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  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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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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  • A61K40/00—Cellular immunotherapy
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  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
  • A61K40/421—Immunoglobulin superfamily
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• • A—HUMAN NECESSITIES
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  • 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
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  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
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  • C12N2500/00—Specific components of cell culture medium
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  • C12N2501/20—Cytokines; Chemokines
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  • C12N2501/20—Cytokines; Chemokines
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  • C12N2501/20—Cytokines; Chemokines
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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  • 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 producing a cell population containing CD4 single positive helper T cells from pluripotent stem cells, particularly induced pluripotent stem cells (iPS cells), a medicine containing the cell population, a killing agent for cells expressing a tumor-associated antigen and a preventive or therapeutic agent for cancer in a mammal, each containing the cell population, and a method for preventing or treating cancer in a mammal, comprising administering an effective amount of the cell population.
• pluripotent stem cells particularly induced pluripotent stem cells (iPS cells)
• a medicine containing the cell population a killing agent for cells expressing a tumor-associated antigen and a preventive or therapeutic agent for cancer in a mammal, each containing the cell population
• a killing agent for cells expressing a tumor-associated antigen and a preventive or therapeutic agent for cancer in a mammal each containing the cell population
• a method for preventing or treating cancer in a mammal comprising administering
• CAR is a fusion protein consisting of the antigen recognition portion of an antibody that specifically recognizes cancer antigens or tumor-associated antigens and an intracellular domain derived from TCR, and CAR-T cells can recognize antigens expressed on the cell surface in a manner that is not restricted by human leukocyte antigens (HLA). For this reason, CAR-T cells are expected to be used in allogeneic transplants in addition to autologous transplants.
• TCR is a receptor used by T cells to recognize antigens, and is composed of a dimer of ⁇ and ⁇ chains, or ⁇ and ⁇ chains. TCR forms a complex with CD3 molecules on the surface of T cells, and activates T cells by recognizing antigen molecules bound to major histocompatibility complex (MHC) molecules.
• MHC major histocompatibility complex
• T cells are broadly classified into killer T cells, which are made up of CD8 positive T cells that directly recognize tumors and induce apoptosis, and helper T cells, which are made up of CD4 positive T cells that enhance the functions of other lymphocytes such as killer T cells and B cells.
• Cancer immunotherapy has focused on CD8 positive T cells that can directly recognize tumor cells. This is because tumor cells that endogenously express MHC class II molecules are direct targets of CD4 positive T cells, but most tumors do not express MHC class II molecules and are not directly recognized by CD4 positive T cells.
• tumor antigens can be captured and cross-presented by tumor stromal cells that express MHC class II molecules (Non-Patent Document 1).
• Non-Patent Document 2 Necrotic tumor cells or vesicles released from tumor cells are taken up by stromal cells and mainly enter the classical processing pathway for MHC class II molecules, so it is speculated that cross-presentation of tumor antigens on MHC class II molecules is more effective than that on MHC class I molecules.
• monocytes/macrophages are the most abundant MHC class II molecule-positive cells.
• CD4 positive T cell subset several different tumor elimination mechanisms have been proposed.
• iPS cells induced pluripotent stem cells
• Non-Patent Document 5 In cancer immunotherapy using genetically modified T cells expressing tumor-reactive TCR or CAR, it has been reported that a synergistic in vivo antitumor effect can be obtained by combining the most potent CD4-positive and CD8-positive CAR-expressing T cell subsets (Non-Patent Document 5). Therefore, when inducing functional T cells from iPS cells, it is important to generate not only CD8-positive T cells but also CD4-positive T cells. However, according to the above-mentioned reported technology, the T cells generated from iPS cells are CD8-positive T cells, and it is not possible to generate CD4-positive T cells or long-lived naive T cells.
• Non-Patent Document 6 In order to generate CD4-positive T cells from iPS cells, a method has been reported in which the CD4 gene is introduced into T cells induced from iPS cells and the cells are cultured in a culture medium containing interleukin-2 and interleukin-15 (Patent Document 5). In addition, a method has been reported in which CD4-positive T cells are generated by culturing iPS cells in an artificial thymic organ (ATO) (Non-Patent Document 6), but the efficiency of generating CD4-positive T cells is low.
• ATO artificial thymic organ
• CAR-T cells Current cancer immunotherapy using autologous transplantation of CAR-T cells shows excellent clinical efficacy. This is because CAR is introduced not only into killer T cells in peripheral blood but also into helper T cells, which then participate in the immune response. On the other hand, in order to perform cancer immunotherapy stably and effectively without relying on autologous cells, the challenge is to stably produce helper T cells from pluripotent stem cells.
• the present invention aims to provide a method for efficiently producing a cell population containing functionally superior CD4 single positive helper T cells from pluripotent stem cells.
• the present invention also aims to provide a medicine containing the cell population produced by the above method, a killing agent for cells expressing a tumor-associated antigen, and a preventive or therapeutic agent for cancer in a mammal, as well as a method for preventing or treating cancer in a mammal, which comprises administering an effective amount of the cell population produced by the above method.
• CD4 single positive helper T cells can be efficiently produced by selecting whether or not to use a T cell differentiation induction factor and a cell adhesion factor for each step in the process of inducing differentiation of pluripotent stem cells, and thus completed the present invention.
• CD4 single positive helper T cells can be produced by culturing cells using a Wnt pathway activator in the process of inducing differentiation of pluripotent stem cells, and by selecting the use or non-use of a T cell differentiation inducer, a cell adhesion factor, and an anti-CD3 antibody for each process, thus completing the present invention.
• T cell differentiation inducer is a Notch ligand.
• the Notch ligand is DLL4 or DLL4-Fc.
• the cell adhesion factor is fibronectin or a fragment thereof, or Retronectin (registered trademark).
• the Wnt pathway activator is a GSK-3 inhibitor.
• the GSK-3 inhibitor is CHIR-99021.
• a pharmaceutical comprising a cell population produced by the method according to [1] or [9].
• the pharmaceutical according to [31] for use in the prevention or treatment of cancer.
• a method for killing cells expressing a tumor-associated antigen comprising the cell population produced by the method according to [1] or [9].
• a method for preventing or treating cancer in a mammal comprising administering to the mammal an effective amount of a cell population produced by the method according to [1] or [9].
• a method for preventing or treating cancer in a mammal comprising administering to the mammal an effective amount of the medicament according to [31].
• a method for preventing or treating cancer in a mammal comprising administering to the mammal an effective amount of the killing agent described in [33].
• a preventive or therapeutic agent for cancer in a mammal comprising a cell population produced by the method according to [1] or [9].
• the method described in [1] is referred to as the first method, and the method described in [9] is referred to as the second method.
• CD4 single positive helper T cells are necessary for enhancing tumor immunity, but based on the techniques reported so far, T cells produced from pluripotent stem cells are mainly CD8 single positive killer T cells, and CD4 single positive helper T cells cannot be produced efficiently.
• hematopoietic stem cells induced to differentiate from pluripotent stem cells are cultured in the presence of a T cell differentiation inducer, then in the presence of a T cell differentiation inducer and a cell adhesion factor, and finally in the presence of a cell adhesion factor but in the absence of a T cell differentiation inducer, thereby producing a cell population containing CD4 single positive helper T cells without introducing a CD4 gene from outside.
• a cell population containing CD4 single positive helper T cells and CD8 positive killer T cells can be produced.
• the proportion of CD4 single positive helper T cells in the cell population can be increased by culturing the hematopoietic stem cells in the presence of a Wnt/ ⁇ -catenin pathway activator throughout the entire culture period.
• hematopoietic stem cells induced to differentiate from pluripotent stem cells are cultured in the presence of a T cell differentiation inducer and a Wnt/ ⁇ -catenin pathway activator, then in the presence of a cell adhesion factor, a Wnt pathway activator and an anti-CD3 antibody but in the absence of a T cell differentiation inducer, and finally in the presence of a cell adhesion factor and a Wnt pathway activator but in the absence of a T cell differentiation inducer and an anti-CD3 antibody, thereby producing a cell population containing CD4 single positive helper T cells and CD8 positive killer T cells.
• the CD4 single positive helper T cells produced by the method of the present invention have superior cell proliferation properties compared to CD8 single positive killer T cells, and also have superior killing effects against cancer cells and in vivo tumor suppression effects.
• a medicine containing a cell population including the CD4 single positive helper T cells produced by the method of the present invention, or a killing agent for cells expressing tumor-associated antigens, is useful for the prevention or treatment of cancer.
• FIG. 1 shows the results of a flow cytometric analysis of the effects of coating a cell culture plate with DLL4-Fc and RetroNectin in steps (1) and (2) on the generation of CD4 single-positive helper T cells from iPS cells.
• FIG. 1 is a diagram comparing the coating conditions in steps (1) and (2) and the amount of CD4 single-positive helper T cells generated from iPS cells.
• FIG. 1 shows the effect of a Wnt pathway activator (CHIR-99021) on the generation of CD4 single positive helper T cells from iPS cells.
• FIG. 1 shows the effects of a Wnt pathway activator (CHIR-99021) on the generation of CD4 single positive helper T cells from iPS cells.
• This figure shows the expression of Thpok and PLZF in CD4 single positive helper T cells and CD8 single positive killer T cells generated from iPS cells by DLL4-Fc/retroNectin switching in the coating of cell culture plates, measured by TaqMan qPCR, and compared with the results in adaptive immune system primary T cells obtained from peripheral blood.
• This figure shows the results of measuring Thpok and Runx3 expression in CD4 single-positive helper T cells and CD8 single-positive killer T cells that have undergone different maturation processes by TaqMan qPCR.
• FIG. 1 shows the results of measuring the proliferation of CD4 single positive helper T cells and CD8 single positive killer T cells that have undergone different maturation processes.
• This figure shows flow cytometry analysis of naive markers (CD28) and adaptive immune markers (CD2 and CD5) expressed by CD4 single-positive helper T cells and CD8 single-positive killer T cells generated from iPS cells by DLL4-Fc/retroNectin switching in the coating of cell culture plates after feeder-free expansion, and compares the analysis results with those of the T cells and fresh human peripheral blood mononuclear cells before expansion.
