US20220184128A1 - Method for producing car gene-introduced nk cells and use thereof - Google Patents

Method for producing car gene-introduced nk cells and use thereof Download PDF

Info

Publication number
US20220184128A1
US20220184128A1 US17/598,916 US202017598916A US2022184128A1 US 20220184128 A1 US20220184128 A1 US 20220184128A1 US 202017598916 A US202017598916 A US 202017598916A US 2022184128 A1 US2022184128 A1 US 2022184128A1
Authority
US
United States
Prior art keywords
cells
cancer
car
ink
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US17/598,916
Other languages
English (en)
Inventor
Yee Sook Cho
Han-Seop KIM
Jae Yun Kim
Binna Seol
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Korea Research Institute of Bioscience and Biotechnology KRIBB
Original Assignee
Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Korea Research Institute of Bioscience and Biotechnology KRIBB filed Critical Korea Research Institute of Bioscience and Biotechnology KRIBB
Assigned to KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY reassignment KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY ASSIGNMENT OF ASSIGNOR'S INTEREST Assignors: CHO, YEE SOOK, KIM, HAN-SEOP, KIM, JAE YUN, SEOL, Binna
Publication of US20220184128A1 publication Critical patent/US20220184128A1/en
Assigned to CEFO CO., LTD. reassignment CEFO CO., LTD. LICENSE Assignors: KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K40/4255—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • 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
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K2239/54—Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2312—Interleukin-12 (IL-12)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2318—Interleukin-18 (IL-18)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2321—Interleukin-21 (IL-21)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • 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
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • C12N2506/115—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells from monocytes, from macrophages
    • 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
    • C12N2510/00—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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18811—Sendai virus
    • C12N2760/18841—Use of virus, viral particle or viral elements as a vector
    • C12N2760/18843—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a method for producing induced natural killer (iNK) cells into which a chimeric antigen receptor (CAR) gene encoding a CAR is introduced, iNK cells produced by the method, and a cell therapy composition and a pharmaceutical composition for preventing or treating cancer, including the iNK cells.
  • iNK induced natural killer
  • CAR chimeric antigen receptor
  • NK (natural killer) cells are key innate immune cells that perform the body's primary defense (innate immunity) function by immediately recognizing and eliminating infections of viruses, bacteria and parasites, and abnormal autologous cells (especially cancer cells).
  • T cells which recognize target cells by expressing antigen-specific receptors
  • NK cells perceive abnormal changes in target cells (especially cancer cells), such as balance of inhibitory or activating receptors, i.e., killer immunoglobulin receptors (KIR), natural cytotoxicity receptors (NCR), DNAM-1 (DNAX accessory molecule-1), and NKG2D (NK group 2 member D, and loss of surface MHC (major histocompatibility complex) class I antigen, without antigen specificity and human leukocyte antigen (HLA) matching, and have contact-dependent cytotoxicity.
  • KIR killer immunoglobulin receptors
  • NCR natural cytotoxicity receptors
  • DNAM-1 DNAX accessory molecule-1
  • NKG2D NK group 2 member D
  • NK cells can kill cancer cells by directly mediating target cancer cell apoptosis through secretion of cytokines, such as perforin (Prf1), granzyme B (GzmB), interferon- ⁇ (IFN- ⁇ ), interleukin (tumor necrosis factor- ⁇ ; TNF- ⁇ ), etc., and activation of apoptosis-inducing receptors (Fas, tumor necrosis factor-related apoptosis-inducing ligand; TRAIL).
  • cytokines such as perforin (Prf1), granzyme B (GzmB), interferon- ⁇ (IFN- ⁇ ), interleukin (tumor necrosis factor- ⁇ ; TNF- ⁇ ), etc.
  • IFN- ⁇ interferon- ⁇
  • TNF- ⁇ interleukin
  • TRAIL apoptosis-inducing receptors
  • NK cells that express Fc receptor-FcR ⁇ IIIa can effectively eliminate cancer cells directly or indirectly by inducing acquired immunity as well as innate immunity, such as mediating antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • NK cells do not cause side effects such as graft-versus-host disease (GVHD), and thus have gained attention as a safe cell source that can be used for the development of not only autologous but also allogeneic anticancer immune cell therapeutics.
  • GVHD graft-versus-host disease
  • human NK cells are produced by isolating and proliferating a small amount of cells present in the human body [peripheral blood, bone marrow, umbilical cord blood, etc.] through a primary culture method, or can be obtained multidimensionally from stem cells having the ability to differentiate into NK cells [hematopoietic stem cells (HSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs)] through differentiation induction culture.
  • HSCs hematopoietic stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • CAR-T cells which are produced by introducing a cancer cell target chimeric antigen receptor (CAR) gene as a method for promoting specificity and activation for target cancer cells and consequently enhancing the efficacy of anticancer treatment, have an enhanced anticancer effect in the treatment of blood cancer, and thus have received much interest in the development of CAR-immune cell therapeutics.
  • CRS cytokine release syndrome
  • IFN interferon
  • TNF tumor necrosis factor
  • IL-6 factors secreted by CAR-T cells is recognized as a serious problem that needs to be solved.
  • CAR-T cells are known to inhibit cancer cell cytotoxicity by the immune checkpoint protein PD (programmed death ligand)-1, etc. in the tumor microenvironment (TME) of solid tumors, and accordingly, there is a need for technology development to improve the efficacy of solid cancer treatments.
  • PD programmed death ligand
  • NK cells secrete IL-3 and granulocyte-macrophage colony-stimulating factor, they are less likely to cause CRS [K. Rezvani, R. Rouce, E. Liu et al., Engineering Natural Killer Cells for Cancer Immunotherapy, Mol. Ther., 25 (2017), pp. 1769-1781]. Additionally, the level of PD-1 secreted by NK cells is substantially low, and almost no immunosuppression is induced, and also, it is known that NK cells enable migration of dendritic cells to tumors, thereby enhancing anti-PD-1 immunotherapy [K. C. Barry, J. Hsu, M. L. Broz, et al.
  • a Natural Killer-dendritic Cell Axis Defines Checkpoint Therapy-responsive Tumor Microenvironments, Nat. Med., 24 (2016), pp. 1178-1191], and thus, the excellence of CAR-NK development has been demonstrated. Further, it was confirmed from the results of preclinical and clinical trials for CAR-NK cell therapy that NK cells can effectively remove blood cancer and solid cancer cells [E. L. Siegler, Y. Zhu, P. Wang, et al. Off-the-shelf CAR-NK Cells for Cancer Immunotherapy, Cell Stem Cell, 23 (2016), pp. 160-161], and thus have been recognized to have the potential to be developed as anticancer immune cell therapeutics in a wide range, including for solid cancers.
