WO2011096482A1 - Procédé de reconstruction du système immunitaire par utilisation de cellules souches pluripotentes - Google Patents

Procédé de reconstruction du système immunitaire par utilisation de cellules souches pluripotentes Download PDF

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WO2011096482A1
WO2011096482A1 PCT/JP2011/052260 JP2011052260W WO2011096482A1 WO 2011096482 A1 WO2011096482 A1 WO 2011096482A1 JP 2011052260 W JP2011052260 W JP 2011052260W WO 2011096482 A1 WO2011096482 A1 WO 2011096482A1
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cells
cell
human
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gene
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啓光 中内
新 金子
聡修 西村
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国立大学法人東京大学
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Priority to US13/565,558 priority patent/US9206394B2/en
Priority to US14/928,556 priority patent/US10787642B2/en
Priority to US16/991,758 priority patent/US20210032595A1/en

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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to a method for reconstructing immune function using pluripotent stem cells. More specifically, the present invention relates to a method for producing human T cells, a pharmaceutical composition containing T cells produced by the method, and immunity utilizing the method. It relates to a method of cell therapy.
  • Non-Patent Documents 1-2 CD34 positive cells, which are bone marrow progenitor cells, naive T lymphocytes, and the like are used as gene transfer cells, but these have low self-regenerative ability in Ex-vivo and low gene transfer efficiency. It has many problems such as difficulty in controlling differentiation by gene transfer.
  • ES cells embryonic stem cells
  • iPS cells inducible pluripotent stem cells
  • ES cells embryonic stem cells
  • iPS cells inducible pluripotent stem cells
  • IPS cells can be established by reprogramming somatic cells in various ways.
  • Reprogrammed iPS cells carry over the genetic information used for reprogramming as they are, so even those cells of the immune system, especially B lymphocytes and T lymphocytes, which have been rearranged and terminally differentiated are also reprogrammed. Whether it is possible was a subject of great interest. Under such circumstances, the establishment of iPS cells from mouse B lymphocytes was reported in 2008 by Jaenisch et al. (Non-patent Document 3), and the establishment of iPS cells from mouse T lymphocytes was reported in 2009 by Yamanaka et al. (Non-Patent Document 4). However, there is no report that iPS cells were established from human T lymphocytes.
  • T lymphocytes In order to realize immunotherapy using T lymphocytes, in addition to establishing iPS cells from human T lymphocytes, the TCR that the original human T lymphocytes had had established iPS cells. Although it is necessary to induce differentiation into functional T lymphocytes while maintaining the recombination structure of the gene, such a technique has not yet been established.
  • the present invention has been made in view of the above-described problems of the prior art, and its purpose is to establish iPS cells from human T lymphocytes, and to further establish the established iPS cells from the original human T lymphocytes.
  • An object of the present invention is to provide a method capable of inducing differentiation into functional T lymphocytes while maintaining the recombination structure of the TCR gene.
  • a further object of the present invention is to provide a pharmaceutical composition containing the T cells thus produced, and a method of immune cell therapy using the T cells thus produced.
  • the present inventors have succeeded in establishing iPS cells from human T cells while maintaining the same TCR reconstitution state as the original human T lymphocytes. did. Furthermore, the present inventors have functions of producing cytokines and the like from the thus established human T cell-derived iPS cells (TiPS cells) while maintaining the same TCR reconstitution state as the original human T lymphocytes. Succeeded in inducing differentiation of typical T cells. And it discovered that an ideal immune cell therapy could be performed by transplanting the human T cell obtained in this way into a patient's body, and came to complete this invention.
  • the present invention provides the following inventions.
  • a method for producing a human T cell comprising a step of inducing iPS cells from human T cells, and a step of differentiating the iPS cells into T cells.
  • the human T cell is a T cell expressing at least one molecule selected from the group consisting of CD4 and CD8.
  • the method according to (1) or (2), wherein the T cells induced into iPS cells have antigen specificity.
  • the method according to (3), wherein the antigen specificity of T cells induced into iPS cells and T cells differentiated from iPS cells are the same.
  • a pharmaceutical composition comprising the human T cell according to (5). (7) isolating T cells having a desired antigen specificity from humans; Inducing iPS cells from T cells having the desired antigen specificity; Differentiating the iPS cells into T cells; Administering the obtained iPS cell-derived T cells into a human body.
  • Lymphocytes that acquire structural changes in the TCR and BCR gene base sequences during development are extremely unique cells as the source of iPS cell establishment.
  • iPS cells with TCR reconstituted from mouse T cells and B cells can be established, suggesting that the TCR reconstitution mode is not lost by nuclear transfer or reprogramming. It had been.
  • mice these are all examples in mice.
  • the present invention shows for the first time that iPS cells can be obtained from human peripheral blood T lymphocytes, and further, when T lineage cells are derived from iPS cells having at least one in-frame TCR reconstruction, the TCR It has been revealed for the first time that human T-lineage cells having the above appear in a monoclonal (monoclonal) manner.
  • TCR T lineage cells from iPSCs (iPS cells) that have only one in-frame rearrangement with both the TCR ⁇ chain and ⁇ chain
  • the appeared TCR is identical to the base sequence conserved in iPS cells ( identity) and completely monoclonal.
  • identity base sequence conserved in iPS cells
  • CD34 positive hematopoietic stem cells that must always take into account the inhibitory effect (interfere) due to the expression of endogenous TCR ⁇ chain ⁇ chain (especially ⁇ chain incompletely controlled by allelic exclusion)
  • T cells having monoclonal TCR ⁇ against the target antigen can be induced as efficiently as compared with the method of introducing the TCR gene into naive T cells.
  • the present invention it is possible to efficiently produce human T lymphocytes specific to the target antigen.
  • human T lymphocytes thus produced, it is possible to provide an immune cell therapy method with few problems of rejection, which is the biggest barrier for regenerative medicine and transplantation medicine.
  • FIG. 1 shows the outline of preparation of the human iPS cell from a peripheral blood T lymphocyte.
  • “Tapering” indicates a period during which the tape was gradually replaced with human iPS cell medium (hiPS medium, etc.).
  • the upper part shows colonies obtained by introducing the 3/4 factor in the mountains into CD3 T cells, and the lower part shows, from the left
  • Day 6 cells reseeded on MEF cells
  • Day 15 ES cell-like colonies.
  • Formed cells Day 24: each cell that has completed colony formation (see FIG. 1 for Day).
  • the scale bar indicates 200 ⁇ m.
  • TkT3V1-7 human T-iPS cells
  • PB CD4 T-cell active CD4T lymphocytes
  • FIG. 7 is a scatter diagram showing a comparison result (right side) between TkT3V1-7 and human ES cells (KhES3) in FIG.
  • KhES3 human ES cells
  • two parallel lines in the figure indicate that there was a 5-fold difference between the corresponding samples.
  • 2 is a photomicrograph showing the karyotype of two typical examples of T-iPS cells (TkT3V1-7 and TkT4V1-3). The passage number of the examined T-iPS cells was 10, and no chromosomal abnormality was observed in any of them.