• CD28 naive markers
• CD2 and CD5 adaptive immune markers
• This figure shows the results of flow cytometry analysis of EGFR expression in the cells obtained after retroviral gene transfer of anti-CD19 CAR (anti-CD19 CAR-IRES-EGFR) into a cell population containing CD4 single positive helper T cells and CD8 single positive killer T cells generated from iPS cells by DLL4-Fc/retronectin switching in the coating of cell culture plates.
• This figure shows the results of measuring the amounts of cytokines (IFN- ⁇ , IL-2, and TNF ⁇ ) in the culture supernatant after co-culturing CD4 single-positive helper T cells and CD8 single-positive killer T cells, which were generated from iPS cells by DLL4-Fc/retroNectin switching in the coating of cell culture plates and transfected with anti-CD19 CAR-IRES-EGFR, with the CD19-positive human pre-B cell leukemia cell line Nalm6 for 24 hours.
• FIG. 13 shows the killing effect of CD4 single positive helper T cells and CD8 single positive killer T cells generated from iPS cells by DLL4-Fc/retroNectin switching in the coating of cell culture plates against the human pre-B cell leukemia cell line Nalm6.
• FIG. 13 shows the effects of IL-7 and IL-15 on the killing activity of CD4 single-positive helper T cells and CD8 single-positive killer T cells generated from iPS cells by DLL4-Fc/retroNectin switching in the coating of cell culture plates against the human pre-B cell leukemia cell line Nalm6.
• This figure shows the results of measuring the cytotoxic activity against the CD19-positive human pre-B cell leukemia cell line Nalm6 after introducing anti-CD19 CAR into CD4 single-positive helper T cells and CD8 single-positive killer T cells generated from iPS cells by DLL4-Fc/retroNectin switching in the coating of cell culture plates, T cells generated from iPS cells generated by artificial thymus organ (ATO) culture, and primary T cells derived from healthy individuals.
• ATO thymus organ
• This figure shows the results of measuring the cytotoxic activity against the CD19-negative human leukemia cell line K562 after introducing anti-CD19 CAR into CD4 single-positive helper T cells and CD8 single-positive killer T cells generated from iPS cells by DLL4-Fc/retronectin switching in the coating of cell culture plates, T cells generated from iPS cells generated by artificial thymus organ (ATO) culture, and primary T cells derived from healthy individuals.
• ATO thymus organ
• FIG. 17B shows Kaplan-Meier survival curves for NSG mice described in FIG. 17A.
• FIG. 1 shows the results of flow cytometry analysis of CD4 single positive helper T cells generated from hematopoietic stem cells derived from human iPS cells in the presence of a Wnt pathway activator (CHIR-99021).
• FIG. 18B shows the results of flow cytometric analysis of the expression of CD5, TCR ⁇ chain/ ⁇ chain, and CD1a in the CD4 single-positive T cell subset shown in FIG. 18A.
• FIG. 1 shows the results of flow cytometry analysis of the effect of a Wnt pathway activator (CHIR-99021) on the generation of CD4 single-positive helper T cells from hematopoietic stem cells derived from iPS cells.
• FIG. 1 shows the results of flow cytometry analysis of the effect of a Wnt pathway activator (CHIR-99021) on the generation of CD4 single-positive helper T cells from hematopoietic stem cells derived from iPS cells.
• FIG. 1 shows the percentage of mature CD8 single positive killer T cells and cell yield in T cells generated from hematopoietic stem cells derived from iPS cells in the presence of a Wnt pathway activator (CHIR-99021).
• FIG. 1 shows the percentage of mature CD4 single positive helper T cells and cell yield in T cells generated from hematopoietic stem cells derived from iPS cells in the presence of a Wnt pathway activator (CHIR-99021).
• FIG. 1 shows the percentage of mature CD8 single positive killer T cells and cell yield in T cells generated from hematopoietic stem cells derived from iPS cells in the presence of a Wnt pathway activator (CHIR-99021).
• FIG. 1 shows the results of flow cytometric analysis of CD4 and CD8 expression in mature T cells generated from hematopoietic stem cells derived from iPS cells in the presence of a Wnt pathway activator (CHIR-99021), and the results of flow cytometric analysis of CD4 and CD8 expression in CD4 and CD8 single positive T cells after flow cytometric sorting and subsequent expansion using anti-CD3 and anti-CD28 antibody-bound beads.
• FIG. 13 shows the results of flow cytometry analysis of CD40L expression in CD4 single positive helper T cells generated from hematopoietic stem cells derived from iPS cells in the presence of a Wnt pathway activator (CHIR-99021), with or without stimulation with an anti-CD3 antibody (OKT3).
• FIG. 1 shows the results of flow cytometric analysis of CD4 and CD8 expression in mature T cells generated from hematopoietic stem cells derived from iPS cells in the presence of a Wnt pathway activator (CHIR-99021),
• cytokines IFN- ⁇ and IL-2
• CD4 single positive helper T cells CD8 single positive killer T cells, which were generated from hematopoietic stem cells derived from human iPS cells in the presence of a Wnt pathway activator (CHIR-99021), with PMA and ionomycin in the presence of momensin.
• CHIR-99021 Wnt pathway activator
• This figure shows the results of flow cytometry analysis of anti-CD19 CAR expression in the resulting cells after retroviral gene transfer of anti-CD19 CAR (anti-CD19 CAR-4-1BB-CD3zeta) into CD4 single-positive helper T cells and CD8 single-positive killer T cells generated from hematopoietic stem cells derived from human iPS cells in the presence of a Wnt pathway activator (CHIR-99021).
• This figure shows the results of measuring the cytotoxic activity against the CD19-positive human pre-B cell leukemia cell line Nalm6 after introducing anti-CD19 CAR (anti-CD19 CAR-4-1BB-CD3zeta) into CD4 single-positive helper T cells and CD8 single-positive killer T cells generated from hematopoietic stem cells derived from human iPS cells in the presence of a Wnt pathway activator (CHIR-99021).
• anti-CD19 CAR anti-CD19 CAR-4-1BB-CD3zeta
• the present invention is a method for producing a cell population containing CD4 single positive and CD8 negative helper T cells.
• the first method of the present invention includes a step (1) of culturing hematopoietic stem cells, which have been induced to differentiate from pluripotent stem cells, in the presence of a T cell differentiation inducer; a step (2) of culturing the cells obtained in step (1) in the presence of a T cell differentiation inducer and a cell adhesion factor; and a step (3) of culturing the cells obtained in step (2) in the presence of the cell adhesion factor but in the absence of the T cell differentiation inducer.
• the second method of the present invention includes the steps of: (1) culturing hematopoietic stem cells induced to differentiate from pluripotent stem cells in the presence of a T cell differentiation inducer and a Wnt/ ⁇ -catenin pathway activator; (2) culturing the cells obtained in step (1) in the presence of a cell adhesion factor, a Wnt pathway activator, and an anti-CD3 antibody, but in the absence of a T cell differentiation inducer; and (3) culturing the cells obtained in step (2) in the presence of a cell adhesion factor and a Wnt pathway activator, but in the absence of a T cell differentiation inducer and an anti-CD3 antibody.
• pluripotent stem cells are stem cells that have both the ability to grow almost infinitely (self-proliferation ability) and the ability to differentiate into almost all cells that constitute an individual (multipotency).
• Pluripotent stem cells are not particularly limited, but include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), multipotent germline stem cells (mGS cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (nuclear transfer embryonic stem cells (ntES cells)), and pluripotent stem cells isolated from fibroblasts and bone marrow mesenchymal cells (Multi-lineage differentiating stress enduring cells (Muse cells)).
• iPS cells may be used, focusing on the fact that they do not involve the destruction of embryos and eggs, and have high differentiation ability.
• iPS cells are generated by initializing somatic cells of a mammal. Examples of mammals include humans, monkeys, pigs, dogs, cats, rats, mice, etc., but humans are preferred.
• the somatic cells are not particularly limited, but cells isolated from peripheral blood can be used.
• the somatic cells are peripheral blood mononuclear cells from which B cells and T cells have been removed, or T cells.
• Peripheral blood mononuclear cells from which B cells and T cells have been removed may be obtained by isolating mononuclear cells from whole blood using a mononuclear cell separation solution, and then removing the B cells and T cells using surface antigens expressed on the B cells and T cells.
• Examples of the mononuclear cell separation solution include Lymphoprep (registered trademark).
• Lymphoprep registered trademark
• antibodies against CD19, CD20, CD22, or B cell receptor, which are surface antigens of B cells, and CD3, CD4, or CD8, which are surface antigens of T cells may be used, for example, by flow cytometry or magnetic beads such as MACS (registered trademark) beads.
• T cells can be isolated from peripheral blood by removing non-T cells such as monocytes, neutrophils, eosinophils, B cells, stem cells, dendritic cells, NK (natural killer) cells, and erythrocytes by negative sorting using specific antibodies against surface antigens (CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD234a (Glycophorin A), etc.) carried by these cells.
• T cells may also be isolated from peripheral blood mononuclear cells by positive selection.
• the source of T cells is preferably peripheral blood because of its low invasiveness, but is not limited thereto.
• Other sources include cancer tissue or tumor tissue or other tissue, 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), nasal discharge, urinary pleural cavity, peritoneal cavity, cranial cavity, or spinal canal exudates (pleural fluid, ascites, etc.).
• cancer tissue or tumor tissue examples include tissues derived from ovarian cancer, hepatoblastoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, malignant melanoma, non-small cell lung cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid cancer, colon cancer, head and neck cancer, brain tumor, multiple myeloma, or B-cell non-Hodgkin's lymphoma.
• iPS cells can be induced by introducing cellular reprogramming factors into peripheral blood mononuclear cells or T cells from which cells and T cells have been depleted.
• cell reprogramming factors include genes or gene products such as Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, klf4, klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, ⁇ -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 may be introduced into the peripheral blood mononuclear cells or T cells from the viewpoint of efficiently establishing iPS cells.
• the method of introducing the cell reprogramming factor into the peripheral blood mononuclear cells or T cells there is no particular limitation on the method of introducing the cell reprogramming factor into the peripheral blood mononuclear cells or T cells, and any method known in the art can be used.