  • a method for introducing a CAR gene specific for a cancer antigen into a single NK cell line NK-92, iPSC, etc.
  • NK-92, iPSC, etc. a single NK cell line
  • the most used primary cultured NK-92 cells are cells derived from patients with malignant non-Hodgkin's lymphoma and have the potential to induce secondary tumorigenesis and EB (Epstein-Barr) virus sensitivity after injection.
  • a lethal irradiation process is essential before clinical application, but this process shortens the in vivo survival time of CAR-NK-92 cells, and consequently, the anticancer effect is reduced.
  • the method of producing NK cells by introducing a CAR gene into induced pluripotent stem cells (iPSCs) and inducing differentiation essentially requires not only the CAR-iPSCs process, and isolation and amplification process, but also a complex differentiation process that involves much time and cost, and carries the possibility of tumor formation, and thus, it is essential to develop a technology that can secure and maintain safety.
  • the present inventors have made extensive efforts to develop a method for efficiently producing CAR-NK cells, and as a result, they have developed a CAR-NK cell-specific reprogramming medium and reprogramming culture conditions, and confirmed that CAR-NK cells can be produced from human somatic cells by a method that does not require a differentiation process without limiting the initial cell resources, and accordingly, the produced CAR-NK cells exhibit an excellent cancer cell killing ability and thus can be applied to the prevention or treatment of cancer, thereby completing the present invention.
  • a reprogramming factor and a CAR chimeric antigen receptor
  • the method according to the present invention can produce iNK cells, into which a CAR gene is introduced, from the isolated cells without a differentiation process through direct reprogramming, thereby producing CAR-NK cells with an enhanced cancer cell killing function with high efficiency without limitation of the initial cells, and has an effect of reducing cost and time by simplifying the production process.
  • NK cells can be produced without passing through induced pluripotent stem cells produced through a conventional reprogramming technique, there is an excellent NK cell production effect which is enhanced and distinguished in terms of safety.
  • the CAR gene-introduced iNK cells produced by the above method have an excellent cancer cell killing ability, and thus can be effectively utilized as a cell therapy composition or a pharmaceutical composition for preventing or treating cancer.
  • FIG. 1 shows a diagram showing the constitutional domains of four CAR genes.
  • FIG. 2 is a schematic diagram showing a method for producing CAR-iNK by direct somatic cell reprogramming and CAR gene introduction.
  • FIG. 3 is a result confirming CD19-CAR-iNK cells produced from PBMC.
  • FIG. 4 is a result confirming MSLN-CAR-iNK cells produced from PBMC.
  • FIG. 5 is a result confirming HER2-CAR-iNK cells produced from PBMC.
  • FIG. 6 shows a result confirming the CD19-CAR-iNK cells produced according to the introduction period of CAR-expressing lentivirus (day 0, day 14, day 24), and (B) shows a result confirming the insertion of the CD19-CAR gene in the iNK cells produced from PBMC.
  • FIG. 7 is a result confirming the CAR-iNK production efficiency according to the composition or incubation time of the CAR-iNK first medium and third medium.
  • FIG. 8 is a result confirming the frequency of CD107a-expressing cells during co-culture of the produced CD19-CAR-iNK cells and cancer cells.
  • FIG. 9 is a result confirming the frequency of CD107a-expressing cells during co-culture of the produced CD19-CAR-iNK cells and cancer cells.
  • FIG. 10 is a result confirming the frequency of CD107a-expressing cells during co-culture of the produced MSLN-CAR-iNK cells and cancer cells.
  • FIG. 11 is a result confirming the frequency of CD107a-expressing cells during co-culture of the produced HER2-CAR-iNK cells and cancer cells.
  • FIG. 12 is a result confirming the frequency of IFN-gamma-expressing cells during co-culture of the produced CD19-CAR-iNK cells and cancer cells.
  • FIG. 13 is a result confirming the frequency of IFN-gamma-expressing cells during co-culture of the produced CD19-CAR-iNK cells and cancer cells.
  • FIG. 14 is a result confirming the frequency of IFN-gamma-expressing cells during co-culture of the produced MSLN-CAR-iNK cells and cancer cells.
  • FIG. 15 is a result confirming the frequency of IFN-gamma-expressing cells during co-culture of the produced HER2-CAR-iNK cells and cancer cells.
  • FIG. 16 is a result confirming the cancer cell killing ability of the produced CD19-CAR-iNK cells.
  • FIG. 17 is a result confirming the cancer cell killing ability of the produced MSLN-CAR-iNK cells.
  • FIG. 18 is a result confirming the cancer cell killing ability of the produced HER2-CAR-iNK cells.
  • FIG. 19 is a result confirming the tumor growth inhibitory effect of MSLN-CAR-iNK cells in a pancreatic cancer cell xenograft mouse animal model.
  • one aspect of the present invention provides a method for producing CAR-iNK (induced natural killer) cells, including: culturing isolated cells, into which a reprogramming factor and a CAR (chimeric antigen receptor) gene are introduced, sequentially in a) a first medium containing growth factors, cytokines, and a GSK3 ⁇ (glycogen synthase kinase 3 beta) inhibitor; b) a second medium containing growth factors, cytokines, and an AHR (aryl hydrocarbon receptor) antagonist; and c) a third medium containing cytokines, a GSK3 ⁇ inhibitor, and an AHR antagonist, and thereby directly reprogramming into NK cells.
  • a reprogramming factor and a CAR (chimeric antigen receptor) gene sequentially in a) a first medium containing growth factors, cytokines, and a GSK3 ⁇ (glycogen synthase kinase 3 beta) inhibitor
  • NK naturally killer cell
  • innate immunity first defense
  • cancer cells abnormal autologous cells
  • NK cells have been proven to be effective in preventing the occurrence, proliferation, metastasis, and recurrence of various types of cancers including not only blood cancer but solid cancers, and thus have received attention as a useful cellular resource for anticancer therapeutics and relapse inhibitors.
  • Efforts to develop NK cells isolated and cultured from the human body and NK cells differentiated from stem cells as anticancer immune cell therapeutics are rapidly increasing, but low NK production efficiency and anticancer treatment efficacy are still recognized as problems to be overcome.
  • NK (CAR-NK) cells expressing a chimeric antigen receptor (CAR) specific to various cancer antigens were produced, and accordingly, studies are being actively conducted to investigate the enhancement of cancer cell killing ability and its therapeutic efficacy.