  • FIG. 6 is a photomicrograph showing a representative HE-stained section of teratoma formed in NOD-Scid mice 8 weeks after injection of T-iPS cells.
  • Tissues derived from the three germ layers (neural tissue and neural plate as ectoderm, retinocytes), cartilage and myocytes as mesoderm, and stomach as endoderm It shows the presence of gut-like mucosa and secretory glands / ducts.
  • the reconstituted TCRG was identified by a PCR band in the effective size range (about 200 bp). It is an electrophoresis photograph which shows the typical result of PCR analysis for detecting TCRB reconstruction.
  • FIG. 13 is a diagram showing a sequence result of a PCR product derived from TkT3V1-7 shown in FIGS. 11 and 12.
  • the functional (Productive) rearrangement in one allele of the TCRA gene is shown in the upper part.
  • the functional rearrangement (V (D) J ⁇ rearrangement) in one allele of the TCRB gene is shown in the middle.
  • Unproductive rearrangement (DJ ⁇ rearrangement) in the other allele of the TCRB gene is shown at the bottom.
  • CD3 + CD56 ⁇ peripheral blood T cells are first divided into a CD4 expression subset and a CD8 expression subset, and then na ⁇ ve T cells (Naive, CD45RA + CD62L + ), central memory T cells (Cemtral Memory, CD45RA ⁇ CD62L + ), effector memory T FIG.
  • FIG. 6 is a scatter diagram showing the results of classification into cells (Effect Memory, CD45RA ⁇ CD62L ⁇ ) or terminal effector T cells (Effector, CD45RA + CD62L ⁇ ). It is a graph which shows the result of having evaluated the reprogramming efficiency in each T cell subset based on the number of ALP + colonies obtained from the 1 * 10 ⁇ 5 > T cell subset replated on MEF. In the figure, the vertical axis represents the average number ⁇ S. Of ALP + colonies in three independent experiments. D. Indicates.
  • (A) is a figure which shows the outline of T lineage cell preparation from a pluripotent cell by culture
  • “Cytokine cocktail” indicates that hIL-7, hFlt-3L, and hSCF were added.
  • (B) is a scatter diagram showing the results of examining the expression of CD45, CD3, CD4, CD8, and TCR ⁇ using flow cytometry for T lineage cells prepared from each pluripotent cell.
  • ES indicates a T lineage cell derived from ES cells
  • skin iPS indicates a T lineage cell derived from skin iPS cells
  • T-iPS indicates a T lineage cell derived from T-iPS cells
  • PB peripheral blood (control data).
  • A is a diagram showing an outline of preparation of T lineage cells from pluripotent cells such as T-iPS cells by culturing on OP9 and OP9-DL1 stromal cell layers.
  • pluripotent cells were cultured on an irradiated OP9 cell layer for 10 to 14 days in an essential medium containing no cytokine and containing ⁇ -MEM, and then the induced hematopoietic cells were treated with OP9-DL1 cells.
  • FIG. 4 shows that the cells were cultured in an essential medium based on ⁇ MEM supplemented with hIL-7, hFlt-3L, hSCF and the like for 3 or 4 weeks.
  • B collects floating cells in the medium at each culture time point and examines the expression of T lineage markers CD45, CD34, CD3, CD4, CD8, TCR ⁇ using flow cytometry, and at least 3 independent It is a scatter diagram which shows the typical result in 1 experiment.
  • FIG. 3 is a scatter plot showing representative results in at least three independent experiments, in which the expression of T lineage cell markers was analyzed by flow cytometry.
  • the numerical values shown in each scatter diagram indicate the percentage (%) of cells in each category, “Human ESC” indicates the result in human ES cells (KhES3), and “CB-iPS” indicates umbilical cord blood CD34 positive.
  • FIG. 5 is a graph showing CD4 and CD8 expression in CD3 + T lineage cells as assessed by flow cytometry. In the figure, the vertical axis represents the relative distribution ⁇ S. D. Indicates.
  • “Human ESC” shows the result in human ES cells
  • “CB-iPSC” shows the result in cord blood CD34 positive cell-derived iPS cells
  • “HDF-iPSC” shows the results in human skin fibroblast-derived iPS cells
  • “T-iPSC” indicates the result in T-iPS cells.
  • the column of each cell indicates “DN”, “CD8”, “CD4”, “DP” in order from the left. Further, “DN” indicates CD4 ⁇ CD8 ⁇ , “CD4” indicates CD4 + CD8 ⁇ , “CD8” indicates CD4 ⁇ CD8 + , and “DP” indicates CD4 + CD8 ⁇ . It shows that it is + .
  • FIG. 5 is an electrophoresis photograph showing the results of analyzing the PCR product obtained by amplifying the TCR of TkT3V1-7 by PCR through construction of a cDNA library by SMART.
  • FIG. 25 is a diagram showing the results of sequencing analysis for PCR products in the effective size range (about 750 bp) shown in FIG.
  • FIG. 4 is a diagram showing that no progeny from the DJ ⁇ reconstructed allele was confirmed. Details and results of other samples are shown in Tables 8 and 9. It is a graph which shows the result of having evaluated the expression of CD3 by flow cytometry. In the figure, the vertical axis represents the ratio of CD3 + T lineage cells in CD45 + blood cells (Averages ⁇ SD of independent experiments of 3 or more in each group).
  • “Human ESC” indicates the result in human ES cells
  • “CB-iPSC” indicates the result in cord blood CD34 positive cell-derived iPS cells
  • “HDF-iPSC” indicates the results in human skin fibroblast-derived iPS cells
  • “T-iPSC” indicates the result in T-iPS cells.
  • ** indicates p ⁇ 0.01 for all other samples. It is a graph which shows the average fluorescence intensity (MFI, Mean fluorescence intensity) of CD3 evaluated by flow cytometry. The vertical axis represents CDs / iPS and HDF-iPS for each group 2, and others are averages ⁇ S.
  • “Human ESC” indicates the result in human ES cells
  • “CB-iPSC” indicates the result in cord blood CD34 positive cell-derived iPS cells
  • “HDF-iPSC” indicates human skin fibroblast-derived iPS cells.
  • T-iPSC shows the result in T-iPS cells
  • PB shows the result in peripheral blood mononuclear cells. It is the schematic which shows the arrangement
  • the human TCRG locus spans 135 kb on chromosome 7q14.
  • the forward primer and the reverse primer are each designed in a region having high homology in the V ⁇ segment or the J ⁇ segment, and the reconfiguration of the TCRG gene can be detected by detecting a PCR band of about 200 bp.
  • the human TCRB locus extends over 685 kb on chromosome 7q34.
  • 23 V ⁇ primers, 2 D ⁇ primers, and 13 J ⁇ primers can be annealed to the same conserved region. Designed.
  • the primer was divided into three tubes, each mixed and used for analysis.
  • the present invention provides a method for producing human T cells, comprising the steps of inducing iPS cells from human T cells and differentiating the iPS cells into T cells.