• the gene encoding the cell reprogramming factor e.g., cDNA
• the expression vector can be introduced into the peripheral blood mononuclear cells or T cells by infection, lipofection, liposome, calcium phosphate coprecipitation, DEAE-dextran, microinjection, or electroporation.
• examples of the method include a method using a protein introduction reagent, a method using a protein introduction domain fusion protein, an electroporation method, and a microinjection method.
• examples of the method include a method using an mRNA introduction reagent and a method of adding the mRNA to a culture medium.
• Expression vectors used for gene transfer by infection include, for example, viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus, as well as animal cell expression plasmids. From the viewpoints that insertional mutagenesis is unlikely to occur, gene transfer efficiency is high, and the number of copies of the transferred gene is large, the gene encoding the cellular reprogramming factor may be transferred into the peripheral blood mononuclear cells or T cells using Sendai virus.
• viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus
• Promoters used in expression vectors used when introducing a gene encoding the cellular reprogramming factor into the peripheral blood mononuclear cells or T cells include, for example, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, and ubiquitin promoter. These promoters may be capable of controlling the expression of a gene inserted downstream of the promoter depending on the presence or absence of a drug such as tetracycline.
• the expression vector may contain an enhancer, a polyA addition signal, a selection marker gene (e.g., a neomycin resistance gene), an SV40 replication origin, and the like.
• the T cells When T cells are used to generate iPS cells, the T cells may be stimulated with anti-CD3 and anti-CD28 antibodies in the presence of interleukin-2 (IL-2) or stimulated with a desired antigen peptide to activate them before the cell initialization.
• the stimulation can be performed by adding IL-2, anti-CD3 and anti-CD28 antibodies to a culture medium and culturing the T cells for a certain period of time.
• the anti-CD3 and anti-CD28 antibodies may be in a state coated on cell culture beads or cell culture plates that are dispersed in the culture medium.
• stimulation with an antigen peptide can be performed by adding a peptide consisting of an amino acid sequence of at least 9 residues that constitutes a desired antigen peptide recognized by T cells to a culture medium and culturing the T cells for a certain period of time.
• the culture medium used for culturing iPS cells is not particularly limited, but may be prepared by adding cytokines to a culture medium used for culturing animal cells as a basal culture medium to maintain the undifferentiated ability of iPS cells.
• basal culture medium include IMDM (Iscove's Modified Dulbecco's Medium), Medium 199, EMEM (Eagle's Minimum Essential Medium), ⁇ MEM (alpha Modified Eagle Minimum Essential Medium), DMEM (Dulbecco's modified Eagle's Medium), Ham's F12 culture medium, RPMI1640 culture medium, Fischer's culture medium, Neurobasal Medium (Life Technologies), StemFit (registered trademark) AK03N (Ajinomoto Healthy Supply Co., Ltd.), and mixtures of these.
• the culture medium may contain serum or may be serum-free.
• An example of a cytokine is bFGF, and the concentration of the cytokine in the culture medium is, for example, 1 to 100
• the culture method for iPS cells may be adhesion culture or suspension culture.
• iPS cells When performing suspension culture, iPS cells are cultured in a cell culture dish until they become 80% confluent, and then dissociated into single cells, which can then be used for suspension culture.
• methods for isolating iPS cells include a method of physically isolating them using a cell scraper, or an isolation method using a dissociation solution having protease activity, a dissociation solution having collagenase activity, or a dissociation solution having protease activity and collagenase activity (e.g., Accutase (registered trademark) and Accumax (registered trademark), etc.).
• hematopoietic stem cells refer to cells that can differentiate into blood cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes.
• Hematopoietic stem cells and hematopoietic progenitor cells are not distinguished from each other and refer to the same cell unless otherwise specified.
• Hematopoietic stem cells/progenitor cells are recognized, for example, by being positive for the surface antigen CD34 or by being double positive for CD34 and CD43.
• iPS cells which are pluripotent stem cells, into hematopoietic stem cells
• Vitamin C refers to L-ascorbic acid and its derivatives.
• L-ascorbic acid derivatives refer to substances that are converted to vitamin C by enzymatic reactions in vivo. Examples of L-ascorbic acid derivatives include vitamin C phosphate, ascorbic acid glucoside, ascorbyl ethyl, vitamin C ester, ascorbyl tetrahexyldecanoate, ascorbyl stearate, and ascorbyl-2-phosphate-6-palmitate.
• vitamin C phosphate examples include L-ascorbate phosphates such as sodium L-ascorbate phosphate or magnesium L-ascorbate phosphate.
• Vitamin C is contained in the culture medium at a concentration of, for example, 5 to 500 ⁇ g/mL.
• the culture medium used for inducing differentiation of iPS cells into hematopoietic stem cells is not particularly limited, but may be prepared by adding vitamin C or the like to a culture medium used for culturing animal cells as a basal culture medium.
• basal culture medium include IMDM (Iscove's Modified Dulbecco's Medium), Medium 199, EMEM (Eagle's Minimum Essential Medium), ⁇ MEM (alpha Modified Eagle Minimum Essential Medium), DMEM (Dulbecco's modified Eagle's Medium), Ham's F12 culture medium, RPMI1640 culture medium, Fischer's culture medium, Neurobasal Medium (Life Technologies), StemPro34 (Life Technologies), and mixed culture mediums thereof.
• IMDM Iscove's Modified Dulbecco's Medium
• Medium 199 EMEM (Eagle's Minimum Essential Medium)
• ⁇ MEM alpha Modified Eagle Minimum Essential Medium
• DMEM Dulbecco's modified Eagle's Medium
• the culture medium used to induce differentiation of iPS cells into hematopoietic stem cells may further contain a cytokine selected from the group consisting of BMP4 (bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), SCF (stem cell factor), TPO (thrombopoietin), and FLT3L (FMS-like tyrosine kinase ligand).
• BMP4 bone morphogenetic protein 4
• VEGF vascular endothelial growth factor
• bFGF basic fibroblast growth factor
• SCF stem cell factor
• TPO thrombopoietin
• FLT3L FLT3L
• pluripotent stem cells may be co-cultured with feeder cells such as C3H10T1/2 (Takayama N, et al. J Exp Med. 2010; 2817-2830) or heterologous stromal cells (Niwa A, et al. J Ce11 Physiol. 2009; 221:367-77).
• feeder cells such as C3H10T1/2 (Takayama N, et al. J Exp Med. 2010; 2817-2830) or heterologous stromal cells (Niwa A, et al. J Ce11 Physiol. 2009; 221:367-77).
• Hematopoietic stem cells can be prepared from embryoid bodies formed by culturing iPS cells.
• embryoid bodies are three-dimensional sac-like structures with internal spaces derived from iPS cells, formed from endothelial cell populations, etc., and containing hematopoietic stem cells.
• T cell differentiation inducing factor a Notch ligand for a Notch molecule expressed on a cell membrane can be used as a T cell differentiation inducer.
• Notch 1 to 4 are four Notch molecules, Notch 1 to 4, in mammals.
• the binding induces the Notch signal, which is involved in the control of cell survival, proliferation and differentiation (Artavanis-Tsakonas S, et al. Science. 1999; 284:770-776).
• DLL Delta-Like-Protein
• Jagged 1 and 2 Jagged 1 may be used.
• DLL4 which is an endogenous Notch1 ligand in mammals, particularly humans
• the DLL4 may be a partial peptide of DLL4, so long as it induces the Notch signal.
• the extracellular domain of human DLL4 (residue numbers 1 to 524 or residue numbers 27 to 529 (including mutations)
• DLL4-Fc A recombinant protein (DLL4-Fc) in which the Fc domain of human IgG1 has been added to the C-terminus of DLL4) can also be used.
• Wnt/ ⁇ -catenin pathway activators Signal transduction in the Wnt/ ⁇ -catenin pathway is involved in numerous cellular processes and plays an important role in both animal development and regeneration (Gao J, et al. Cell Regeneration. 2021; 10:11). Wnt proteins mainly bind to the Frizzled receptor, a seven-transmembrane receptor protein, to initiate signal transduction. Binding of Wnt proteins to the receptor induces the accumulation of ⁇ -catenin, and as a result, ⁇ -catenin functions as an intracellular mediator of the Wnt signal. Wnt/ ⁇ -catenin pathway activators have the effect of enhancing signal transduction in this pathway.
• Wnt/ ⁇ -catenin pathway activators are not particularly limited as long as they enhance the signal transduction, but examples of such activators include glycogen synthase kinase 3 (GSK-3) inhibitors, which are serine/threonine kinases. After being phosphorylated by GSK-3 and the like, ⁇ -catenin is ubiquitinated and degraded by the proteasome, and a GSK-3 inhibitor inhibits the degradation of ⁇ -catenin.
• GSK-3 glycogen synthase kinase 3
• GSK-3 has two isoforms, GSK-3 ⁇ and GSK-3 ⁇ .
• any of GSK-3 ⁇ inhibitors, GSK-3 ⁇ inhibitors, and GSK-3 ⁇ / ⁇ dual inhibitors may be used as long as they enhance the signal transduction of the Wnt/ ⁇ -catenin pathway.
• the GSK-3 inhibitor CHIR-99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile) may be used.
• CHIR-99021 is also called laduviglusib and is a compound with CAS number 252917-06-9.
• the concentration of the GSK-3 inhibitor added to the culture medium may be 0.01 to 100 ⁇ M.
• Retronectin is a recombinant protein that contains a cell adhesion domain (C-domain) that binds to ⁇ 5 ⁇ 1 integrin (VLA-5) in human fibronectin, a heparin -binding domain (H-domain), and a CS-1 site that binds to ⁇ 4 ⁇ 1 integrin (VLA-4).
• C-domain cell adhesion domain
• H-domain heparin -binding domain
• VLA-4 CS-1 site that binds to ⁇ 4 ⁇ 1 integrin
• the cell culture in steps (1) to (3) of the first and second methods of the present invention may be adhesion culture or suspension culture.
• a cell culture plate can be used in the case of adhesion culture
• cell culture beads can be used in the case of suspension culture.
• step (1) is performed in the presence of the T cell differentiation inducer
• step (2) is performed in the presence of the T cell differentiation inducer and the cell adhesion factor
• step (3) is performed in the presence of the cell adhesion factor and in the absence of the T cell differentiation inducer.