  • CAR-NK cells As a method for producing the CAR-NK cells, a method of introducing cells into a single NK cell line (NK-92, iPSCs, etc.) that is relatively easy to culture and proliferate is mainly used, but low production efficiency, a complicated process, and safety issues (possibility of tumor formation, etc.) have been recognized as problems to be overcome.
  • the present inventors have attempted to produce CAR-NK cells using a method that is applicable to various human somatic cells that are easy to collect under ex vivo culture conditions and does not require a differentiation process, and a result, they have identified for the first time a method for directly inducing and producing CAR-NK cells from isolated human somatic cells through direct reprogramming.
  • CAR-iNK CAR-induced natural killer cell
  • NK natural killer
  • the term “reprogramming” refers to a method of converting a lineage into a target cell having completely different characteristics by controlling the global gene expression pattern of a specific cell.
  • Reprogramming may include dedifferentiation of cells, direct reprogramming or direct conversion, or direct trans-differentiation, but is not limited thereto.
  • the reprogramming may be performed by introducing a vector containing a foreign gene or DNA into a cell.
  • transformation refers to the change of a cell to a different state
  • differentiation refers to a phenomenon in which daughter cells produced by cell division acquire a function different from that of the original parent cell
  • conversion and “differentiation” can be used interchangeably with “induction”.
  • NK cells which are target cells
  • conventional reprogramming techniques 1) induced pluripotent stem cells were produced from isolated somatic cells; 2) hematopoietic stem (progenitor) cells, the intermediate, were subjected to primary differentiation and production from induced pluripotent stem cells; 3) and subsequently, NK cells, which are target cells, were subjected to secondary differentiation and production from differentiated stem (progenitor) cells.
  • the conventional technique has the disadvantages of low production efficiency and high time and cost consumption, because it has to sequentially go through a complex culture process.
  • NK cells are produced via induced pluripotent stem cells with pluripotency, the remaining undifferentiated cells have the potential to form tumors, and thus safety is an important issue to be verified.
  • the present invention produces NK cells, which are the target cells, directly from isolated somatic cells through direct reprogramming, thereby providing reduced production time and cost, and excellent efficiency and safety, and thus can be distinguished from the prior art and can provide an alternative that can overcome the problems.
  • the direct reprogramming may be used interchangeably with direct dedifferentiation, direct differentiation, direct conversion, direct cross-differentiation, cross-differentiation, etc., and as used herein, the direct reprogramming may mean direct dedifferentiation or cross-differentiation into NK cells from isolated somatic cells, but is not limited thereto.
  • the term “differentiated cells” refers to a state in which cells with specialized structures or functions, that is, cells, tissues, etc. of living organisms, have changed into a form and function suitable for performing the role assigned thereto.
  • the differentiated cells broadly refer to ectodermal, mesodermal, and endodermal cells derived from pluripotent stem cells such as embryonic stem cells, and narrowly to red blood cells, white blood cells, platelets, etc. derived from hematopoietic stem cells.
  • the “lineage-conversion cell” is a cell which is converted to a cell type with different lineage characteristics due to the change in the intrinsic lineage characteristics of the cell embryologically or artificially (e.g., reprogramming, etc.), thereby having the characteristics of a cell type that are completely different from the characteristics of the cell type before conversion.
  • the lineage-conversion cell may be a target cell.
  • non-NK lymphocyte cells in peripheral blood mononuclear cells may be converted to NK cells in a reprogramming medium, but are not limited thereto.
  • the method may be performed by culturing isolated cells, into which a reprogramming factor and a CAR gene are introduced, sequentially in a) a first medium containing growth factors, cytokines, and a GSK3 ⁇ (glycogen synthase kinase 3 beta) inhibitor; b) a second medium containing growth factors, cytokines, and an AHR (aryl hydrocarbon receptor) antagonist; and c) a third medium containing cytokines, a GSK3 ⁇ inhibitor, and an AHR antagonist, and thereby directly reprogramming into NK cells.
  • a first medium containing growth factors, cytokines, and a GSK3 ⁇ (glycogen synthase kinase 3 beta) inhibitor
  • a third medium containing cytokines, a GSK3 ⁇ inhibitor, and an AHR antagonist, and thereby directly reprogramm
  • isolated cells are not particularly limited, but specifically refer to cells whose lineage has already been specified, such as germ cells, somatic cells, or progenitor cells.
  • the “somatic cells” refer to all cells in which differentiation constituting animals and plants has been completed except for germ cells.
  • progenitor cells refer to a mother cell which does not express a differentiated character, but has a differentiation fate, if it has been found that a cell corresponding to its progeny expresses a certain differentiation character.
  • nerve cells nerve cells
  • nerve fibroblasts neurovascular stem cells
  • myoblasts correspond to the precursor cell.
  • the isolated cells may be cells derived from a human, but are not limited thereto, and cells derived from various individuals may also fall within the scope of the present invention.
  • the isolated cells of the present invention may include both in vivo or ex vivo cells.
  • the isolated cells may be somatic cells, and more specifically, somatic cells other than NK cells, but are not limited thereto.
  • reprogramming factor refers to a gene (or polynucleotide) that can be introduced into a cell to induce reprogramming, or a protein encoded therefrom.
  • the reprogramming factor may vary depending on the target cell to be obtained through reprogramming, and the type of cell before reprogramming.
  • the reprogramming factor introduced into the isolated somatic cells may include any one or more selected from the group consisting of Lin28, Asc11, Pitx3, Nurr1, Lmx1a, Nanog, Oct4, Oct3, Sox2, Klf4, Myc, and a combination thereof, and specifically Oct4, Sox2, Klf4, and Myc, but is not limited thereto, and may include any factor known in the art as long as it is a reprogramming factor that can induce the isolated somatic cells into NK cells.
  • the reprogramming using the reprogramming factor is the induction of conversion to a target cell by controlling the entire gene expression pattern of the cell, and the cell may be reprogrammed into a target cell having a gene expression pattern of a desired type of cell by introducing the reprogramming factor into the cell and culturing the same for a certain period of time.
  • the “introduction of reprogramming factor” may be a method of administering a reprogramming factor to a culture solution of cells; a method of directly injecting a reprogramming factors into cells; a method of increasing the expression level of a reprogramming factor present in a cell; a method of transforming a cell with an expression vector containing a gene encoding a reprogramming factor; a method of modifying a gene sequence to increase the expression of a gene encoding a reprogramming factor; a method of introducing an exogenously expressed gene encoding a reprogramming factor; a method of treating a substance having an effect of inducing expression of the reprogramming factor; and a method of increasing the expression level of a reprogramming factor in a cell through a combination thereof, but is not limited thereto as long as it can increase the expression level of the reprogramming factor.