  • the “human” from which T cells are isolated is not particularly limited. Even a healthy person may be a person who has a reduced immune function or a person suffering from a malignant tumor, an infectious disease, an autoimmune disease or the like.
  • the human from whom T cells obtained by the present invention are administered is compared with the human to which the T cells obtained by the present invention are administered.
  • T cell means a cell expressing an antigen receptor called T cell receptor (TCR) on the surface.
  • TCR T cell receptor
  • human T cells induced into iPS cells and “human T cells differentiated from iPS cells” are not particularly limited, but preferably express CD3 and are selected from the group consisting of CD4 and CD8. T cells expressing at least one selected molecule.
  • human T cells include helper / regulatory T cells that are CD4 positive cells, cytotoxic T cells that are CD8 positive cells, naive T cells (CD45RA + CD62L + cells), central memory T cells ( CD45RA ⁇ CD62L + cells), effector memory T cells (CD45RA ⁇ CD62L ⁇ cells), and terminal effector T cells (CD45RA + CD62L ⁇ cells).
  • human T cells differentiated from iPS cells have the same antigen specificity as human T cells induced into iPS cells.
  • antigen specificity in T cells is provided by antigen-specific, rearranged TCR genes.
  • human T cells induced into iPS cells can be isolated from human tissues by a known technique.
  • the human tissue is not particularly limited as long as it is a tissue containing the T cell, and examples thereof include peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and lesioned tissue.
  • peripheral blood and umbilical cord blood are preferable from the viewpoint of low invasiveness to humans and easy preparation.
  • a known technique for isolating human T cells includes, for example, flow cytometry using an antibody against a cell surface marker such as CD4 and a cell sorter as shown in the Examples described later.
  • desired T cells can be isolated using cytokine secretion or functional molecule expression as an index.
  • T cells have different cytokines secreted depending on Th1 type or Th2 type. Therefore, T cells having a desired Th type can be isolated by selecting such cytokines as indicators. .
  • cytotoxic (killer) T cells can be isolated using secretion or production of granzyme or perforin as an index.
  • a desired tissue is obtained from a human tissue containing “T cells having desired antigen specificity”. A method of purification using an affinity column or the like on which an antigen is immobilized can be employed.
  • MHC tetramer major histocompatibility gene complex
  • T a desired antigen specificity from a human tissue.
  • a method of purifying “cells” can also be employed.
  • the “iPS cell” in the present invention is a cell also called an induced pluripotent stem cell or an inducible pluripotent stem cell, and is induced by introducing a cell reprogramming factor into the T cell. can do.
  • the “cell reprogramming factor” is a factor that can impart differentiation pluripotency to a somatic cell by being introduced into the T cell alone or in cooperation with other differentiation pluripotency factors.
  • Oct3 / 4 c-Myc, Sox2, Klf4, Klf5, LIN28, Nanog, ECAT1, ESG1, Fbx15, Eras, ECAT7, ECAT8, Gdf3, Sox15, ECAT15-1, ECAT15-2, It is preferably at least one protein selected from the group consisting of Fthl17, Sal14, Rex1, Utf1, Tcl1, Stella, ⁇ -catenin, Stat3 and Grb2. Furthermore, among these proteins, it is more preferable to introduce Oct3 / 4, c-Myc, Sox2 and Klf4 (4 factors) into the somatic cells from the viewpoint that iPS cells can be established efficiently with few factors. Further, from the viewpoint of reducing the risk of canceration of the resulting pluripotent stem cells, it is more preferable to introduce Oct3 / 4, Sox2 and Klf4 (3 factors) excluding c-Myc into the somatic cells.
  • OCT4, SOX2, KLF4, C-MYC, and NANOG are introduced into human CD8 positive T cells from the viewpoint of further increasing the efficiency of induction into iPS cells.
  • OCT4, SOX2, KLF4, C-MYC, NANOG, and LIN28 are more preferably introduced into human CD8-positive T cells.
  • the method of “introducing a cell reprogramming factor into T cells” is not particularly limited, and a known method can be appropriately selected and used.
  • the nucleic acid (eg, cDNA) encoding the cell reprogramming factor contains a promoter that functions in the T cell. And can be introduced into cells by infection, lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, electroporation method .
  • Examples of the “expression vector” according to the present invention include viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, and animal cell expression plasmid.
  • the promoter used in such an expression vector examples include SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter and the like.
  • a promoter may be one that can control the expression of a gene inserted downstream of the promoter depending on the presence or absence of a drug (eg, tetracycline).
  • the expression vector may further contain an enhancer, a poly A addition signal, a selection marker gene (for example, neomycin resistance gene), an SV40 replication origin, and the like.
  • the T cells are treated with interleukin-2 (IL-2) before the introduction of the cell reprogramming factor.
  • IL-2 interleukin-2
  • Such stimulation can be performed, for example, by adding IL-2, anti-CD3 antibody and anti-CD28 antibody to the medium and culturing the T cells for a certain period of time, as shown in Examples described later.
  • the anti-CD3 antibody and the anti-CD28 antibody may be ones to which magnetic beads or the like are bound, and instead of adding these antibodies to the medium, the anti-CD3 antibody and the anti-CD28 antibody are bound to the surface.
  • Stimulation may be given by culturing the T cells for a certain period on a cultured dish.
  • stimulation may be given by adding an antigen peptide recognized by human T cells to a medium together with feeder cells.
  • the concentration of IL-2 added to the medium is not particularly limited, but is preferably 1 to 200 ng / ml.
  • the concentration of the anti-CD3 antibody and anti-CD28 antibody added to the medium for such stimulation is not particularly limited, but it is preferably 1 to 10 times the culture amount of the T cells.
  • the concentration of anti-CD3 antibody and anti-CD28 antibody bound on the surface of the culture dish in such stimulation is not particularly limited, but the concentration at the time of coating is 1 to 100 ⁇ g / ml for anti-CD3 antibody, and anti-CD28 antibody. In this case, it is preferably 0.1 to 10 ⁇ g / ml.
  • the culture period for performing such stimulation is a period sufficient to give such stimulation to the T cells, and the T cells can be expanded to the number of cells necessary for introduction of the cell reprogramming factor.
  • the period is not particularly limited, but is usually 2 to 14 days, and preferably 2 to 7 days from the viewpoint of gene transfer efficiency. Furthermore, from the viewpoint of increasing gene transfer efficiency, it is preferable to culture on a culture dish coated with retronectin.
  • Examples of a medium for culturing the T cell and adding IL-2, anti-CD3 antibody, anti-CD28 antibody, etc. include, for example, a known medium suitable for culturing the T cell (more specifically, IL-2 etc. Roswell Park Memorial Institute (RPMI) 1640 medium, minimal essential medium ( ⁇ -MEM), Dulbecco's modified Eagle medium (DMEM), F12 medium, etc.) containing the above-mentioned cytokines, fetal calf serum (FCS) .
  • the medium may contain amino acids necessary for culture (eg, L-glutamine) and antibiotics (eg, streptomycin, penicillin). .