• Examples of the basal culture medium used for cell culture in steps (1) to (3) of the first and second methods of the present invention include IMDM (Iscove's Modified Dulbecco's Medium), Medium 199, EMEM (Eagle's Minimum Essential Medium), ⁇ MEM (alpha Modified Eagle Minimum Essential Medium), DMEM (Dulbecco's modified Eagle's Medium), Ham's F12 culture medium, RPMI1640 culture medium, Fischer's culture medium, Neurobasal Medium (Life Technologies), and mixed culture mediums thereof.
• the culture medium may contain serum or may be serum-free. Serum may be added, for example, fetal bovine serum at 5 to 20% (v/v).
• the cell culture in steps (1) and (2) is carried out in the presence of interleukin-7 (IL-7) and FLT3L (FMS-like tyrosine kinase ligand).
• IL-7 and FLT3L FMS-like tyrosine kinase ligand.
• concentrations of IL-7 and FLT3L in the culture medium are 0.1 to 50 ng/mL, and may even be 1 to 10 ng/mL.
• the cell culture in steps (1) and (2) is carried out in the presence of IL-7, FLT3L, 2-phospho-L-ascorbic acid, and SDF-1 ⁇ (stromal cell-derived factor 1).
• concentrations of IL-7, Flt3L, and 2-phospho-L-ascorbic acid in the culture medium are 0.1 to 200 ⁇ g/mL, and may be 1 to 100 ⁇ g/mL.
• concentration of SDF-1 ⁇ in the culture medium is 0.1 to 100 nM, and may be 1 to 20 nM.
• the cell culture in steps (1) and (2) is carried out in the presence of IL-7, FLT3L, 2-phospho-L-ascorbic acid, and a p38 inhibitor.
• concentrations of IL-7, Flt3L, and 2-phospho-L-ascorbic acid in the culture medium are 0.1 to 200 ⁇ g/mL, and may be 1 to 100 ⁇ g/mL.
• concentration of the p38 inhibitor in the culture medium is 0.1 to 100 ⁇ M, and may be 1 to 10 ⁇ M.
• the cell culture in steps (1) and (2) is carried out in the presence of IL-7, FLT3L, a p38 inhibitor and SDF-1 ⁇ .
• concentrations of IL-7 and Flt3L in the culture medium are 0.1 to 50 ng/mL, and may be 1 to 10 ng/mL.
• concentration of the p38 inhibitor in the culture medium is 0.1 to 100 ⁇ M, and may be 1 to 10 ⁇ M.
• concentration of SDF-1 ⁇ in the culture medium is 0.1 to 100 nM, and may be 1 to 20 nM.
• the cell culture in steps (1) and (2) is performed in the presence of IL-7, FLT3L, 2-phospho-L-ascorbic acid, SDF-1 ⁇ , a p38 inhibitor, and stem cell factor (SCF).
• concentrations of IL-7, Flt3L, and 2-phospho-L-ascorbic acid in the culture medium are 0.1 to 200 ⁇ g/mL, and may be 1 to 100 ⁇ g/mL.
• concentration of SDF-1 ⁇ in the culture medium is 0.1 to 100 nM, and may be 1 to 20 nM.
• the concentration of the p38 inhibitor in the culture medium is 0.1 to 100 ⁇ M, and may be 1 to 10 ⁇ M.
• concentration of SCF in the culture medium is 0.1 to 100 ng/mL, and may be 1 to 20 ng/mL.
• the cell culture in step (3) is performed in the presence of IL-7, 2-phospho-L-ascorbic acid, and SDF-1 ⁇ (stromal cell-derived factor 1).
• concentrations of IL-7 and 2-phospho-L-ascorbic acid in the culture medium are 0.1 to 200 ⁇ g/mL, and may be 1 to 100 ⁇ g/mL.
• concentration of SDF-1 ⁇ in the culture medium is 0.1 to 100 nM, and may be 1 to 20 nM.
• the cell culture in step (3) is carried out in the presence of IL-7, 2-phospho-L-ascorbic acid, and a p38 inhibitor.
• concentrations of IL-7 and 2-phospho-L-ascorbic acid in the culture medium are 0.1 to 200 ⁇ g/mL, and may be 1 to 100 ⁇ g/mL.
• concentration of the p38 inhibitor in the culture medium is 0.1 to 100 ⁇ M, and may be 1 to 10 ⁇ M.
• the cell culture in step (3) is performed in the presence of IL-7, a p38 inhibitor, and SDF-1 ⁇ .
• concentration of IL-7 in the culture medium is 0.1 to 50 ng/mL, and may be 1 to 10 ng/mL.
• concentration of the p38 inhibitor in the culture medium is 0.1 to 100 ⁇ M, and may be 1 to 10 ⁇ M.
• concentration of SDF-1 ⁇ in the culture medium is 0.1 to 100 nM, and may be 1 to 20 nM.
• the cell culture in step (3) is performed in the presence of IL-7, 2-phospho-L-ascorbic acid, SDF-1 ⁇ , a p38 inhibitor, and stem cell factor (SCF).
• concentrations of IL-7 and 2-phospho-L-ascorbic acid in the culture medium are 0.1 to 200 ⁇ g/mL, and may be 1 to 100 ⁇ g/mL.
• concentration of SDF-1 ⁇ in the culture medium is 0.1 to 100 nM, and may be 1 to 20 nM.
• the concentration of the p38 inhibitor in the culture medium is 0.1 to 100 ⁇ M, and may be 1 to 10 ⁇ M.
• the concentration of SCF in the culture medium is 0.1 to 100 ng/mL, and may be 1 to 20 ng/mL.
• the cell culture in step (1) is performed in the presence of IL-7, FLT3L, 2-phospho-L-ascorbic acid, SDF-1 ⁇ , a p38 inhibitor, SCF, and TPO (thrombopoietin).
• concentrations of IL-7, FLT3L, SCF, and TPO in the culture medium are 1 to 500 ng/mL, and may be 10 to 100 ng/mL.
• concentration of 2-phospho-L-ascorbic acid in the culture medium is 1 to 1000 ⁇ g/mL, and may be 5 to 100 ⁇ g/mL.
• the concentration of SDF-1 ⁇ in the culture medium is 0.1 to 500 nM, and may be 1 to 100 nM.
• concentration of the p38 inhibitor in the culture medium is 0.1 to 200 ⁇ M, and may be 1 to 50 ⁇ M.
• the cell culture in steps (2) and (3) is carried out in the presence of IL-7, 2-phospho-L-ascorbic acid, SDF-1 ⁇ and a p38 inhibitor.
• concentration of IL-7 in the culture medium is 1 to 500 ng/mL, and may be 10 to 100 ng/mL.
• concentration of 2-phospho-L-ascorbic acid in the culture medium is 1 to 1000 ⁇ g/mL, and may be 5 to 100 ⁇ g/mL.
• the concentration of SDF-1 ⁇ in the culture medium is 0.01 to 200 nM, and may be 1 to 50 nM.
• the concentration of the p38 inhibitor in the culture medium is 0.1 to 100 mM, and may be 1 to 50 mM.
• isoforms such as SDF-1 ⁇ , SDF-1 ⁇ , SDF-1 ⁇ , SDF-1 ⁇ , or SDF-1 ⁇ may be used in place of SDF-1 ⁇ , or a mixture of these in any ratio may be used.
• the cell culture temperature in steps (1) to (3) of the first and second methods of the present invention is not particularly limited as long as it does not inhibit cell proliferation and survival, but may be about 37°C to about 42°C, and may also be about 37°C to about 39°C.
• the culture period in step (1) of the first and second methods of the present invention can be determined by confirming the formation of T cell precursors.
• the term T cell precursor refers to cells that are CD3, CD5, and CD1a positive and fully committed to the T cell lineage.
• the T cell precursor includes not only CD4/CD8 double positive immature T cells but also CD4 single positive immature T cells. These cell surface antigens can be detected by flow cytometry using fluorescently labeled antibodies against the cell surface antigens.
• the next step (2) may be carried out after confirming the appearance of CD3, CD1a, and CD5 positive T cell among the cells obtained in step (1).
• the step of confirming that the cells obtained in step (1) contain CD3, CD1a, and CD5 positive T cell may be a step of determining whether to proceed from step (1) to step (2).
• the culture period in step (1) is not particularly limited as long as CD3 and CD5 positive cells are obtained, but may be, for example, 10 to 30 days.
• the culture period in step (2) of the first method of the present invention can be determined by confirming the formation of CD3 positive and CD4/CD8 double positive immature T cells.
• the next step (3) may be proceeded to. That is, the step of confirming that the cells obtained in step (2) contain CD3 positive and CD4/CD8 double positive immature T cells can be a step of determining whether to proceed from step (2) to step (3).
• the next step (3) may be proceeded to. That is, the step of confirming that the cells obtained in step (2) are CD3 positive and CD4/CD8 double positive immature T cells and further contain CD5 and CD1a positive cells can be a step of determining whether to proceed from step (2) to step (3).
• the culture period in step (2) is not particularly limited as long as CD3 positive and CD4/CD8 double positive cells are obtained, but may be, for example, 3 to 10 days.
• the culture period in step (3) of the first and second methods of the present invention can be determined by confirming the formation of mature helper T cells that are CD1a and CD8 negative, and CD5 positive and CD4 single positive, or a mixture of the mature helper T cells and mature killer T cells that are CD5 and CD8 positive and CD1a and CD4 negative.
• a mixture of mature helper T cells that are CD1a and CD8 negative, and CD5 positive and CD4 single positive, or a mixture of the mature helper T cells and mature killer T cells that are CD5 and CD8 positive and CD1a and CD4 negative in the cells obtained in step (3), it can be seen that a cell population containing helper T cells that are CD4 single positive and CD8 negative has been obtained.
• mature helper T cells and mature killer T cells may be formed simultaneously.
• the culture period in step (3) is not particularly limited as long as mature helper T cells that are CD1a and CD8 negative, CD5 positive, and CD4 single positive are obtained, but may be, for example, 2 to 20 days.
• the cells obtained in step (3) contain T cells that highly express CD28.