  • the introduction of reprogramming factor may be inducing expression of a reprogramming factor depending on a desired time and conditions.
  • the method of introducing a reprogramming factor into a cell may be a method of administering a reprogramming factor to a cell culture solution, or a method of transforming a cell with an expression vector containing a gene encoding a reprogramming factor, but this not limited.
  • the method of directly injecting a reprogramming factor into a cell may be performed by selecting any method known in the art, but is not limited thereto, and may be performed by appropriately selecting from the methods using microinjection, electroporation, particle bombardment, direct muscle injection, an insulator, and a transposon.
  • the term “vector” refers to a DNA construct containing the nucleotide sequence of a suitable regulatory sequence and the target protein or polypeptide so as to be able to express the target protein or polypeptide in a suitable host cell.
  • the regulatory sequence may include a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer, etc.
  • the vector of the present invention may include a signal sequence or a leader sequence for membrane targeting or secretion, in addition to the regulatory sequence, and can be prepared in various ways depending on the desired purpose.
  • the promoter of the vector may be constitutive or inducible.
  • the vector may include a selective marker for selecting a host cell containing the vector, and in the case of a replicable vector, may include a replication origin. Once transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or may integrate into the genome thereof.
  • the vector used in the present invention is not particularly limited as long as it is able to replicate in the host cell, and any vector known in the art may be used.
  • Examples of the vector conventionally used may include natural or recombinant virus vector, episomal vector, plasmid vector, cosmid vector, etc.
  • the virus vector may include vectors derived from retrovirus such as Sendai virus, lentivirus, HIV (human immunodeficiency virus, MLV (murine leukemia virus), ASLV (avian sarcoma/leukosis), SNV (spleen necrosis virus), RSV (Rous sarcoma virus), MMTV (mouse mammary tumor virus), etc., adenovirus, adeno-associated virus, herpes simplex virus, and more specifically, it may be an RNA-based virus vector, but is not limited thereto.
  • retrovirus such as Sendai virus, lentivirus, HIV (human immunodeficiency virus, MLV (murine leukemia virus), ASLV (avian sarcoma/leukosis), SNV (spleen necrosis virus), RSV (Rous sarcoma virus), MMTV (mouse mammary tumor virus), etc.
  • adenovirus adeno-associated virus
  • herpes simplex virus
  • the episomal vector is a non-viral non-insertable vector, and is known to have a property of expressing a gene included in the vector without being inserted into a chromosome. Accordingly, the cell containing the episomal vector may include both cases in which the episomal vector is inserted into the genome or is present in a cell without being inserted into the genome.
  • operably linked refers to a functional linkage between a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein so as to perform a general function.
  • the operative linkage with the recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation are carried out using enzymes generally known in the art.
  • CAR gene refers to a gene encoding a chimeric antigen receptor consisting of an extracellular domain, a transmembrane domain, and an intracellular domain including genes encoding the extracellular domain, the transmembrane domain, and the intracellular domain including an antibody domain (scFv).
  • the CAR gene may be any one or more selected from the group consisting of a CD19-CAR1 gene or CD19-CAR2 gene including CD19 scFv, a MSLN-CAR gene including MSLN (mesothelin) scFv, and a HER2-CAR gene including HER2 (human epidermal growth factor receptor 2) scFv, but is not limited thereto.
  • CAR target factors for solid tumors include EGFRvIII (Morgan R A, Hum Gene Ther. 2012; 23:1043-1053), MUC-1 (Wilkie S, J Immunol. 2008; 180:4901-4909), MAGE (Willemsen R A, Gene Ther. 2001; 8:1601-1608), CEA (Emtage P C, Clin Cancer Res. 2008; 14:8112-8122), PSMA, GD2, CA125, Her2 and MSLN, FAP, VEGFR (Kakarla S, Cancer J. 2014; 20:151-155), etc.
  • the CD19 is the cluster of differentiation (CD) assigned with number 19 for identifying cell surface molecules according to the immunophenotype, and the CD19 refers to a marker of B lymphocytes.
  • the CD19 is known to be expressed in most B-cell malignant cancer cells and thus provides an ideal target for these carcinomas.
  • the CAR gene may be any one or more selected from the group consisting of:
  • a CAR gene (CD19-CAR1 gene) including CD8 leader, CD19 scFv, CD8 hinge, CD8 transmembrane domain, and Fc- ⁇ receptor;
  • a CAR gene (CD19-CAR2 gene) including CD8 leader, CD19 scFv, CD8 hinge, CD8 transmembrane domain, CD28 intracellular domain, CD3 ⁇ , and IRES (internal ribosome entry site);
  • a CAR gene including CD8 leader, MSLN (mesothelin) scFv, CD8 hinge, CD8 transmembrane domain, CD28 intracellular domain, CD3 ⁇ , and IRE; and
  • a CAR gene including CD8 leader, HER2 (human epidermal growth factor receptor 2) scFv, CD8 hinge, CD8 transmembrane domain, CD28 intracellular domain, CD3 ⁇ , and IRES, but is not limited thereto.
  • the CD8 leader may include a nucleotide sequence of SEQ ID NO: 1, CD19 scFv may include a nucleotide sequence of SEQ ID NO: 2, MSLN scFv may include a nucleotide sequence of SEQ ID NO: 3, HER2 scFv may include a nucleotide sequence of SEQ ID NO: 4, CD8 hinge may include a nucleotide sequence of SEQ ID NO: 5, CD8 transmembrane domain may include a nucleotide sequence of SEQ ID NO: 6, Fc- ⁇ receptor may include a nucleotide sequence of SEQ ID NO: 7, CD28 intracellular domain may include a nucleotide sequence of SEQ ID NO: 8, CD3 ⁇ may include a nucleotide sequence of SEQ ID NO: 9, and the IRES inserted to clone the CAR gene into a vector constituting a double cistron may include a nucleotide sequence of SEQ ID NO: 10, but is not limited there
  • the CAR gene may further include GFP (green fluorescent protein), but is not limited thereto.
  • the GFP may include a nucleotide sequence of SEQ ID NO: 11, but is not limited thereto.
  • nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 11 can be confirmed from NCBI Genbank, a known database.