  • IL-7 or IL-15 When CD8 positive T cells are induced into iPS cells, it is preferable to add IL-7 or IL-15 to the medium from the viewpoint of suppressing apoptosis. There are no particular restrictions on the concentration of IL-7 or IL-15 added, but it is preferably 1 to 100 ng / ml.
  • the T cell into which the cell reprogramming factor has been introduced is cultured on a feeder cell layer. It is preferable to do this.
  • a feeder cell For example, a mouse embryo fibroblast (MEF), STO cell, and SNL cell which stopped cell division by irradiation of radiation or antibiotic treatment are mentioned.
  • the cell reprogramming factor when introducing the cell reprogramming factor into the T cells with basic fibroblast growth factor (bFGF), or Thereafter, it is preferably added to the medium.
  • bFGF basic fibroblast growth factor
  • HDAC histone deacetylase
  • VPA valproic acid
  • trichostatin A sodium butyrate
  • MC 1293, M344 and other small molecule inhibitors siRNA against HDAC
  • G9a histone methyltransferase inhibitors small molecule inhibitors such as BIX-01294, siRNA against G9a, etc.
  • p53 inhibitors small molecule inhibitors such as Pifithrin- ⁇ (PFT- ⁇ ), siRNA against p53, etc. Is preferably added to the medium at the time of introducing the cell reprogramming factor into the T cells or thereafter.
  • protamine sulfate should be added to the medium in order to facilitate the binding of the vector to the T cells. Is preferred.
  • a known medium suitable for culturing the T cells is gradually replaced with a medium suitable for culturing iPS cells. It is preferable to culture while continuing.
  • a medium suitable for culturing such iPS cells a known medium can be appropriately selected and used.
  • knockout serum substitute, L-glutamine, non-essential amino acid, 2-mercaptoethanol, b-FGF and the like can be used. Examples thereof include Dulbecco's modified Eagle medium / F12 medium (human iPS cell medium).
  • 1-1000 ⁇ M Cultivation is preferably performed under low oxygen concentration conditions (oxygen concentration: for example, 5%) in a medium further supplemented with ROCK inhibitor, and 1 to 1000 ⁇ M MEK inhibitor (for example, PD0325901) and 1 to 1000 ⁇ M GSK3 inhibitor (for example, , CHIR99021) is preferably added to the medium until colony formation described below.
  • TiPS cells selection of iPS cells derived from the T cells (hereinafter also referred to as “TiPS cells”) can be performed by appropriately selecting a known technique.
  • known methods include, for example, a method of observing and selecting the morphology of ES cells / iPS cell-like colonies as shown in the examples described later under a microscope, or knowing that they are specifically expressed in iPS cells.
  • Recombinant T cells targeting drug resistance gene or reporter gene (GFP gene, etc.) at the gene locus of the gene are selected using drug resistance or reporter activity as an index.
  • GFP gene drug resistance gene, etc.
  • iPS cells can be confirmed by, for example, undifferentiated cell-specific markers (ALP, SSEA-4, Tra- 1-60, Tra-1-81, etc.) can be detected by immunostaining, RT-PCR, or the like, or selected cells can be transplanted into mice and their teratoma formation observed. .
  • ALP undifferentiated cell-specific markers
  • SSEA-4 Tra- 1-60, Tra-1-81, etc.
  • RT-PCR or the like
  • the confirmation that the cells selected in this way are derived from the T cells can be performed, for example, by detecting the state of TCR gene rearrangement by genomic PCR, as shown in Examples below. it can.
  • the time for selecting and recovering these cells can be appropriately determined while observing the growth state of the colonies, and is generally 14 to 28 days after the introduction of the cell reprogramming factor into the T cells. .
  • the TiPS cells are allowed to contain cytokines on the stromal cells.
  • the stromal cells to be used are preferably OP9 cells and 10T1 / 2 cells subjected to treatment such as irradiation from the viewpoint of facilitating differentiation into hematopoietic system.
  • a medium containing at least one cytokine selected from the VEGF, SCF, TPO, SCF, and FLT3L groups it is preferable to culture in a medium containing at least one cytokine selected from the VEGF, SCF, TPO, SCF, and FLT3L groups. It is more preferable to culture in a medium containing VEGF, SCF and TPO, or in a medium containing VEGF, SCF and FLT3L.
  • the culture period is preferably a period until the formation of a bag-like structure (also referred to as ES-sac (sac)) containing blood cells and the like, and 10 to 14 after the start of cultivation of TiPS cells. Preferably it is days.
  • Cells contained in the sac obtained as described above should be cultured on stromal cells in ⁇ -MEM medium containing cytokines, FCS, etc. Is preferred.
  • the cell which exists in the inside of this sac-like structure can be isolate
  • the stromal cells used for this culture are OP9-DL1 cells, OP9-DL4 cells, 10T1 / 2/10 treated with radiation and the like from the viewpoint of inducing differentiation into T lymphocytes via a notch signal. DL4 cells are preferred.
  • cytokines added to the medium examples include IL-7, FLT3L, VEGF, SCF, TPO, IL-2, and IL-15.
  • IL-7 and FLT3L are preferable from the viewpoint of helping differentiation of early T cells.
  • SCF is preferably not contained in the medium.
  • the concentration of IL-7 added to the medium is 0 from the viewpoint that CD3-positive CD56-negative T lineage cells can be easily obtained and differentiation into CD4 single-positive (SP) T cells or CD8SPT cells is easily induced. It is preferably 1 to 4 ng / ml.
  • T cells thus obtained can be isolated by appropriately selecting a known technique.
  • a known technique for example, flow cytometry using an antibody against a cell surface marker such as CD4 and a cell sorter as shown in the Examples described later can be mentioned.
  • a method of purification using an affinity column or the like on which a desired antigen is immobilized can be employed.
  • a method of purifying “T cells having desired antigen specificity” using an MHC tetramer to which a desired antigen is bound may be employed.
  • Human T cell, pharmaceutical composition, immune cell therapy method Since human T cells produced by the method of the present invention have an antigen-specific immune function, they can be used, for example, for the treatment or prevention of diseases such as tumors, infectious diseases, and autoimmune disorders.
  • the present invention provides a human T cell produced by the method of the present invention, a pharmaceutical composition containing the human T cell, and a method of immune cell therapy using the human T cell.
  • the pharmaceutical composition of the present invention can be prepared by formulating human T cells produced by the method of the present invention by a known pharmaceutical method.
  • it can be used mainly parenterally as capsules, liquids, film coating agents, suspensions, emulsions, injections (intravenous injections, infusions, etc.).
  • a pharmacologically acceptable carrier or medium specifically, sterilized water or physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, vehicle, Combined with preservatives, binders, diluents, tonicity agents, soothing agents, extenders, disintegrating agents, buffering agents, coating agents, lubricants, colorants, solubilizers or other additives as appropriate.
  • a pharmacologically acceptable carrier or medium specifically, sterilized water or physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, vehicle, Combined with preservatives, binders, diluents, tonicity agents, soothing agents, extenders, disintegrating agents, buffering agents, coating agents, lubricants, colorants, solubilizers or other additives as appropriate.