• the "high expression” means that the CD28 expression level is equivalent to that of primary T cells obtained from peripheral blood. Since CD28 is a co-stimulatory receptor essential for T cell activation, it is one of the indicators that the obtained cells are functional helper T cells.
• the step of confirming that the cells obtained in step (3) contain T cells that highly express CD28 may be a step of determining whether the helper T cells obtained by the method of the present invention are functional helper T cells.
• the cells obtained in step (3) show a CD28 expression level equivalent to that of mature T cells present in human peripheral blood.
• the "equivalent” means that the CD28 expression level is close to the CD28 expression level of a naive T cell fraction that expresses CD28 most highly in flow cytometry analysis, to a degree that it is difficult to distinguish.
• the cell culture in steps (1) and (2) is performed while maintaining the cell density at subconfluence.
• Confluence is a state in which the cell adhesion surface of the cell culture substrate is completely covered with cultured cells, leaving no room for the cells to grow as a monolayer.
• subconfluent refers to a state in which 50-90% of the adhesion surface of the culture vessel is occupied by cultured cells, and the occupancy rate of the adhesion surface of the culture vessel by the cultured cells may be 60-80%.
• the first and second methods of the present invention can produce a cell population containing CD4 single positive and CD8 negative helper T cells.
• the term "cell population” means that the cell population may contain a variety of cells, such as cells classified as CD4 single positive/CD8 negative helper T cells, cells with different cell surface antigen expression patterns, and CD4 negative/CD8 positive killer T cells.
• the obtained CD4 positive/CD8 negative helper T cells and CD4 negative/CD8 positive killer T cells may be used after being isolated individually or as a mixture. When isolating the cells, a method well known in the art can be used.
• a method of labeling with antibodies against CD4, CD8, CD3 and/or CD45 and isolating using a flow cytometer, or a method of purifying using an affinity column on which a desired antigen is immobilized, etc. can be mentioned.
• a nucleic acid encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR) molecule reactive to a tumor-associated antigen can be introduced from outside the cell into the pluripotent stem cells or the iPS cells, the T precursor cells, the hematopoietic stem cells, the immature T cells, or the mature T cells.
• CAR chimeric antigen receptor
• TCR T cell receptor
• cDNAs encoding the TCR ⁇ and ⁇ chains or cDNAs encoding the ⁇ and ⁇ chains can be prepared and incorporated into an expression vector. These cDNAs may be incorporated into a viral vector or a non-viral vector (transposon vector) using, for example, the Gibson assembly system.
• a gene in which cDNAs encoding the TCR ⁇ and TCR ⁇ chains are linked via a T2A sequence is linked downstream of a ubiquitin promoter, and a marker gene such as EGFR with the ligand binding site and intracellular domain removed (EGFRt, truncated EGFR) or CD19 lacking the intracellular domain is linked to the IRES (internal ribosome entry site) sequence downstream of the gene, and this construct may be incorporated into a viral or non-viral vector.
• the cDNAs encoding the TCR ⁇ and ⁇ chains may each be incorporated into a separate expression vector.
• cDNA encoding CAR can be prepared and incorporated into an expression vector.
• total RNA is extracted from lymph nodes of an animal immunized with a desired tumor-associated antigen, and cDNA is synthesized using the total RNA as a template; the light and heavy chains of the variable regions of a monoclonal antibody against the tumor-associated antigen are amplified separately, linked using a flexible linker, and amplified by assembly PCR; a CAR construct can be prepared by linking the sequences encoding the fused light and heavy chains to a sequence encoding the intracellular domain of the CAR molecule via a sequence encoding the transmembrane domain of the CAR molecule.
• EGFR EGFRt
• the flexible linker that connects the light and heavy chains of the variable regions of a monoclonal antibody is a linker peptide of about 5 to 20 amino acid residues, and is known in the art (see, for example, Ueda T, et al. Cancer Sci. 2020; 111:1478-1490. Kawasaki Tomomi et al., SCEJ 72nd Annual Meeting (Kyoto, 2007) H209.).
• the antigen-binding domain to be incorporated into the CAR may be prepared using phage display without immunizing animals with the antigen.
• an antibody that specifically binds to a desired tumor-associated antigen may be screened from a phage display antibody library expressing the Fab regions of a large number of human antibodies.
• a TCR or CAR reactive to a tumor-associated antigen may be introduced into the cells alone, or a TCR and a CAR may be introduced simultaneously.
• the expression vectors may be the same or different.
• the TCR and the CAR may be reactive to the same tumor-associated antigen, or may be reactive to different tumor-associated antigens.
• a viral vector and a non-viral vector can be used.
• the viral vector include lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus, as well as animal cell expression plasmids.
• Lentivirus or retrovirus can be preferably used.
• retrovirus or lentivirus infection is performed, a spin-infection method or the like may be used.
• An example of a non-viral vector is the piggyBac (registered trademark) vector, which is a transposon vector.
• a genome editing technique may be used.
• Examples of such genome editing techniques include the CRISPR/Cas9 method, the CRISPR/MAD method, and the CRISPR/CAS3 method.
• Methods for gene introduction using non-viral vectors or methods for introducing guide RNA and donor DNA for genome editing include the lipofection method, the liposome method, the calcium phosphate coprecipitation method, the DEAE dextran method, the microinjection method, and the electroporation method.
• Gene introduction of TCR and/or CAR may be performed at the TCR locus or other loci (e.g., the ⁇ 2-microglobulin locus). When modification or destruction of existing genes including TCR by the introduced gene is not desired, gene introduction may be performed at a safe harbor locus.
• An example of the safe region is the AAVS1 (Adeno-associated virus integration site 1) region in the human genome.
• methods for gene introduction targeting the safe region include the CRISPR/Cas9 method and the TALEN method.
• Tumor-associated antigens are antigens that are expressed specifically or non-specifically in tumors, including antigens derived from proteins overexpressed in tumor cells and their mutants, antigens derived from tumor viruses, certain types of differentiation antigens, and novel tumor-associated antigens (neoantigens) resulting from genetic mutations and splice abnormalities. In the case of protein antigens, they may be fragmented peptides (peptide fragments).
• Antigens that are specifically or non-specifically expressed in tumors include WT1, GPC3, BCMA, XAGE1, MUC1, MUC5A1, MUC6, EGFRvIII, HER-2/neu, MAGE-A1, MAGE-A3, 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, gene
• neoantigens include, but are not limited to, unmutated p53, p53 with genetic mutations, p53 mutants, NY-ESO-1, PSMA, ETV6-AML, CEA, PSA, AFP, hTERT, EpCAM, ALK, androgen receptor, EphA2, CYP1B1, OY-TES-1, MAD-
• Viral antigens include, but are not limited to, HBV, HBs, HPV, EBV, LMP1, EBV, LMP2, EBNA, HPV-E1, HPV-E2, HPV-E6, HPV-E7, HTLV-1 Tax, and HBZ.
• the pharmaceutical containing the cell population produced by the method of the present invention can be used as a preventive or therapeutic agent for cancer in mammals.
• the pharmaceutical of the present invention may be produced by a method commonly used in the field of formulation technology.
• the pharmaceutical of the present invention may contain a pharma- ceutical acceptable additive. Examples of the additive include a cell culture medium, physiological saline, and a suitable buffer solution (e.g., a phosphate buffer solution).
• the pharmaceutical of the present invention can be produced by suspending the cells of the present invention in physiological saline or a suitable buffer (e.g., phosphate buffer), etc.
• the amount of the cells to be administered may contain, for example, 1 ⁇ 10 7 or more, 1 ⁇ 10 8 or more, or 1 ⁇ 10 9 or more cells.
• the content of the cells can be adjusted in consideration of the sex, age, weight, condition of the affected area, and cell condition of the subject to be administered.
• the pharmaceutical of the present invention may contain dimethyl sulfoxide (DMSO) and serum albumin for the purpose of protecting the cells. It may also contain antibiotics and the like to prevent bacterial contamination.
• DMSO dimethyl sulfoxide
• serum albumin for the purpose of protecting the cells. It may also contain antibiotics and the like to prevent bacterial contamination.
• the pharmaceutical of the present invention may contain other components that are acceptable for formulation (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.).
• the pharmaceutical containing the cells of the present invention can be stored frozen.
• the storage temperature is not particularly limited as long as it is suitable for storing cells. Examples include -20°C, -80°C, and -120 to -196°C, with -150°C or lower being preferred.
• the cells may be stored in an appropriate container such as a cryovial or a cryobag.
• the pharmaceutical agent of the present invention is used for the prevention or treatment of cancer.
• cancer include, but are not limited to, ovarian cancer, hepatoblastoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, malignant melanoma, non-small cell lung cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid cancer, colorectal cancer, head and neck cancer, brain tumor, multiple myeloma, and B-cell non-Hodgkin's lymphoma.
• the cells of the present invention can kill cells expressing tumor-associated antigens, and can therefore be used as a killing agent for cells expressing tumor-associated antigens.
• the killing agent can be manufactured and used in the same manner as the pharmaceutical.
• iPS cells were generated from CD3 positive T cells isolated from human peripheral blood using the method described in Non-Patent Document 3.
• a total T cell isolation kit Pan T Cell Isolation Kit, Miltenyi Biotec, catalog number: 130-096-535) was used to isolate the T cells.
• the T cell differentiation culture medium was prepared by adding 15% fetal bovine serum (Nacalai Tesque, 01863-48), PSG (penicillin-streptomycin-L-glutamine) solution (100x, Sigma-Aldrich, catalog number: G1146-100ML), ITS (insulin-transferrin-sodium selenite) supplement (100x, Invitrogen, catalog number: 41400045), 2-phospho-L-ascorbic acid (final concentration 50 ⁇ g/mL, Sigma-Aldrich, catalog number: A8960-5G), human recombinant IL-7 (final concentration 5 ng/mL, PeproTech, catalog number: 200-0 7), human recombinant Flt3L (final concentration 5 ng/mL, PeproTech, catalog number: 300-19), human recombinant SCF (final concentration 10 ng/mL, R&D Systems, catalog number: 255-SC), human recombinant SDF-1 ⁇ (final concentration 10 nM
• the obtained cells were analyzed by flow cytometry, and it was confirmed that the cells differentiated from the iPS cells contained T cell precursors that were positive for both CD3 and CD5.