  • the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 11 may include a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 1 to SEQ ID NO: 11. Additionally, it is apparent that a nucleotide sequence in which a part of the sequence is deleted, modified, substituted, or added may be included within the scope of the present invention as long as the nucleotide sequence has such homology or identity and exhibits a function corresponding to the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 11.
  • homology and identity refers to a degree of relatedness between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. The terms homology and identity may often be used interchangeably with each other.
  • sequence homology or identity of conserved polynucleotides or polypeptide may be determined by standard alignment algorithms and can be used with a default gap penalty established by the program being used. Substantially, homologous or identical sequences are generally expected to hybridize to all or at least about 50%, 60%, 70%, 80%, or 90% or more of the entire length of the sequences under moderate or high stringent conditions. Polynucleotides that contain degenerate codons instead of codons in hybridizing polynucleotides are also considered.
  • the homology or identity of the polypeptide or polynucleotide sequences may be determined by, for example, BLAST algorithm by literature [see Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873(1993)], or FASTA by Pearson (see Methods Enzymol., 183, 63, 1990). Based on the algorithm BLAST, a program referred to as BLASTN or BLASTX has been developed (see: http://www.ncbi.nlm.nih.gov).
  • any two amino add or polynucleotide sequences may be identified by comparing the sequences in a Southern hybridization experiment under stringent conditions as defined, and appropriate hybridization conditions defined are within the skill of the art, and may be determined by a method well known to those skilled in the art (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology ).
  • the CAR gene may be introduced into the cell in the same manner as the above-described method for introducing a reprogramming factor, and in particular, the reprogramming factor and the CAR gene may be introduced simultaneously or sequentially depending on a desired time and conditions.
  • the lentivirus expressing CAR may be transformed into PBMC at the time of conversion into iNK or into cells at the time of reprogramming induction process by inoculating into a culture medium selected from the steps of culturing in the CAR-iNK first medium, CAR-iNK second medium, or CAR-iNK third medium, simultaneously with the reprogramming factor or after introduction of the reprogramming factor.
  • the method according to the present invention is significant in that CAR-expressing NK cells can be produced by introducing the CAR gene and the reprogramming factor into isolated cells other than NK cells.
  • the time of introducing the CAR gene can be determined as desired by those skilled in the art during the reprogramming culturing process.
  • the time of introduction of the CAR gene that is, the time of inoculation of the CAR-expressing lentivirus may be carried out simultaneously with the reprogramming factor (day 0), or at the step of culturing in the CAR-iNK second medium (day 12) or the CAR-iNK third medium (day 24) after introduction of the reprogramming factor, but is not limited thereto ( FIGS. 2 and 6 ).
  • the term “culture” means that the microorganism is grown under appropriately controlled environmental conditions.
  • the culturing process of the present invention may be performed in a suitable culture medium and culture conditions known in the art. Such a culturing process may be easily adjusted for use by those skilled in the art according to the strain to be selected.
  • the culture is a process of converting cells into which reprogramming factors and/or CAR genes have been introduced into target cells of another lineage.
  • the composition of the first medium, the second medium, or the third medium for culturing the cells into which the gene is introduced is a composition suitable for conversion into target cells, and for example, may include growth factors, cytokines, a GSK3 ⁇ inhibitor, or an AHR antagonist, but is not limited thereto.
  • the first medium of a) may include a growth factor, cytokines, and a GSK3 ⁇ inhibitor.
  • growth factor means a polypeptide that promotes the division, growth, and differentiation of various cells.
  • the growth factor may be, for example, EGF (epidermal growth factor), PDGF-AA (platelet-derived growth factor-AA), IGF-1 (insulin-like growth factor 1), TGF- ⁇ (transforming growth factor- ⁇ ), FGF (fibroblast growth factors), SCF (stem cell factor), and FLT3 (FMS-like tyrosine kinase), and may specifically be any one or more selected from the group consisting of SCF, FLT3, and a combination thereof, but is not limited thereto.
  • cytokines are various proteins of relatively small size that are produced in cells and used for cell signaling, and can affect other cells, including themselves. They are generally known to be involved in the immune response to inflammation or infection.
  • the cytokines may be, for example, IL (interleukin)-2, IL-3, IL-5, IL-6, IL-7, IL-11, IL-15, IL-21, IL-12, IL-18, BMP4 (bone morphogenetic protein 4), activin A, notch ligand, G-CSF (granulocyte-colony stimulating factor), SDF-1 (stromal cell-derived factor-1), etc., and may specifically be any one or more selected from the group consisting of IL-3, IL-6 IL-15, IL-7, IL-2, IL-21, IL-12, IL-18, and a combination thereof, but are not limited thereto.
  • the growth factors and cytokines are included in the medium for directly reprogramming the isolated cells into the target cells, and the types of growth factors and cytokines are not particularly limited as long as they can be used for direct reprogramming.
  • GSK3 ⁇ glycogen synthase kinase 3 beta, glycogen synthase kinase-3 ⁇
  • inhibitor means a substance that suppresses or inhibits the activity of GSK3 ⁇ .
  • the GSK3 ⁇ inhibitor may be, for example, 1-azakenpaullone, 2-D08, 3F8, 5-bromoindole, 6-Bio, A 1070722, aloisine A, AR-A014418, alsterpaullone, AZD-1080, AZD2858, bikinin, BIO, BIO-acetoxime, bisindolylmaleimide I, bisindolylmaleimide I hydrochloride, CAS 556813-39-9, malariapaullone, CHIR98014, CHIR98023, CHIR99021 (CT99021), CP21R7, dibromocantherelline, GSK-3 ⁇ inhibitor I, VI, VII, X, XI, XV, GSK-3 inhibitor IX, XVI, hymenidin, hymenialdisine, HMK-32, I3M (indirubin-3-monoxime, indirubin, indole-3-acetamide, IM-12, kenpau
  • the first medium of a) may include SCF, FLT3, IL-3, and IL-6 and CHIR99021, but is not limited thereto.
  • the first medium of a) may further include any one or more selected from the group consisting of fetal bovine serum (FBS), antibiotics, and a combination thereof, but is not limited thereto.
  • FBS fetal bovine serum
  • antibiotics antibiotics, and a combination thereof, but is not limited thereto.
  • the antibiotic may be penicillin/streptomycin, but is not limited thereto.
  • the first medium of a) may include FBS, penicillin/streptomycin, SCF, FLT3, IL-3, and IL-6 and CHIR99021, but is not limited thereto.
