells, was constructed.
  • the immature T cells are T cells that do not express T cell receptors, and after thawing the frozen cells that make up the cell bank, a tumor-specific TCR is introduced.
  • regenerative T cells can be produced by introducing a TCR gene that reacts with tumor antigen X into immature T cells that have been induced to differentiate from iPS cells and cryopreserved in advance. can. For this reason, compared to the case where the step of producing iPS cells, the step of inducing the differentiation of iPS cells into immature T cells, and the step of producing regenerated T cells by introducing a TCR gene into the immature T cells are performed sequentially, the time period is shorter. In (approximately 4 weeks), tumor-specific regenerative T cells can be produced. Therefore, administration of tumor-specific regenerative T cells to patients can be performed rapidly.
  • FIG. 7 shows the analysis results of immature T cells by flow cytometry using cell surface antigen markers. Immature T cells induced from iPS cells were confirmed to express CD45 + TCR ⁇ ⁇ CD3 + CD4 ⁇ CD8 ⁇ + , which is observed to be expressed in mature cytotoxic T cells in vivo.
  • Mononuclear cells were isolated from peripheral blood drawn from patients with hepatocellular carcinoma or hepatoblastoma using the mononuclear cell separation solution Lymphoprep®. From the obtained mononuclear cells, CD19/CD20 positive B cells and CD3/CD4/CD8 positive T cells were depleted using FACS or MACS beads to obtain non-T non-B cells or monocytes.
  • Sendai virus (CytoTune (registered trademark) 2.0) carrying Yamanaka 4 factors (Oct3/4, Sox2, Klf4 and c-Myc) and Sendai Viruses were infected at an MOI (multiplicity of infection) of 5-20. Note that the SV40 may be omitted.
  • 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 DLL4 protein and the Fc region of immune immunoglobulin, to induce differentiation into T cells.
  • FcDLL4 is a fusion protein of DLL4 protein and the Fc region of immune immunoglobulin
  • the frequency of immature cytotoxic T cells obtained after the above 21 days of culture was verified by the CD8 ⁇ -chain/ ⁇ -chain double-positive rate, and clones with the highest appearance frequency of CD8 ⁇ -chain/ ⁇ -chain double-positive cells were selected.
  • iPS cell clones with good differentiation efficiency into T cells are cultured for 2 weeks in an iPS cell maintenance medium, then dispensed into cell storage containers and cryopreserved to construct a master cell bank. did.
  • T-cell receptor ⁇ -chain and ⁇ -chain whose tumor antigen specificity was confirmed for iPS cell clones derived from non-T non-B cells or monocytes with good differentiation efficiency into T cells obtained in Example 2 was introduced using the electroporation method.
  • iPS cells expressing the desired T-cell receptor ⁇ -chain and ⁇ -chain were isolated by a cell sorter using the expression of the marker molecule CD19 as an indicator.
  • 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 an FcDLL4-coated plate for about 21 days to induce differentiation into T cells.
  • the CD8 ⁇ -chain/ ⁇ -chain double-positive immature T cells obtained after the above 21 days of culture were isolated and purified using a cell sorter.
  • immature T cells were co-expressed in the presence of PHA (phytohemagglutinin) and peripheral blood mononuclear cells as feeder cells, in the presence of Retronectin (registered trademark) and anti-CD3 antibody, or in the presence of anti-CD3 antibody and anti-CD28 antibody. cultured and induced into mature cytotoxic T cells. These stimulations were performed one or more times. The performance of the obtained T cells was confirmed by GPC3-specific target cell cytotoxic activity, IFN- ⁇ production and antigen-binding ability.
  • TCR gene into mature T cells differentiated from iPS cell clones For mature T cells differentiated via hematopoietic stem cells and immature T cells from non-T non-B cells or monocyte-derived iPS cell clones with good differentiation efficiency into T cells obtained in Example 3, A gene (cDNA) encoding the GPC3 antigen-specific T-cell receptor ⁇ -chain ⁇ -chain was introduced in the same manner as in Example 4 using the piggyBac (registered trademark) system.
  • Fig. 8 shows the results of analyzing the phenotype of mature T cells derived from transfected iPS cells using a flow cytometer.
  • No EP is the mature T cell used for gene transfer, the analysis result of the mature T cell expressing the WT1 antigen-specific T cell receptor ⁇ chain ⁇ chain
  • EGFP is the analysis result of the mature T cells introduced with an expression vector incorporating a tracer gene (EGFP (enhanced green fluorescent protein) gene) as an indicator of gene introduction manipulation
  • "Empty-CD19” is an intracellular defect as a tracer gene.
  • Fig. 3 shows the analysis results of the mature T cells introduced with a piggyBac (registered trademark) transposon vector in which defective human CD19 gene is tandemly integrated.