• RetroNectin 150 ⁇ L of PBS containing 5 ⁇ g/mL human DLL4-Fc and RetroNectin (RetroNectin, registered trademark, Takara Bio) was added to each well of a new 48-well cell culture plate and left to stand overnight at 4°C to coat the cell culture plate with human DLL4-Fc and RetroNectin. RetroNectin was used as a cell adhesion factor.
• Process (2) (15th day) The cell culture plate coated with DLL4-Fc and retronectin was washed once with 1 mL/well of PBS, and the T cell differentiation medium was added at 500 ⁇ L/well. A cell suspension (2-4 ⁇ 10 cells/mL) containing the above-mentioned T cell differentiation medium was added at 500 ⁇ L/well. These cells were cultured by replacing half of the culture medium with fresh T cell differentiation medium every 2-3 days. The cells were cultured at 37° C. in a 5% CO 2 environment while the medium was being exchanged. During the culture, the cell density was maintained at about 70-80% confluence or less.
• the obtained cells were analyzed by flow cytometry, and it was confirmed that the cells differentiated from the T precursor cells were CD3 positive, CD4/CD8 double positive, and further contained CD5 and CD1a positive T cells.
• Retronectin solution diluted to 5 ⁇ g/mL with PBS was added to each well of a new 48-well cell culture plate, and the plate was left to stand overnight at 4°C to coat the cell culture plate with Retronectin.
• Process (3) (22nd day) A culture medium was prepared by removing human recombinant Flt3L from the T cell differentiation culture medium (Flt3L-free T cell differentiation culture medium). The new cell culture plate coated with RetroNectin was washed once with 1 mL/well of PBS. After washing, the Flt3L-free T cell differentiation medium was added at 500 ⁇ L/well. The Flt3L-free T cell differentiation medium was added at 500 ⁇ L/well. The cells were then incubated at 37° C. for 5 min while replacing half of the medium with fresh Flt3L-free T cell differentiation medium every 2 to 3 days. The cells were cultured in a 25% CO2 environment.
• new 48-well cell culture plates were coated with Retronectin.
• the new 48-well cell culture plate coated with RetroNectin was washed once with 1 mL/well of PBS, and the Flt3L-free T cell differentiation medium was added at 500 ⁇ L/well.
• the culture medium in the wells containing the T cells cultured in the cell culture plate was then reduced to 500 ⁇ L, and all the cells in each well were transferred together with the culture medium to the wells of the new 48-well cell culture plate, and further cultured at 37° C. in a 5% CO 2 environment.
• step (1) [Effect of coating cell culture plates with DLL4-Fc and retronectin on the generation of CD4 single positive helper T cells from iPS cells (1)]
• step (3) (days 22 to 35) shown in Example 1
• cells were cultured by changing the combination of coating of the cell culture plate in each step.
• the coating of DLL4-Fc and RetroNectin, the culture of iPS cells, and the culture conditions were performed in accordance with Example 1.
• Table 1 shows the combinations of coating of cell culture plates with DLL4-Fc and Retronectin in each step.
• coating combination (c) is the coating condition used in Example 1. That is, in coating combination (c), cells were cultured using a cell culture plate coated with DLL4-Fc only in step (1) (days 1 to 14), a cell culture plate coated with DLL4-Fc and Retronectin in step (2) (days 15 to 21), and a cell culture plate coated with Retronectin only in step (3) (days 22 to 35).
• the results of flow cytometric analysis of the generation of CD4 single-positive helper T cells in coating combinations (a) to (h) are shown in FIG. 1.
• Figure 1 and Table 1 show that the presence or absence of DLL4-Fc (T cell differentiation inducer) and retronectin (cell adhesion factor) in each step is important for producing CD4 single positive helper T cells from iPS cells. That is, by applying a combination of DLL4-Fc single coating in step (1) (days 1 to 14), double coating of DLL4-Fc and retronectin in step (2) (days 15 to 21), and retronectin single coating in step (3) (days 22 to 35) (coating combination (c)), the proportion of CD4 single positive helper T cells in all cells was found to be significantly higher than other combinations. On the other hand, as shown in Figure 1, it was shown that the presence or absence of coating of the cell culture plate is not important for producing CD8 single positive killer T cells from iPS cells.
• DLL4-Fc T cell differentiation inducer
• retronectin cell adhesion factor
• Table 2 shows the combination of coating of the cell culture plate with DLL4-Fc and Retronectin in step (3).
• the coating conditions in steps (1) and (2) were the same.
• “coating combination (c)” is the coating condition used in Example 1.
• the results of flow cytometry analysis of the generation of CD4 single positive helper T cells in coating combinations (i), (c), and (d) are shown in FIG. 2.
• “coating combination (c)” in which the cell culture plate was coated with Retronectin showed a higher generation of CD4 single positive helper T cells than "coating combination (i)” in which the cell culture plate was not coated and “coating combination (d)” in which the cell culture plate was double coated with DLL4-Fc and Retronectin.
• Example 3 [Effect of coating cell culture plates with DLL4-Fc and retronectin on the generation of CD4 single positive helper T cells from iPS cells (3)]
• the results obtained in Example 3 showed that coating the culture plate with RetroNectin alone in step (3) resulted in a large amount of CD4 single positive helper T cells generated.
• the effects of the combination of coating the cell culture plate with DLL4-Fc and RetroNectin in step (1) (days 1 to 14) and step (2) (days 15 to 21) on the generation of CD4 single positive helper T cells from iPS cells were examined. Specifically, the effect of coating RetroNectin in addition to DLL4-Fc in steps (1) and (2) was examined.
• the coating of DLL4-Fc and RetroNectin, the culture of iPS cells, and the culture conditions were performed in accordance with Example 1.
• the percentage of CD3 and CD5 positive/CD1a negative/CD4 single positive mature helper T cells in the cell population obtained on day 35 of the culture schedule was compared in the presence and absence of CHIR-99021. Four independent tests were performed, and the results are shown in Figure 6. It was shown that the percentage of CD4 single positive mature helper T cells was statistically significantly higher in the presence of CHIR-99021 than in the absence of CHIR-99021. Statistical analysis was performed using paired t-test.
• CD40L expression by TCR stimulation of CD4 single positive helper T cells CD4 single positive helper T cells (CD4SP iPSC-T cells) generated from iPS cells (1)
• CD40L CD40 ligand
• CD40L expression of a cell population containing CD4SP iPSC-T cells matured by DLL4-Fc/retronectin switching in the coating of a cell culture plate was analyzed by flow cytometry, and compared with conventional CD4SP iPSC-T cells matured from iPS cells using an anti-CD3 antibody (OKT3) according to a previous report (International Publication WO 2018/135646).
• the flow cytometry analysis of the CD4SP iPSC-T cells matured by DLL4-Fc/retronectin switching was performed 4 days after TCR stimulation with an anti-CD3 antibody coated on a cell culture plate in the presence of IL-7, IL-12, IL-15, IL-18, and IL-21.
• the analysis results are shown in FIG. 7.
• the upper panel shows the analysis results for conventional CD4SP iPSC-T cells matured with an anti-CD3 antibody
• the lower panel shows the analysis results for CD4SP iPSC-T cells matured by DLL4-Fc/retronectin switching.
• CD4SP iPSC-T cells matured by the DLL4-Fc/retronectin switching showed higher CD40L expression upon TCR stimulation compared to CD4SP iPSC-T cells matured with anti-CD3 antibody. That is, CD4SP iPSC-T cells matured by the DLL4-Fc/retronectin switching contained 24.3% highly CD40L expressing cells, whereas CD4SP iPSC-T cells matured with anti-CD3 antibody contained only 2.91% highly CD40L expressing cells. This suggests that CD4SP iPSC-T cells matured by the DLL4-Fc/retronectin switching also have superior helper T cell function compared to conventional CD4SP iPSC-T cells matured with anti-CD3 antibody.
• CD4 single positive helper T cells ATO-CD4SP iPSC-T cells
• CD8 single positive killer T cells ATO-CD8SP iPSC-T cells
• ATO artificial thymic organ
• CD4 single positive helper T cells and CD8 single positive killer T cells were isolated from primary T cells derived from peripheral blood of healthy subjects. CD40L expression of these cells was analyzed by flow cytometry.
• Flow cytometry analysis was performed 24 hours after stimulation of each cell with anti-CD3 antibody (1 ⁇ g/mL) immobilized on a cell culture plate in the presence of IL-7 (5 ng/mL), IL-12 (50 ng/mL), IL-15 (5 ng/mL), IL-18 (50 ng/mL), and IL-21 (20 ng/mL).
• Thpok is a master transcription factor for helper T cell differentiation and is essential for helper T cells to exert their functions.
• PLZF is a master transcription factor for innate immune T cell differentiation such as natural killer T (NKT) cells.
• the expression of the two transcription factors in these T cells was compared with that of adaptive immune system primary T cells obtained from peripheral blood.
• the expression of the transcription factors was measured by TaqMan qPCR (quantitative PCR).
• Assay ID: Hs00757087_g1 (Gene: Zbtb7b) (Thermo Fisher Scientific, Catalog No.: 4331182) was used as a reagent set.
• the measurement results were normalized by the expression level of ⁇ -actin.
• the results are shown in Figure 9.
• CD4 single positive T cells had higher expression levels of Thpok compared to CD8 single positive T cells.
• CD4SP iPSC-T cells had higher expression levels of Thpok compared to CD8SP iPSC-T cells, suggesting that they exhibit a phenotype related to helper T cells.
• both CD4SP iPSC-T cells and CD8SP iPSC-T cells expressed PLZF, which is significantly different from primary adaptive immune T cells obtained from peripheral blood, which do not express PLZF at all.
• PLZF which is significantly different from primary adaptive immune T cells obtained from peripheral blood, which do not express PLZF at all.
• CD4SP iPSC-T cells and CD8SP iPSC-T cells have a unique innate immune-like phenotype.
• Thpok is a master transcription factor for helper T cell differentiation, and is an essential transcription factor for helper T cells to exert their functions.
• Runx3 is a master transcription factor for killer T cell differentiation.