  • the dosage is appropriately selected according to the age, weight, symptom, health condition of the subject, the type of composition (pharmaceutical, food and drink, etc.) and the like.
  • the product (pharmaceutical product) of the composition of the present invention or the instructions thereof may have a label indicating that it is used for treating or preventing a decrease in immune function.
  • label indicating that it is used for treating or preventing a decrease in immune function means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to.
  • the method of immune cell therapy of the present invention comprises a step of isolating T cells having a desired antigen specificity from a human, a step of inducing iPS cells from T cells having the desired antigen specificity, and the iPS cells.
  • the method comprises the step of differentiating T cells into T cells and the step of administering the obtained iPS cell-derived T cells into the human body.
  • the human from whom the T cells obtained by the present invention are administered has the same type of HLA as the human from whom the T cells are isolated. It is preferable that the T cell obtained by the present invention is the same person as the human being administered.
  • human T cells to be administered human T cells produced by the method of the present invention may be administered as they are, or may be administered in the form of a pharmaceutical composition formulated as described above.
  • Flow cytometry> The flow cytometry analysis was performed using MoFlo (manufactured by Dako Cytomation), FACSAria (registered trademark, manufactured by BD Bioscience), or FACSCanto II (registered trademark, manufactured by BD Bioscience). Analysis of the obtained data was performed using FlowJo software (manufactured by Treestar). Moreover, the antibody used for the flow cytometry analysis is as follows.
  • Anti-human CD3-APC antibody (manufactured by BD Bioscience), anti-human CD4-FITC antibody (manufactured by BD Bioscience), anti-human CD8-PerCP / Cy5.5 antibody (manufactured by BD Bioscience), anti-human CD56-PE antibody ( BD Bioscience), anti-human CD45RA-Pacific Blue antibody (Caltag Laboratories), anti-human CD62L-PE-Cy7 antibody (Biolegend), anti-human CD45-Alexa 405 antibody (Molecular Probes-Invitrogen-Invitrogen-Invitrogen) USA), anti-human CD34-PE antibody (BD Bioscience), anti-human CD38-PerCP / Cy5.5 antibody (Biolegend), Human CD1a-APC antibody (manufactured by Biolegend), anti-human CD5-PE / Cy7, -CD7-FITC antibody (manufactured by Biolegend), anti-human CD5-PE / Cy7 antibody (manufactured by Biolegend), anti
  • PBMCs Peripheral blood mononuclear cells
  • Ficoll density gradient centrifugation Ficoll-Paque PLUS (registered trademark), 17-1440-02, manufactured by GE Healthcare
  • MoFlo The product was purified by DAKO Cytomation.
  • T cells were isolated using the CD3 + CD56 ⁇ cell population as a gate to avoid natural killer T (NKT) cell contamination.
  • a subset of T cells set an additional gate and segregated into the CD4 (CD4 + CD8 ⁇ ) and CD8 (CD4 ⁇ CD8 + ) cohorts.
  • CD4 and / or CD8 cells were further classified as naive (CD45RA + CD62L + ), central memory (CD45RA ⁇ CD62L + ), effector memory (CD45RA ⁇ CD62L ⁇ ), or terminal effector (CD45RA + CD62L ⁇ ).
  • the cells thus sorted were 10% fetal bovine serum (GIBCO-Invitrogen), 100 U / ml penicillin, 100 ng / ml streptomycin, 2 mM L-glutamine, and 20 ng / ml human interleukin 2 (hIL-2,
  • the cells were first cultured in Roswell Park Memorial Institute Medium (RPMI) 1640 medium (GIBCO-Invitrogen) supplemented with Novartis Vaccines & Diagnostics. Then, these cells were activated by adding anti-CD3 / CD28-bound magnetic beads (Dynabeads (registered trademark), ClinExVivo (registered trademark) CD3 / CD28, manufactured by Invitrogen) three times the amount of such T cells.
  • T-iPS cells iPS cells derived from T cells
  • this activated day is defined as day 0 (hereinafter, see FIG. 1).
  • CD3 + cells magnetically captured from PBMCs were isolated and stimulated with anti-CD3 / CD28 coupled magnetic beads.
  • day 2 and day 3 cells were retrovirused by spinoculation on a retronectin (registered trademark) coating plate (manufactured by Takara) supplemented with 10 ⁇ g / ml protamine sulfate (manufactured by Sigma-Aldrich).
  • Kaneko S. et al., Blood, 2009, 113, 1006-1015
  • the complete synthetic medium for T cell culture was changed daily.
  • infected cells were harvested and transferred onto 3 ⁇ 10 5 irradiated MEF cell layers on 6 cm dishes. Thereafter, for 4 days (day 6 to day 10), half of the medium was replaced with human iPS cell medium every day.
  • the composition of the human iPS cell culture medium is as follows (see Takayama, N. et al., Blood, 2008, 111, 5298-5306).
  • VPA HDAC inhibitor valproic acid
  • Chromosome G band analysis was outsourced to the Japan Institute of Genetics and performed according to a predetermined method.
  • ALP staining human ES / iPS cell-like colonies were fixed with ice-cold fixative (90% methanol, 10% formaldehyde), and ALP staining kit (Vector Laboratories) was used, following the manufacturer's instructions. Stained.
  • immunocytochemical staining human ES / iPS cell-like colonies were fixed with 5% paraformaldehyde and permeabilized with 0.1% Triton X-100.
  • the colonies pretreated in this way were incubated with the primary antibody.
  • the primary antibodies used and the dilution ratios are as follows. PE-conjugated anti-SSEA-4 (FAB1435P, manufactured by R & D Systems, dilution ratio: 1/50), anti-Tra-1-60 (MAB4360, manufactured by Millipore, dilution ratio: 1/100), anti-Tra- 1-81 (MAB4381, manufactured by Millipore, dilution ratio: 1/100). Further, for the detection of Tra-1-60 and Tra-1-81, Alexa Fluor 488-conjugate goat anti-mouse antibody (dilution ratio: 1/500, A11029, manufactured by Molecular Probes-Invitrogen) is used as a secondary antibody. Using.
  • PCR was performed using ExTaq HS (manufactured by Takara) at 30 cycles for housekeeping genes (GAPDH or ACTB) and 35 cycles for all pluripotency genes or T cell-related genes.
  • GPDH or ACTB housekeeping genes
  • T cell-related genes The target genes and the sequences of the PCR primers used for the analysis are shown in Tables 1 and 2.
  • RNA Human genome cells (KhES3), T cell-derived iPS cells (TkT3V1-7), and CD3 / CD28-stimulated CD3 + CD4 + CD8 ⁇ cells were subjected to expression analysis of whole genome genes.
  • total RNA (2 ⁇ g) was extracted from each cell and subjected to probe preparation.
  • the fluorescence-labeled complementary RNA and Whole Human Genome Microarray 4 ⁇ 44K (G4112F, manufactured by Agilent Technologies) were hybridized by a one-color method (contractor: Takara Bio Inc).
  • the array was scanned, and the spot image was detected with an Agilent Feature Extraction apparatus (manufactured by Agilent Technologies).