  • the first medium of a) may be StemSpan SFEM II containing 8% to 12% FBS, 0.1% to 2% penicillin/streptomycin, 80 ng/mL to 120 ng/mL human SCF, 80 ng/mL to 120 ng/mL human FLT3, 10 ng/mL to 30 ng/mL human IL-3, 10 ng/mL to 30 ng/mL human IL-6, and 2 ⁇ M to 7 ⁇ M CHIR99021, and more specifically StemSpan SFEM II containing 10% FBS, 1% penicillin/streptomycin, 100 ng/mL human SCF, 100 ng/mL human FLT3, 20 ng/mL human IL-3, 20 ng/mL human IL-6, and 5 ⁇ M CHIR99021, but is not limited thereto.
  • the second medium of b) may include a growth factor, cytokines, and an AHR (aryl hydrocarbon receptor) antagonist.
  • growth factor and “cytokine” are the same as described above.
  • AHR aryl hydrocarbon receptor
  • TCDD dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin)
  • the AHR antagonist may be, for example, StemRegenin I (SRI; 4-(2-((2-benzo[b]thiphen-3-yl)-9-isopropyl-9H-purin-6-yl)amino)ethyl)phenol hydrochloride], CH-223191(1-methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide), etc., and specifically StemRegenin I, but is not limited as long as it plays a role in increasing the efficiency of direct reprogramming.
  • the second medium of b) may include SCF, FLT3, IL-15, IL-7, IL-2, and StemRegenin I, but is not limited thereto.
  • the second medium of b) may further include any one or more selected from the group consisting of FBS, antibiotics, and a combination thereof, but is not limited thereto.
  • the antibiotic may be penicillin/streptomycin, but is not limited thereto.
  • the second medium of b) may include FBS, penicillin/streptomycin, SCF, FLT3, IL-15, IL-7, IL-2, and StemRegenin I, but is not limited thereto.
  • the second medium of b) may include 8% to 12% FBS, 0.1% to 2% penicillin/streptomycin, 10 ng/mL to 30 ng/mL human SCF, 10 ng/mL to 30 ng/mL human FLT3, 100 IU/mL to 500 IU/mL human IL-2, 10 ng/mL to 30 ng/mL human IL-7, 10 ng/mL to 30 ng/mL human IL-15, and 1 ⁇ M to 3 ⁇ M StemRegenin I, and more specifically 10% FBS, 1% penicillin/streptomycin, 20 ng/mL human SCF, 20 ng/mL human FLT3, 200 IU/mL human IL-2, 20 ng/mL human IL-7, 20 ng/mL human IL-15, and 2 ⁇ M StemRegenin I, but is not limited thereto.
  • the third medium of c) may include cytokines, a GSK3 ⁇ inhibitor, and an AHR antagonist.
  • cytokines cytokines
  • GSK3 ⁇ inhibitor cytokines
  • AHR antagonist cytokines
  • the third medium of c) may include IL-2, IL-15, IL-21, IL-12, IL-18, CHIR99021, and StemRegenin I, but is not limited thereto.
  • the third medium of c) may further include any one or more selected from the group consisting of FBS, antibiotics, and a combination thereof, but is not limited thereto.
  • the antibiotic may be penicillin/streptomycin, but is not limited thereto.
  • the third medium of c) may include FBS, penicillin/streptomycin, IL-2, IL-15, IL-21, IL-12, IL-18, CHIR99021, and StemRegenin I, but is not limited thereto.
  • the third medium of c) may be RPMI 1640 containing 8% to 12% FBS, 0.1% to 2% penicillin/streptomycin, 100 IU/mL to 500 IU/mL human IL-2, 40 ng/mL to 60 ng/mL human IL-12, 10 ng/mL to 30 ng/mL human IL-15, 90 ng/mL to 110 ng/mL human IL-18, 10 ng/mL to 30 ng/mL human IL-21, 1 ⁇ M to 3 ⁇ M CHIR99021, and 1 ⁇ M to 3 ⁇ M StemRegenin I, and more specifically RPMI 1640 containing 10% FBS, 1% penicillin/streptomycin, 200 IU/mL human IL-2, 50 ng/mL human IL-12, 20 ng/mL human IL-15, 100 ng/mL human IL-18, 20 ng/mL human IL-21, 2 ⁇ M CHIR99021, and 2 ⁇
  • the isolated cells into which the reprogramming factor and the CAR gene are introduced may be cultured in the first medium of a) for 4 to 7 days, and in the second medium of b) for 10 to 14 days, and then in the third medium of c) for 14 days or more, but is not limited thereto.
  • the cells were cultured for different culture periods (3, 5, 7, and 9 days) in the CAR-iNK first medium, and then cultured in the CAR-iNK second medium and the third medium under the same conditions as in Example 1, and the CAR-iNK production yield was analyzed by comparison. As a result, it was confirmed that the highest CAR-iNK production yield of about 80% was obtained when the cells were cultured in the CAR-iNK first medium for 5 days ( FIG. 7A ).
  • the CAR-iNK yield efficiency was increased when the cells were cultured in the CAR-iNK third medium containing 20 ng/mL human IL-21, 2 ⁇ M CHIR99021, 2 ⁇ M StemRegenin I (SR1), 50 ng/mL human IL-12, and 100 ng/mL human IL-18 ( FIG. 7B ).
  • the CAR-iNK yield efficiency was increased up to 15 times when cultured for 14 days ( FIG. 7B ).
  • CAR-iNK cells exhibited the highest production efficiency (CD19-CAR-iNK cells: maximum 45.9% ( FIG. 6A ), MSLN-CAR-iNK cells: 43.7% ( FIG. 4 )), HER2-CAR-iNK cells: 47.8% ( FIG. 5 ) when cultured in the CAR-iNK first medium for 5 days, in the CAR-iNK second medium for 12 days, and in the CAR-iNK third medium for 2 to 3 weeks.
  • the CAR-iNK cells produced according to the method of the present invention may express any one or more selected from the group consisting of CD56+, CD16+, CD3 ⁇ , and a combination thereof, but is not limited thereto.
  • CD56+”, CD16+, and CD3 ⁇ are indicators on the surface of NK cells, and in the present invention, the expression of CD56+, CD16+, and CD3 ⁇ was analyzed through flow cytometry to determine whether CAR-iNK cells were produced ( FIGS. 3 to 6 ).
  • the CAR-iNK cells produced according to the method of the present invention may have an excellent killing ability against various cancer cells.