• CD4SP iPSC-T cells and CD8SP iPSC-T cells were isolated from a cell population containing CD4SP iPSC-T cells and CD8 single positive killer T cells (CD8SP iPSC-T cells) that were matured without using feeder cells by DLL4-Fc/retronectin switching in the coating of cell culture plates.
• CD4 single positive helper T cells ATO-CD4SP iPSC-T cells
• ATO-CD8SP iPSC-T cells were isolated from T cells produced from iPS cells by artificial thymic organ (ATO, see Non-Patent Document 6) culture on feeder cells.
• CD4 single positive helper T cells and CD8 single positive killer T cells were isolated from primary T cells derived from peripheral blood of healthy subjects. Total mRNA was isolated from these cells immediately after the maturation process, and Thpok and Runx3 expression was measured by TaqMan qPCR (quantitative PCR).
• Thpok the reagent set used was Assay ID: Hs00757087_g1 (Gene: Zbtb7b) (Thermo Fisher Scientific, Catalog No.: 4331182).
• Runx3 the reagent set used was Assay ID: Hs01091094_m1 (Gene: Runx3) (Thermo Fisher Scientific, Catalog No.: 4331182).
• CD4SP iPSC-T cells matured without the use of feeder cells, as well as ATO-CD4SP iPSC-T cells and CD4 single-positive helper T cells derived from peripheral blood-derived primary T cells, had statistically significantly higher Thpok expression levels than the corresponding CD8 single-positive killer T cells, suggesting that they exhibit a phenotype associated with helper T cells. Furthermore, the Thpok expression levels of feeder-free CD4SP iPSC-T cells were significantly higher than those of peripheral blood-derived CD4 single-positive helper T cells, suggesting that they have high helper T cell function. On the other hand, no significant difference in Runx3 expression was observed between CD4 single-positive helper T cells and CD8 single-positive killer T cells.
• CD4 single positive helper T cells CD4SP iPSC-T cells
• CD8 single positive killer T cells CD8SP iPSC-T cells generated from iPS cells
• CD4SP iPSC-T cells and CD8SP iPSC-T cells were isolated from a cell population containing CD4SP iPSC-T cells and CD8 single positive killer T cells (CD8SP iPSC-T cells) that were matured without using feeder cells by DLL4-Fc/retroNectin switching in the coating of a cell culture plate.
• CD4 single positive helper T cells ATO-CD4SP iPSC-T cells
• CD8 single positive killer T cells ATO-CD8SP iPSC-T cells
• ATO artificial thymic organ
• CD4 single positive helper T cells and CD8 single positive killer T cells were isolated from primary T cells derived from peripheral blood of a healthy individual.
• IL-7 5 ng/mL
• IL-12 50 ng/mL
• IL-15 5 ng/mL
• IL-18 50 ng/mL
• IL-21 20 ng/mL
• the proliferation rates of the above cells are shown in Figure 11.
• a line connects pairs of independent (CD4 and CD8) tests (shown as dots).
• the proliferation rate of feeder-free CD4SP iPSC-T cells was significantly higher than that of feeder-free CD8SP iPSC-T cells.
• the proliferation rate of CD8 single positive killer T cells was significantly higher than that of CD4 single positive helper T cells.
• the proliferation rates of ATO-CD4SP iPSC-T cells and ATO-CD8SP iPSC-T cells were almost the same.
• CD4 single positive helper T cells CD4 single positive helper T cells
• CD8 single positive killer T cells CD8SP iPSC-T cells
• an anti-CD3 antibody (OKT3) diluted to 1 ⁇ g/mL with PBS was added to each well of a 96-well flat-bottom cell culture plate, and the plate was left to stand overnight at 4° C., followed by washing once with PBS to prepare an anti-CD3 antibody-coated cell culture plate.
• the cell population was suspended in ⁇ MEM containing 20% fetal bovine serum, PSG (penicillin-streptomycin-L-glutamine) solution (100x), ITS (insulin-transferrin-sodium selenite) supplement (100x), 2-phospho-L-ascorbic acid (50 ⁇ g/mL), human recombinant IL-7 (5 ng/mL), human recombinant IL-15 (5 ng/mL), human recombinant IL-21 (20 ng/mL), IL-12 (50 ng/mL) and IL-18 (50 ng/mL), and added to the anti-CD3 antibody-coated cell culture plate (2 ⁇ 10 4 cells/200 ⁇ L/well), and then cultured at 37° C.
• PSG penicillin-streptomycin-L-glutamine
• ITS insulin-transferrin-sodium selenite
• 2-phospho-L-ascorbic acid 50 ⁇ g/mL
• the culture medium used for the culture medium replacement was ⁇ MEM containing 20% fetal bovine serum, PSG solution (100 ⁇ ), ITS supplement (100 ⁇ ), 2-phospho-L-ascorbic acid (50 ⁇ g/mL), human recombinant IL-7 (5 ng/mL), and human recombinant IL-15 (5 ng/mL).
• the cell surface markers of the post-expansion cell population were analyzed by flow cytometry and compared with the pre-expansion cell population.
• Fresh human peripheral blood mononuclear cells (PBMCs) were also analyzed by flow cytometry for cell surface markers.
• PBMCs were prepared using lymphocyte separation tubes (Greiner Bio-One) according to the manufacturer's instructions. The results are shown in Figure 12.
• CD2 and CD5 are well-established adaptive immune markers expressed in all T cells.
• CD28 is a naive marker.
• Figure 12 shows that CD4SP iPSC-T cells expressed and maintained the expression of adaptive and naive markers even after expansion, compared with CD8SP iPSC-T cells.
• CD5 and CD28 in CD4SP iPSC-T cells were shown to be equivalent to that of fresh PBMCs from healthy individuals even after expansion.
• These adaptive immune and naive markers are highly expressed in young T cells with fewer exhausted traits, and are known to positively correlate with the efficacy of immune cell therapy.
• CD4 single positive helper T cells CD4SP iPSC-T cells
• CD8 single positive killer T cells CD8SP iPSC-T cells
• Anti-CD19 CAR anti-CD19 CAR-IRES-EGFR
• Example 1 a retrovirus as previously reported (Ueda T, et al. Nat Biomed Eng. 2023; 7:24-37), and the EGFR expression of the resulting cells was analyzed by flow cytometry.
• Figure 13A shows a histogram of EGFR expression, and EGFR expression was confirmed in both CD4SP iPSC-T cells and CD8SP iPSC-T cells.
• CD4SP iPSC-T cells and CD8SP iPSC-T cells transfected with anti-CD19 CAR-IRES-EGFR were isolated by flow cytometry (FACSAria, Becton Dixon), and 1 ⁇ 10 4 cells each were co-cultured with CD19-positive human pre-B cell leukemia cell line Nalm6 (5 ⁇ 10 4 cells) at 37° C. and 5% CO 2 for 24 hours.
• the co-culture was performed using ⁇ MEM (200 ⁇ L) containing 15% fetal bovine serum, PSG, and ITS in the presence of IL-7 (5 ng/mL) and IL-15 (5 ng/mL).
• IL-7 5 ng/mL
• IL-15 5 ng/mL
• the amount of cytokines (IFN- ⁇ , IL-2, and TNF ⁇ ) in the culture supernatant was measured using the BD (registered trademark) Cytometric Bead Array Human Th1/Th2/Th17 cytokine kit (BD Biosciences).
• BD registered trademark Cytometric Bead Array Human Th1/Th2/Th17 cytokine kit
• CD4SP iPSC-T cells generated according to Example 1 are useful for cancer immunotherapy.
• CD4SP iPSC-T cells CD4 single positive helper T cells
• CD8SP iPSC-T cells CD8 single positive killer T cells generated from iPS cells
• CD4SP iPSC-T cells and CD8SP iPSC-T cells were isolated by flow cytometry (FACSAria, Becton Dickinson) from the cell population containing CD4SP iPSC-T cells and CD8SP iPSC-T cells prepared according to Example 1. These cells were used as effector cells (E) and as target cells (T) to examine the killing effect on the pre-B cell leukemia cell line Nalm6.
• CD4SP iPSC-T cells CD4 single positive helper T cells
• CD8SP iPSC-T cells CD8 single positive killer T cells generated from iPS cells
• CD4SP iPSC-T cells and CD8SP iPSC-T cells were isolated from the cell population containing CD4SP iPSC-T cells and CD8SP iPSC-T cells prepared according to Example 1. These cells were used as effector cells (E) and the effects of IL-7 and IL-15 in killing the pre-B cell leukemia cell line Nalm6 as target cells (T) were examined.
• IL-7 5 ng/mL
• IL-15 5 ng/mL
• CD4SP iPSC-T cells and CD8SP iPSC-T cells on Nalm6 cells were almost equivalent.
• the cell killing effect of CD4SP iPSC-T cells was found to be stronger than that of CD8SP iPSC-T cells.
• the cytotoxic activity of CD4SP iPSC-T cells was less dependent on the homeostatic cytokines IL-7 and IL-15 than that of CD8SP iPSC-T cells. This is a necessary requirement for T cells to maintain their activity even in the tumor microenvironment where the supply of homeostatic cytokines is insufficient.
• anti-CD19 CAR anti-CD19 CAR-IRES-EGFR
• CD4SP T cells and CD8SP T cells expressing anti-CD19 CAR were isolated by flow cytometry (FACSAria, Becton Dixon). These T cells (4 ⁇ 10 3 cells) were used as effector cells (E) and co-cultured with CD19 positive human pre-B cell leukemia cell line Nalm6 or CD19 negative human leukemia cell line K562 (2 ⁇ 10 4 cells) as target cells (T) under 37° C. and 5% CO 2 environment.
• target cells (2 ⁇ 10 4 cells) were added to continue the co-culture, and on the seventh day of co-culture, the proliferation of the cancer cells (target cells) was measured by FACS.
• the co-culture was performed using ⁇ MEM containing 15% fetal bovine serum, PSG, and ITS in the absence of cytokines, in the presence of IL-7 (5 ng/mL), IL-15 (5 ng/mL), and IL-7 and IL-15.
• the number of live cells of Nalm6 cells and K562 cells was measured by FACS. The results when Nalm6 cells were used as target cells are shown in FIG. 16A, and the results when K562 cells were used as target cells are shown in FIG. 16B.