  • the obtained signal data was analyzed using GeneSpring software (manufactured by Agilent Technologies). Two normalization procedures were applied to normalize the obtained data. That is, the signal intensity less than 0.01 was set to 0.01. In addition, each chip was normalized to the 50th percentile of the measured value obtained from the chip. Then, in all three samples, analysis was performed on the gene (36275 gene) with the present flag.
  • Genomic DNA was extracted from about 5 ⁇ 10 6 T-iPS cells using a QIAamp DNA kit (Qiagen). The extracted DNA (40 ng) was used to detect each of TCRG, TCRB, and TCRA gene rearrangement by PCR.
  • PCR (Benhattar, J. et al., Diamol Mol Pathol, 1995, vol. 4, pages 108-112) for detecting TCRG reconstitution was performed using ExTaq HS (Takara).
  • the PCR reaction conditions were 95 ° C for 5 minutes, followed by 5 cycles of reactions with relatively high annealing temperatures, ie, 95 ° C for 45 seconds, 65 ° C for 45 seconds, 72 ° C for 45 seconds, and A reaction having a relatively low annealing temperature, that is, a reaction at 95 ° C. for 45 seconds, 55 ° C. for 45 seconds, and 72 ° C. for 30 seconds was defined as 30 cycles.
  • PCR and heteroduplex analysis are in accordance with the BIOMED-2 protocol (van Dongen, J. Leukemia, 2003, Vol. 17, pp. 2257-2317) with minor modifications. I went.
  • Primer constructs for detecting TCRA reconstitution are Han et al. (See Han, M. et al., J Immunol, 1999, 163, pages 301-311).
  • PCR reaction conditions were 95 ° C for 30 seconds, 68 ° C for 45 seconds, 72 ° C for 6 minutes, 3 cycles of amplification reaction, 95 ° C for 30 seconds, 62 ° C for 45 seconds, and 72 ° C.
  • the amplification reaction of 6 minutes was 15 cycles, 95 ° C for 15 seconds, 62 ° C for 30 seconds, and 72 ° C for 6 minutes for 12 cycles.
  • PCR was performed by LATaq HS (manufactured by Takara).
  • the obtained PCR product was fractionated by gel based on size. Next, main bands within the expected size range were cut out, purified using a QIAquick gel extraction kit (manufactured by Qiagen), and sequenced.
  • sequence reaction PCR amplification and fluorescent dye addition were performed in a multifaceted approach using BigDye terminator kit v3.1 (Applied Biosystems) with V ⁇ , J ⁇ , V ⁇ , D ⁇ , or J ⁇ primer set. .
  • the obtained reaction product was analyzed with an ABI PRISM 3100 automatic sequencer (Applied Biosystems).
  • the results obtained are related to the construction of TCRA or TCRB.
  • the V, D, and J segments are the published sequences and the ImMunoGeneTics (IMGT) database (http://www.cines.fr/). Compared and identified by using a web tool such as v-quest (Lefranc, MP “IMGT databases, web resources and tools for immunoglobulin and T cell receptor sequence./ref. Leukemia, 2003, Vol. 17, pages 260-266). Moreover, the naming of gene fragments (segments) was in accordance with the IMGT nomenclature. The primer sequences used for detection of TCR reconstruction are shown in Tables 3-4.
  • T-iPS cells ⁇ Induction from T-iPS cells to T-lineage cells> Takayama, N., which has been subjected to slight modifications so that T-iPS cells or other pluripotent stem cells have an embryonic stem cell-derived sac (ES-sac) -like structure.
  • ES-sac embryonic stem cell-derived sac
  • Pluripotent cells are on an irradiated OP9 cell layer and contain no ⁇ -MEM based medium (20% fetal bovine serum, 100 U / ml penicillin, 100 ng / ml streptomycin, and 2 mM L-glutamine). ⁇ MEM) was co-cultured for 10 to 14 days (about Day 12).
  • Suspension cells packed in the sac were transferred onto the OP9-DL1 cell layer, and co-cultured to Day 40 in a medium based on ⁇ MEM and supplemented with 10 ng / ml hIL-7 and hFlt-3L.
  • the day when the pluripotent cells are transferred onto the OP9 cell layer is referred to as Day0.
  • the culture medium was changed every 3 days.
  • T lineage cells floating on the OP9-DL1 cell layer and expressing CD45, CD3, and TCR ⁇ were sorted weekly by flow cytometry and analyzed for gene expression.
  • OP9 cells and OP9-DL1 cells were obtained from the RIKEN BioResource Center through the National Bioresource Project (Japan).
  • cytokine production (expression) profile of T lineage cells was modified by Uckert, W. et al. Et al., Cancer Immunol Immunother, 2008, 58, pages 809-822. That is, 1 ⁇ 10 5 floating cells were converted into 10 ng / ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich), 0.4 ⁇ M A23187 calcium ionophore (ionomycin, Sigma-Aldrich) ) And 20 IU / ml hIL-1 ⁇ (Peprotech) for 12 hours. Furthermore, in the last 3 hours, the protein secretion inhibitor brefeldin A (1 mM, manufactured by Sigma-Aldrich) was added and cultured.
  • Such stimulated cells were labeled with anti-CD3 binding magnetic microbeads (Miltenyi Biotec) and held in a magnetic column.
  • an intracellular staining kit Inside stain kit, manufactured by Miltenyi Biotec
  • solid-cell intracellular staining solid-state intracellular cytostaining
  • SMART method a method based on a switching mechanism at the 5 ′ end of the reverse transcript (switch mechanism at the 5′-end of the reverse transcript)
  • switch mechanism at the 5′-end of the reverse transcript
  • SMART method a method based on a switching mechanism at the 5 ′ end of the reverse transcript (switch mechanism at the 5′-end of the reverse transcript)
  • SMART method Du, G. et al., J Immunol Methods, 2006, 308, 19-
  • cDNA was synthesized according to the manufacturer's instructions. That is, using 3 ′ SMART (registered trademark) CDS primer and SMART II A oligo (Super SMART (registered trademark) cDNA synthesis kit) and PrimeScript Reverse Transscriptase (produced by Takara Co., Ltd. at 90 ° C.
  • PCR reaction conditions were 20 cycles of 95 ° C. for 5 seconds, 65 ° C. for 5 seconds, and 68 ° C. for 3 minutes.
  • the amplified cDNA was used as a template in a TCRA-specific amplification reaction or a TCRB-specific amplification reaction. Specifically, amplification was performed by using 25 cycles of reaction of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 3 minutes using a forward primer (2nd 5′-SMART) and a reverse primer. As the reverse primer, 3'-TRAC was used for TCRA amplification and 3'-TRBC was used for TCRB amplification. The obtained PCR product was inserted into a pGEM-T-Easy vector (Promega, made by Madison) and sequenced.
  • iPS cells were established using T cells in human peripheral blood as shown in FIG. That is, peripheral blood mononuclear cells (PBMC) or CD3-positive cells are isolated from the peripheral blood of a healthy person (age (24 to 56 years old) and a plurality of healthy persons with different genders) and stimulated with CD3 / CD28 ClinExVivo beads. did.