  • the frequency (%) of CD107a+ cells and interferon-gamma+cells having an cancer cell lysis ability was increased in cancer cells co-cultured with the CAR-iNK cells ( FIGS. 8 to 15 ), and as a result of confirming the killing ability of iNK cells against blood cancer cell lines, pancreatic cancer cell lines, prostate cancer cell lines, colorectal cancer cell lines, lung cancer cell lines, liver cancer cell lines, gastric cancer cell lines, and melanoma cell lines, it was confirmed that they exhibited an excellent killing ability ( FIGS. 16 to 18 ).
  • Another aspect of the present invention provides CAR-iNK cells produced according to the method above.
  • the terms used herein are the same as described above.
  • the CAR-iNK cells may have an excellent killing ability against various cancer cells.
  • Still another aspect of the present invention provides a cell therapy composition for preventing or treating cancer, including the CAR-iNK cells produced according to the method above, as an active ingredient.
  • prevention refers to all actions that suppress or delay cancer by the administration of the composition.
  • treatment refers to all actions that alleviate or beneficially change the symptoms of cancer by the administration of the composition.
  • cell therapeutic agent refers to a drug for treatment, diagnosis, and prevention (U.S. FDA guidance) containing cells or tissues prepared from humans via isolation, culture, and specialized manipulations, and to a drug for treatment, diagnosis, and prevention prepared by any process including proliferating and selecting autologous, homologous, or heterologous cells ex vivo, or modifying the biological characteristics of cells, so as to restore the function of cells or tissues.
  • the cell therapy composition may have an efficacy of preventing or treating cancer by including the CAR-iNK cells produced according to the method of the present invention.
  • the cell therapy composition may contain the CAR-iNK cells at 1.0 ⁇ 10 4 cells/mL to 1.0 ⁇ 10 10 cells/mL, preferably 1.0 ⁇ 10 5 cells/mL to 1.0 ⁇ 10 9 cells/mL, based on the total weight of the composition, but is not limited thereto.
  • the cell therapy composition may be administered by formulating it into a pharmaceutical formulation in the form of unit dosage suitable for administration to the body of a patient by conventional methods in the pharmaceutical field, and it contains an effective amount by a single dose or in divided doses.
  • a formulation for parenteral administration may preferably include injection formulation such as an injection ampoule, infusion formulation such as an infusion bag, and spray formulation such as an aerosol, etc.
  • the injection ampoule may be mixed with injection solution such as saline solution, glucose, mannitol, and Ringer's solution just before use.
  • the cells can be carried by an infusion bag textured with polyvinyl chloride or polyethylene, and examples thereof may include infusion bags manufactured by Baxter, Becton Dickinson, Medcep, National Hospital Products, or Terumo.
  • the pharmaceutical formulation may additionally include one or more pharmaceutically acceptable inactive carriers in addition to the active ingredient, for example, a preservative, analgesic controller, solubilizer, or stabilizer for injection formulation, and a base, excipient, lubricant, or preservative for topical formulation.
  • a preservative for example, a preservative, analgesic controller, solubilizer, or stabilizer for injection formulation, and a base, excipient, lubricant, or preservative for topical formulation.
  • the thus-produced cell therapy composition of the present invention or a pharmaceutical formulation thereof may be administered in accordance with any conventional method in the art together with other cells used for treatment of cancer, or in the form of a mixture therewith.
  • Direct engraftment or transplantation to the diseased area of a patient in need of treatment, or direct transplantation or injection into the abdominal cavity is preferred, but the method is not limited thereto.
  • both non-surgical administration using a catheter and surgical administration such as injection or transplantation after incision of the diseased area are possible.
  • the composition may also be administered parenterally by the conventional method, for example, transplantation of cells into the hematopoietic system, in addition to direct administration to the lesion.
  • the cell therapy composition of the present invention may be administered in an amount ranging from about 0.0001 mg/kg to 1000 mg/kg, preferably 0.001 mg/kg to 100 mg/kg once per day or in several divided doses per day.
  • amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the disease to be treated, the severity of the disease, the route of administration, and the body weight, age and sex of a patient, and therefore, the above dose should not be intended to limit the scope of the present invention in any way.
  • Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, including the CAR-iNK cells produced according to the method above, as an active ingredient.
  • the cancer may be a cancer showing prevention or treatment results due to an immune response of CAR-iNK cells, etc.
  • the cancer may be, for example fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma cancer, osteogenic sarcoma, myeloma, myeloma, myelodysplasia, lymphoma, non-Hodgkin's lymphoma, blood cancer, melanoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovial sarcoma, mesothelioma, Ewing's sarcoma, gastric cancer, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colon cancer, colorectal cancer, rectal cancer, pancreatic cholangiocarcinoma, pancreatic cancer,
  • the pharmaceutical composition may have an efficacy of preventing or treating cancer by including the CAR-iNK cells produced according to the method of the present invention.
  • the pharmaceutical composition of the present invention may contain the CAR-iNK cells at 1.0 ⁇ 10 4 cells/mL to 1.0 ⁇ 10 10 cells/mL, preferably 1.0 ⁇ 10 5 cells/mL to 1.0 ⁇ 10 9 cells/mL, based on the total weight of the composition, but is not limited thereto.
  • the pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical compositions, and the carrier may include a carrier which does not occur naturally.
  • the carriers, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and minerals.
  • the pharmaceutical composition may be formulated according to a conventional method into a tablet, a pill, a powder, a granule, a capsule, a suspension, a solution for internal use, an emulsion, a syrup, a sterilized aqueous solution, a non-aqueous solution, a suspension, an emulsion, a lyophilized preparation, a transdermal preparation, a gel, a lotion, an ointment, a cream, a patch, a cataplasma form, a paste, a spray, a skin emulsion, a skin suspension, a transdermal patch, a drug-containing bandage, or a suppository for use.
  • the preparation may be formulated with commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.
  • Solid formulations for oral administration may include tablets, pills, powders, granules, capsules, etc., but are not limited thereto.
  • Such solid formulations may be prepared by mixing with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc.
  • lubricants such as magnesium stearate or talc may also be used.
  • Liquid formulations for oral administration may be prepared by adding various excipients, for example, wetting agents, flavoring agents, aromatics, preservatives, etc. in addition to liquid paraffin.
  • Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.
  • the non-aqueous solutions and the suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyloleate, etc.
  • the base for suppositories may include witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc.
  • the pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount which is sufficient to treat diseases at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the effective dosage level may be determined depending on factors including a kind of a subject and severity, age, sex, activity of a drug, drug sensitivity, administration time, administration route, excretion rate, duration of treatment, drugs used concurrently, and other factors known in the medical field.