• Modified feeder-free iPSC-T cells exhibited cytotoxic activity against CD19-positive Nalm6 cells similar to that of ATO-iPSC-T cells. Both CD4SP T cells derived from modified feeder-free iPSC-T cells and ATO-iPSC-T cells exhibited superior target-specific cytotoxic activity to CD8SP T cells. In contrast, in primary T cells, in vitro cytotoxic activity was observed exclusively in CD8SP T cells. Unlike CD8SP iPSC-T cells generated as previously reported (Maeda T, et al. Cancer Res. 2016; 76:6839-6850, Kawai Y, et al. Molecular Therapy.
• the modified feeder-free iPSC-T cells, ATO-iPSC-T cells, and primary T cells did not show NK activity against NK (natural killer)-sensitive K562 cells. This suggests that, unlike the T cells generated as previously reported, there is a low risk of graft-versus-host disease due to unintended NK activity.
• CD4 single positive helper T cells CD4SP iPSC-T cells
• CD8 single positive killer T cells CD8SP iPSC-T cells
• a human pre-B cell leukemia cell line Nalm6 (2 ⁇ 10 5 cells per mouse) expressing luciferase was intravenously transplanted into NOD-SCID IL2Rgc null (NSG) mice (day 0).
• CD4SP iPSC-T cells or CD8SP iPSC-T cells (5 ⁇ 10 6 cells per mouse) prepared according to Example 1, expanded without feeder cells, and transfected with anti-CD19 CAR were administered by intravenous injection.
• a culture solution not containing the T cells was administered intravenously.
• luciferase luminescence was observed by in vivo imaging on days 14, 21, and 28. The results are shown in FIG. 17A. In the control group, luciferase luminescence increased over time, indicating that the transplanted Nalm6 had taken root and proliferated.
• the luciferase luminescence level was lower in the CD4SP iPSC-T cell-administered group compared to the control group, revealing that Nalm6 proliferation was suppressed.
• the Nalm6 proliferation inhibitory effect in the CD4SP iPSC-T cell-administered group was also stronger than that in the CD8SP iPSC-T cell-administered group.
• CD4SP iPSC-T cells showed significantly improved survival rates compared to CD8SP iPSC-T cells, indicating that they have superior in vivo tumor suppression effects.
• iPS cells were generated from CD3 positive T cells isolated from human peripheral blood using the method described in Non-Patent Document 3.
• a total T cell isolation kit Pan T Cell Isolation Kit, Miltenyi Biotec, catalog number: 130-096-535) was used to isolate the T cells.
• DLL4-Fc is a recombinant protein in which the Fc domain of human IgG1 is added to the C-terminus of the extracellular domain (residues 1 to 524) of human DLL4 (Delta-Like Protein 4), and was used as a T cell differentiation inducer.
• Day 1 to Day 21 Process (1) (Day 1) Embryoid bodies formed by suspension culture of iPS cells for 14 days were collected and dispersed into single cells by repeated aspiration and ejection six times using a syringe equipped with a 21-gauge needle. Cell debris was removed using a cell strainer. The obtained cells were suspended in T cell differentiation medium. The cell culture plate coated with DLL4-Fc was washed with 1 mL/well of PBS. After washing once, the T cell differentiation medium was added at 500 ⁇ L/well. The cell suspension containing 2 ⁇ 10 3 cells was added at 500 ⁇ L/well. The cells were cultured at 37° C. in a 5% CO 2 environment, with half of the culture medium being replaced with fresh T cell differentiation medium every 2 to 3 days.
• the T cell differentiation culture medium contained 15% fetal bovine serum (Corning), ITS (insulin-transferrin-sodium selenite) supplement (100x), 2-mercaptoethanol (final concentration 55 ⁇ M, Thermo Fisher Scientific), GSK-3 inhibitor CHIR-99021 (1 ⁇ M, Wako), 2-phospho-L-ascorbic acid (final concentration 50 ⁇ g/mL), and GlutaMAX (100x, registered trademark).
• ⁇ MEM (Thermo Fisher Scientific) containing human recombinant SCF (final concentration 50 ng/mL), human recombinant TPO (final concentration 50 ng/mL), human recombinant IL-7 (final concentration 50 ng/mL), human recombinant Flt3L (final concentration 50 ng/mL), human recombinant SDF-1 ⁇ (final concentration 10 nM, PeproTech) and the p38 inhibitor SB203580 (final concentration 15 ⁇ M, Tocris Bioscience) was used.
• the obtained cells were analyzed by flow cytometry, and it was confirmed that the cells differentiated from the iPS cells were CD4/CD8 double positive.
• Process (2) (22nd day) PBS containing 5 ⁇ g/mL RetroNectin (registered trademark, Takara Bio) was added to each well of a new 48-well cell culture plate, and the plate was incubated at 37° C. for 2 hours to inoculate the cell culture plate with RetroNectin. Retronectin was used as a cell adhesion factor.
• RetroNectin registered trademark, Takara Bio
• the culture medium used was ⁇ MEM containing 15% fetal bovine serum, ITS (insulin-transferrin-sodium selenite) supplement (100x), GlutaMAX (100x), 2-phospho-L-ascorbic acid (50 ⁇ g/mL), PSG (penicillin-streptomycin-L-glutamine) solution (x100, Sigma-Aldrich), GSK-3 inhibitor CHIR-99021 (1 ⁇ M, Wako), IL-7 (50 ng/mL), SDF-1 ⁇ (10 nM), and p38 inhibitor SB203580 (10 mM).
• Process (3) (24th day) The cells obtained in step (2) were collected, washed, and then seeded on a freshly prepared cell culture plate coated with RetroNectin. The cells were cultured in a culture medium having the same composition as that used in step (2). (but without anti-CD3 antibody) for 4 to 11 days in a 37°C, 5% CO2 environment.
• the cells obtained in step (3) were analyzed by flow cytometry, confirming the presence of CD4 single positive helper T cells (Figure 18A). Further analysis of the CD4 single positive helper T cell subset by flow cytometry showed high expression of CD5 and TCR ⁇ / ⁇ chains, but little expression of CD1a ( Figure 18B).
• the GSK-3 inhibitor CHIR-99021 functions as a Wnt pathway activator.
• steps (1) to (3) of Example 17 the generation of CD4 single positive helper T cells from hematopoietic stem cells derived from iPS cells in the presence or absence of CHIR-99021 was examined. The results of flow cytometry analysis of the obtained cells are shown in FIG. 19. As a result, the proportion of CD4 single positive helper T cells was 3.08% in the absence of CHIR-99021, but 10.3% in the presence of CHIR-99021, demonstrating the effect of adding CHIR-99021 on the generation of CD4 single positive helper T cells.
• CD4 and CD8 expression after expansion of CD4 and CD8 single positive T cells obtained in the presence of CHIR-99021 According to Example 17, the CD4 and CD8 expression of mature T cells generated from hematopoietic stem cells derived from iPS cells in the presence of CHIR-99021 was analyzed by flow cytometry. The results are shown in FIG. 21 (Before expansion). The results confirmed the generation of CD4 single positive T cells and CD8 single positive T cells. The CD4 and CD8 single positive T cells were sorted by flow cytometry and expanded for 14 days using anti-CD3 and anti-CD28 antibody-bound beads (Dynabeads, registered trademark), after which the CD4 and CD8 expression was analyzed by flow cytometry. The results are shown in FIG. 21 (After sorting and expansion). The results showed that both the obtained CD4 single positive T cells and CD8 single positive T cells could be expanded and maintained the expression of co-receptors.
• CD40L expression by TCR stimulation of CD4 single positive helper T cells (CD4SP iPSC-T cells) generated from iPS cells in the presence of CHIR-99021)
• CD4SP iPSC-T cells CD4 single positive helper T cells
• CHIR-99021 CHIR-99021
• a cell population containing CD4SP iPSC-T cells was prepared from iPS cells.
• the CD40L expression of this cell population was analyzed by flow cytometry before and after TCR stimulation with an anti-CD3 agonist antibody coated on a cell culture plate in the presence of IL-7, IL-12, IL-15, IL-18, and IL-21.
• FIG. 22 CD40L-expressing cells increased from 2.18% to 62.5% by TCR stimulation.
• CD40L-expressing cells were significantly increased by TCR stimulation. It is suggested that the CD4SP iPSC-T cells prepared according to Example 17 fully exert the function as helper T cells by TCR stimulation. Statistical analysis was performed by paired t-test.
• CD4 single positive helper T cells CD4SP iPSC-T cells
• CD8 single positive killer T cells CD8SP iPSC-T cells
• the CD4SP iPSC-T cells and CD8SP iPSC-T cells prepared according to Example 17 were treated with PMA (phorbol 12-myristate 13-acetate) and ionomycin in the presence of monensin for 4 hours, and then stained with anti-IFN- ⁇ antibody and anti-IL-2 antibody to measure cytokine production ability. The results are shown in FIG. 23.
• CD4SP iPSC-T cells produced higher levels of IFN- ⁇ and IL-2 than the CD8SP iPSC-T cells.
• These cytokines are known not only to exhibit direct antitumor activity, but also to be capable of modifying the immunosuppressive tumor microenvironment. Therefore, it is suggested that the CD4SP iPSC-T cells prepared according to Example 17 are useful for cancer immunotherapy.
• Anti-CD19 CAR (anti-CD19 CAR-4-1BB-CD3zeta) was introduced into the CD4 single positive helper T cells and CD8 single positive killer T cells prepared according to Example 17 using a retrovirus as previously reported (Ueda T, et al. Nat Biomed Eng. 2023; 7:24-37). The expression of anti-CD19 CAR in the obtained cells was analyzed by flow cytometry, and the results are shown in FIG. 24. It was confirmed that anti-CD19 CAR was expressed to the same extent in both CD4SP iPSC-T cells and CD8SP iPSC-T cells.
• CD4 single positive helper T cells CD4SP iPSC-T cells
• CD8 single positive killer T cells CD8SP iPSC-T cells
• Anti-CD19 CAR anti-CD19 CAR-4-1BB-CD3zeta
• CD4SP iPSC-T cells prepared according to Example 17 to prepare effector cells (E).
• CD19-positive human pre-B cell leukemia cell line Nalm6 was used as target cells (T), and the killing effector cells against target cells was examined.

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