  • PBMC peripheral blood mononuclear cells
  • CD3-positive cells are isolated from the peripheral blood of a healthy person (age (24 to 56 years old) and a plurality of healthy persons with different genders) and stimulated with CD3 / CD28 ClinExVivo beads. did.
  • iPS medium human iPS cell culture medium
  • the T cell medium was completely replaced with the iPS medium 4 days after seeding.
  • a cobblestone-like cell cluster was observed around 11 days after dissemination (5 days after seeding), and after several days (18-24 days after the start of culture), a human ES cell-like appearance was exhibited (FIG. 2). : See colony (photo of human ES cell-like colony).
  • CD3-derived pluripotent cells human ES cells (KhES3) and peripheral blood T cells were compared by microarray analysis.
  • the overall pattern of gene expression in CD3-derived iPS cells was similar to that in human ES cells, but different from that in T cells (see FIG. 6).
  • iPS cells established from human peripheral blood T cells have pluripotency.
  • Example 2 In order to confirm that the established iPS cells were derived from T cells, reconstitution of the TCR gene was confirmed. Three groups of TCR ⁇ , TCR ⁇ , and TCR ⁇ start reconstitution from an early stage (almost in this order). Reconstitution of the TCR ⁇ gene was observed in 2 out of 4 clones derived from 4 factors and 6 out of 9 clones derived from 3 factors (see FIG. 9: electrophoresis).
  • Example 3 Further, as in Example 2, in order to confirm that the established iPS cells were derived from T cells, reconstitution of the TCR gene was confirmed. That is, humans have four TCR genes (TCRA, TCRB, TCRG, and TCRD), and these DNA rearrangements are known to be involved in the development of normal T lymphocytes in the thymus. Yes. In addition, TCR gene rearrangement is an irreversible genetic phenomenon specific to T lineage cells, and this genetic phenomenon gives T cells a genetic signature and is characterized. Therefore, by examining these traces, it can be retrospectively confirmed whether the iPS cells obtained in Example 1 are derived from mature peripheral T lymphocytes of a healthy donor.
  • TCRA TCRA
  • TCRB TCRB
  • TCRG TCRG
  • TCRD TCRD
  • TCRG or TCRB rearrangement was detected by PCR analysis using a TCR primer set designed by Benhatter et al. And BIOMED-2 Consortium (see FIGS. 28 and 29, Benhattar, J. et al., Diamol Mol Pathol, 1995). 4, pp. 108-112, van Dongen, JJ, Leukemia, 2003, 17, pp. 2257-2317).
  • TCRG or TCRB reconstitution was identified in all examined iPS cell lines, and it was confirmed that all examined iPS cell lines were derived from peripheral blood T lymphocytes (FIGS. 10 and 11). .
  • the TCR ⁇ chain forms a heterodimer with the TCR ⁇ chain, and most peripheral blood T cells express TCR ⁇ (Davis, MM et al., Nature, 1998, 334, 395- (See page 402).
  • the TCRA locus is complex.
  • the TCRA locus is a region spanning over 1000 kb, not only containing the TCRD locus, but also including 103 V ⁇ segments, 61 J ⁇ segments, and C ⁇ segments. Therefore, in order to detect TCRA gene rearrangement in genomic DNA, a set of 34 forward primers for the V ⁇ segment and 12 reverse primers for the J ⁇ segment was designed.
  • TCR binding to the HLA-peptide complex is inevitably determined by the three-dimensional structure of the antigen recognition site consisting of three types of complementarity determining regions (CDR1, 2, and 3).
  • CDR3 is the most dispersible because it spans the V (D) J binding region into which various random nucleotides (N-nucleotides or P-nucleotides) are inserted.
  • D V
  • Peripheral T lymphocytes are mainly derived from two functional subsets: CD4 + helper / regulatory cells and CD8 + cytotoxic cells.
  • CD4 + helper / regulatory cells CD4 + helper / regulatory cells
  • CD8 + cytotoxic cells CD4 + helper / regulatory cells
  • peripheral T lymphocytes are divided into CD4 + T cells and CD8 + T cells and reprogrammed into iPS cells. Is there a difference in the efficiency of reprogramming into iPS cells depending on the difference in the derived T cells? I checked. The induction conditions for each cell are shown below.
  • CD4-positive T cells A CD3-positive CD56-negative CD4-positive T cell fraction (hereinafter referred to as CD4-positive T cells) of peripheral blood mononuclear cells obtained by blood collection is collected at a flow site, and anti-CD3 antibody and anti-CD28 antibody (bead binding, solidification). , Regardless of the addition to the medium). About 20 ng / ml of human IL-2 was added to the medium, and Yamanaka factor gene was introduced multiple times on a 24-well plate coated with retronectin 48 to 96 hours after stimulation. The Yamanaka factors used at that time are OCT4, SOX2, KLF4, and C-MYC, or OCT4, SOX2, and KLF4.
  • CD8-positive T cell A CD3-positive CD56-negative CD8-positive T cell fraction (hereinafter referred to as CD8-positive T cell) of peripheral blood mononuclear cells obtained by blood collection is collected at a flow site, and anti-CD3 antibody and anti-CD28 antibody (bead binding, solidification). , Regardless of the addition to the medium).
  • human IL-2 (20 ng / ml), IL-7 (10 ng / ml), and IL-15 (about 10 ng / ml) were added, and multiple times on a 24-well plate coated with retronectin 48 to 96 hours after stimulation. Introduced Yamanaka factor gene.
  • the Yamanaka factors used at that time are OCT4, SOX2, KLF4, C-MYC, NANOG, and LIN28, or OCT4, SOX2, KLF4, C-MYC, and NANOG.
  • Two to five days after the last gene transfer 1 to 5 ⁇ 10 5 gene-transferred T cells were placed on irradiated MEFs on a 6 cm dish.
  • the CD8 positive T cells were not sorted and used at the flow site on the first day, the CD8 positive T cells were sorted by the flow site at this timing and placed on the MEF.
  • T-iPS cells can be obtained from CD4 + T lymphocytes in 7 or 8 trials without introducing NANOG.
  • CD4 + T lymphocytes were further analyzed based on the expression of CD45RA and CD62L / CCR7, naive (CD45RA + CD62L +), central memory (CD45RA-CD62L +), effector memory (CD45RA-CD62L-), and terminal effector
  • the cells were divided into subsets of T cells (CD45RA + CD62L ⁇ ) and induced (reprogrammed) into iPS cells (see Sallusto, F. et al., Nature, 1999, 401, pages 708-712, see FIG. 14).
  • the average reprogramming efficiency in three healthy donors was 0.0041% for naive T cells, 0.017% for central memory T cells, 0.0036% for effector memory T cells, and terminal effector It was clarified that T-iPS cells were established relatively better than central memory T cells, which was 0.0008% in T cells (see FIG. 15). Note that the high reprogramming efficiency of CD4 + central memory T cells may reflect the sensitivity to anti-CD3 / CD28 stimulation.