  • TCR-CD19 In the iPS cell-derived mature T cells indicated by "TCR-CD19", in addition to the expression of the CD3 gene and the intracellularly defective human CD19 gene, which is a tracer gene incorporated in the piggyBac (registered trademark) transposon vector, GPC3 antigen-specific Binding to the GPC3 peptide/HLA complex (GPC3-Dex) recognized by the target T-cell receptor ⁇ -chain ⁇ -chain was detected.
  • GPC3 antigen-specific Binding to the GPC3 peptide/HLA complex (GPC3-Dex) recognized by the target T-cell receptor ⁇ -chain ⁇ -chain was detected.
  • GPC3 antigen-specific T cell receptor ⁇ -chain ⁇ -chain gene by introducing a GPC3 antigen-specific T cell receptor ⁇ -chain ⁇ -chain gene into iPS cell-derived mature T cells using the piggyBac (registered trademark) system, GPC3 antigen-specific T expressed on the cells
  • the cell receptor ⁇ -chain ⁇ -chain functions as a molecule that recognizes the GPC3 peptide/HLA complex (GPC3-Dex). Therefore, novel tumor-associated antigens (neoantigens) and other tumor-associated antigen-specific T-cell receptor ⁇ -chain ⁇ -chain genes can be used as T-cell receptor ⁇ -chain ⁇ -chain genes to be introduced into iPS cell-derived mature T cells. demonstrated that iPS cell-derived mature T cells that recognize these antigens can be generated.
  • FIG. 9 shows a method of 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 are isolated from the peripheral blood of a subject infected with EBV (Epstein-Barr virus), and the isolated mononuclear cells are stimulated with EBV antigen in vitro to generate a CD8-positive T cell population that recognizes the EBV antigen. Isolated.
  • EBV Epstein-Barr virus
  • iPS cell clones were isolated from the obtained iPS cell population, each clone was examined for its ability to differentiate into T cells, and iPS cell clones with high differentiation efficiency into T cells were selected.
  • T cells that recognize EBV antigens are cells that are less likely to produce allo-reactivity even when allografts are performed.
  • FIG. 10 shows that in the process of producing regenerated T cells from iPS cells reprogrammed from peripheral blood T cells, genome editing is performed on iPS cell clones selected as cells with high differentiation efficiency into T cells, and regenerated T cells are produced. A method of manufacturing cells is shown.
  • iPS cell clones derived from CD8-positive T cells that recognize EBV antigens and have high differentiation efficiency into T cells are ⁇ 2M gene and CIITA gene involved in the expression of MHC class I and MHC class II, natural killer (NK) cells
  • the PVR gene which is involved in the activation of T cell receptors, as well as the Rag2 gene, which is involved in rearrangement of the T cell receptor, were deleted using CRISPR/Cas9, while the inhibitory ligand of NK cells, ⁇ 2 ⁇ /binding peptide/HLA -E fusion gene (HLA-E * ) was expressed to make iPS cells immune to attack by host T cells and NK cells.
  • iPS cell-derived regenerated T cells may be preserved 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 from the host T cells.
  • the host T cells can be used as materials for producing regenerative T cells into which a TCR gene or CAR (chimeric antigen receptor) gene has been introduced. Since the host T cells are T cells that recognize EBV antigens, they are unlikely to cause an alloreaction even when transferred into the body. "Host T cells” are used as starting materials for the manufacture of cancer preventive or therapeutic agents containing regenerated T cells as active ingredients, although the cells themselves are not used for treatment of patients. means T cells.
  • FIG. 11 shows a method for producing regenerative T cells that recognize cancer antigens from the host T cells.
  • host T cells that recognize EBV antigen gene replacement by genome editing using CRISPR / Cas9, rearranged T cell receptor ⁇ chain that recognizes EBV antigen T cells that recognize cancer antigens 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. 12 summarizes the methods shown in FIGS.
  • Regenerated T cells produced from allogeneic universal iPS cells reprogrammed peripheral blood T cells (iPS-T cells) can be used as a starting material for producing T cells introduced with the TCR gene or CAR gene.
  • "Universal iPS cells” are very low immunogenicity, so they can be used in patients with any type of MHC (Major Histocompatibility Complex) without causing rejection. It means iPS cells that can be used. That is, iPS cells that can be administered to patients without consideration of MHC matching.
  • Universal iPS cells can be generated by knocking out MHC class I or MHC class II molecules and expressing ligands that suppress NK cells.
  • a viral vector used for producing T cells used in conventional T cell replacement therapy or regenerative therapy may be used to introduce the TCR gene or CAR gene.
  • iPS-T cells as a starting material, it becomes possible to produce desired T cells, which are active ingredients of cancer preventive or therapeutic agents, in a short period of time.
  • it is easy to introduce a TCR that recognizes a neoantigen and by introducing a plurality of TCR genes that recognize different neoantigens, it is possible to produce iPS-T cells while maintaining polyclonality.

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