  • Example 5 ⁇ Functional analysis of T lineage cells differentiated from TiPS cells> Transplantation of CD34 positive cells obtained from human ES cells into SCID-hu mice induces differentiation of the T cell lineage in the transplanted human fetal thymus (Galic, Z et al., PNAS, 2006, 103, 31). No., pages 11742-7). In vivo, ES cells are induced by OP9-DL1 cells into CD3 positive and TCR positive cells mainly CD4 / CD8 double positive (Timermans et al., JI, 2009, 182, 6879-6888). ).
  • T-iPS cells T cell-derived iPS cells
  • T-iPS cells T cell-derived iPS cells
  • T-iPS cells cultured on OP9 in the presence of IL-7, Flt3L (human FMS-like tyrosine kinase 3 ligand) or SCF (human stem cell factor, hSCF) are ES-sac (Sac) (Takayama et al., Blood, 2008 The structure of No. 111, No.
  • CD45 positive blood cells were induced therein by day 14 (see (a) of FIG. 16).
  • Blood cells were transferred to OP9-DL1 on days 12 to 14 and the culture was repeated every 3 days to continue the culture.
  • CD3-positive cells were collected by FACS and T cell-related genes were collected. The expression of was evaluated.
  • the CD3 positive cells on the 40th day were mainly CD4 / 8 negative cells, but some CD8 single positive cells were observed in part (see FIG. 16 (b)).
  • CD3 + TCR ⁇ + cells were in the conegative (DN) stage, but in some cell groups, only CD4 positive cells (8. 5 ⁇ 8.2%) and cells positive only for CD8 (15.5 ⁇ 15.9%) were also detected (see FIGS. 18 and 19).
  • TCR-expressing T lineage cells can also respond to TCR stimulation and produce cytokines at the thymocyte stage (Fischer, M. et al., J Immunol, 1991, 146, (See pages 3452-3456). Therefore, the floating cells were stimulated with OKT-3 and IL-2, and the activation and number of CD3-expressing cells were examined. That is, as a result of stimulating floating cells with 30 ng / ml OKT-3 and 600 IU / ml hIL-2 for 5 days, the number of CD3 + cells increased, and CD3 expressing cells were activated by morphological criteria. It was observed (see FIG. 20). However, it was confirmed that the actual CD3 + cells were not changed.
  • the cytokine production ability was evaluated as follows. That is, as a result of stimulation with PMA and calcium ionophore (ionomycin), 12.5% of T lineage cells produce IFN- ⁇ , 1.0% of T lineage cells produce IL-2, 6.2% It was revealed that the T-lineage cells produced both of them (see FIG. 21). Therefore, it was revealed that CD3 + TCR ⁇ + cells derived from T-iPS cells contributed to induction into T lineage cells both morphologically and functionally.
  • TCR expression of the induced CD3 cells was analyzed, only one type of TCR ⁇ chain appeared in TkT3V1-3 and TkT3V1-7, and the sequence centered on the CDR3 region was the same as that of iPS cells before induction. (See FIG. 22). Most of the TCR ⁇ chains found diversity were derived from reconstituted ⁇ chains (see FIG. 23). It was revealed that reprogrammed T cells express clonal TCR when induced again into T cells.
  • CD3 high TCR ⁇ high T lineage cells redifferentiated from 4 T-iPS cells derived from two donors were analyzed. That is, the cDNA library of these cells was constructed by SMART-mediated reverse transcription reaction (see Du, G., et al., J Immunol Methods, 2006, 308, 19-35), and the TCR gene was amplified. The amplified TCR gene was inserted into a cloning vector, cloned and analyzed.
  • transcripts were derived from a functional chain, and there were also transcripts derived from a non-functional chain (see Table 8). However, transcripts different from sequences derived from functional or non-functional rearrangements were not confirmed (see Table 9).
  • iPS Clone indicates an established T cell-derived iPS cell line
  • Productivity indicates whether the reconstituted T cell receptor has a function, ie, a complex with ⁇ receptor.
  • ES cells human ES cells, Human ESC
  • skin-derived iPS cells human skin fibroblast-derived iPS cells, HDF-iPS
  • CD34-positive cell-derived iPS cells umbilical cord blood CD34-positive cell-derived iPS cells, CB
  • CD3-positive cells were induced in both iPSC) and T-iPS cells (T-iPSC), but T-iPS cells were compared to iPS and ES cells derived from skin cells and CD34-positive cells. It was clearly found that they were easily induced into T cells (see FIGS. 16, 17, 26, and 27).
  • Example 6 ⁇ Induction of iPS cells established from human T cells into functional T cells 2> Using T-iPSC clones (6 clones) derived from CD4 T cells or CD8 T cells of multiple donors, an attempt was made to induce a T cell population having a monoclonal TCR. Specifically, 3 ⁇ 10 5 T-iPS cells were seeded on irradiated OP9 cells or 10T1 / 2 cells spread in a 10 cm dish on the day of induction, and co-cultured for about 14 days using a differentiation medium. From 12 to 14 days, blood cells that entered the bag-like structure appeared during the co-culture.
  • CD34 cells CD34 positive hematopoietic stem cells
  • the seeds were co-cultured in ⁇ -MEM medium containing 20% FBS in the presence of IL-7 and Flt3L. SCF was not used because it strongly promotes differentiation into NK cells.
  • IL-7 5-10 ng / ml a CD3-positive CD56-positive NKT-like cell population appeared, and again most were DN.
  • CD3-positive CD56-negative T lineage cells accounted for the majority, and differentiation from DN to DP, CD4SP, and CD8SP was confirmed.
  • peripheral blood T cell differentiation markers about 80% of CD8SP was CD45RA + CD62L + naive T cells, and most of CD4SP was CD45RA ⁇ CD62L + central memory T cells. That is, it was suggested that T cells that have advanced to the effector memory stage can rejuvenate to naive T cells (in the case of CD8SP) and central memory T cells (in the case of CD4) via T-iPS cells.
  • the method for producing human T lymphocytes of the present invention is particularly suitable for producing human T lymphocytes that are specific to the target antigen, and thus specific to the target antigen, and desired human tissue compatibility. It is excellent in the production of sex antigen (HLA) -restricted CD4 single positive cells or CD8 single positive cells. Therefore, the present invention can greatly contribute to the treatment or prevention of various intractable diseases such as chronic intractable infections, malignant tumors, and autoimmune diseases.
  • HLA sex antigen

Abstract

L'invention porte sur un procédé de préparation de lymhocytes T humains, qui comprend une étape d'induction de cellules iPS à partir de lymhocytes T humains et une étape de différenciation des cellules iPS en lymhocytes T ; sur une composition pharmaceutique qui contient des lymhocytes T qui sont préparés par le procédé ; et sur un procédé destiné au traitement de cellules immunes par utilisation du procédé décrit ci-dessus.
PCT/JP2011/052260 2010-02-03 2011-02-03 Procédé de reconstruction du système immunitaire par utilisation de cellules souches pluripotentes WO2011096482A1 (fr)

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