  • the pharmaceutical composition may be administered in a daily dosage of 0.0001 mg/kg to 1000 mg/kg, and specifically 0.001 mg/kg to 100 mg/kg, and the dose may be administered once per day or in several divided doses per day.
  • the pharmaceutical composition may be administered alone as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with existing therapeutic agents.
  • the composition may be administered in a single or multiple dosage form. It is important to administer the composition in a minimum amount that may exhibit a maximum effect without causing side effects, considering all of the above-described factors. The amount may be readily determined by those skilled in the art.
  • the term “administration” means introducing the composition of the present invention into a subject by any suitable method.
  • the administration route of the composition may be administered through any general route as long as it can reach the target tissue, including intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, and intranasal administration, but is not limited thereto.
  • the term “subject” refers to any animal, including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs having cancer or at risk of having cancer. As long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention into a subject, any type of subject may be included without limitation.
  • Even another aspect of the present invention provides a method for treating cancer, including administering the cell therapy composition or the pharmaceutical composition to a subject except a human.
  • the present invention provides a simplified production process because CAR-iNK cells are produced from isolated cells through direct reprogramming and a production time of at least 32 days, which is shorter than that obtained by the method in which the CAR gene was introduced after producing NK cells using conventional reprogramming techniques, thereby reducing costs, has excellent NK cell production efficiency of 47.8% at maximum, and ensures safety as the cells are produced without passing through pluripotent stem cells, thereby showing an excellent CAR-iNK cell production effect that is distinguished from that of the conventional reprogramming techniques.
  • the present invention is distinguished from the conventional method of obtaining NK cells through direct harvesting, stem cell differentiation, etc. in that NK cells can be directly produced by culturing cells, which have a different cell lineage from NK cells and are easy to collect, in a reprogramming medium, and thus, the present invention is meaningful in that it can provide a wide range of options for cell types and quality.
  • the CAR-iNK cells produced according to the method of the present invention have an excellent cancer cell killing ability as described above, and thus can be provided as a cell therapy composition and a pharmaceutical composition for preventing or treating cancer including the same.
  • CAR chimeric antigen receptor
  • HER2 human epidermal growth factor receptor 2
  • MSLN mouse epidermal growth factor receptor 2
  • scFv antibody domain
  • CD19-CAR1 gene including CD8 leader (SEQ ID NO: 1), CD19 scFv (SEQ ID NO: 2), CD8 hinge (SEQ ID NO: 5), CD8 transmembrane (TM) domain (SEQ ID NO: 6), Fc- ⁇ (gamma) receptor (SEQ ID NO: 7), and GFP (green fluorescent protein) (SEQ ID NO: 11);
  • CD19-CAR2 gene including CD8 leader (SEQ ID NO: 1), CD19 scFv (SEQ ID NO: 2), CD8 hinge (SEQ ID NO: 5), CD8 transmembrane domain (SEQ ID NO: 6), CD28 intracellular domain (SEQ ID NO: 8), CD3 (zeta) (SEQ ID NO: 9), IRES (internal ribosome entry site) (SEQ ID NO: 10), and GFP (SEQ ID NO: 11);
  • MSLN-CAR gene including CD8 leader (SEQ ID NO: 1), MSLN (mesothelin) scFv (SEQ ID NO: 3), CD8 hinge (SEQ ID NO: 5), CD8 transmembrane domain (SEQ ID NO: 6), CD28 intracellular domain (SEQ ID NO: 8), CD3 (SEQ ID NO: 9), IRES (SEQ ID NO: 10), and GFP (SEQ ID NO: 11); and
  • HER2-CAR gene including CD8 leader (SEQ ID NO: 1), HER2 (human epidermal growth factor receptor 2) scFv (SEQ ID NO: 4), CD8 hinge (SEQ ID NO: 5), CD8 transmembrane domain (SEQ ID NO: 6), CD28 intracellular domain (SEQ ID NO: 8), CD3 (SEQ ID NO: 9), IRES (SEQ ID NO: 10), and GFP (SEQ ID NO: 11).
  • the IRES was inserted for cloning the CAR gene into a vector constituting a double cistron.
  • each domain of FIG. 1 was synthesized from CAR1 (Addgene ID: 113014), and CAR-expressing lentiviruses were constructed through overlap PCR (Gibson assembly).
  • PBMC peripheral blood mononuclear cells
  • stempro SFEM II containing 2.5% Stem Pro-34 nutrient supplement, 2 mM Glutamax I, 1% penicillin/streptomycin, 20 ng/mL human IL (interleukin)-3, 20 ng/mL human IL-6, 100 ng/mL human stem cell factor (SCF), and 100 ng/mL human FLT3 (FMS-like tyrosine kinase)
  • SCF human stem cell factor
  • FLT3 FMS-like tyrosine kinase
  • the Sendai virus system [Oct4, Sox2, Klf4, and Myc-expressing RNA-based Sendai virus (CytoTune 2.0 Sendai Reprogramming Kit, Thermo Scientific); OSKM-SeV] expressing the reprogramming factors and the 4 types of lentiviruses expressing the 4 types of CARs of Example 1 were used.
  • the cells were cultured in a standard culture medium (SCM medium) containing the Sendai virus (5 MOI), PBMC and polybrene (4 ⁇ g/mL) for 1 day, and then sequentially cultured in the first medium, second medium, and third medium shown below.
  • SCM medium standard culture medium
  • Sendai virus 5 MOI
  • PBMC peripheral blood mononuclear cells
  • polybrene 4 ⁇ g/mL
  • the cells transformed by the method (2 ⁇ 10 5 cells/48-well plate) were cultured in the first CAR-iNK medium (StemSpan SFEM II containing 10% FBS, 1% penicillin/streptomycin, 100 ng/mL human SCF, 100 ng/mL human FLT3, 20 ng/mL human IL-3, 20 ng/mL human IL-6, and 5 ⁇ M CHIR99021) including the GSK3 ⁇ (glycogen synthase kinase 3 ⁇ ) inhibitor for 5 to 6 days, and then cultured in the CAR-iNK second medium (StemSpan SFEM II containing 10% FBS, 1% penicillin/streptomycin, 20 ng/mL human SCF, 20 ng/mL human FLT3, 200 IU/mL human IL-2, 20 ng/mL human IL-7, 20 ng/mL human IL-15, and 2 ⁇ M StemRegenin I) including the AHR (aryl hydrocarbon receptor) antagonist for 12 days
  • the CAR-expressing lentiviruses were inoculated into a culture medium selected from the step of culturing in the CAR-iNK first medium (day 4), the CAR-iNK second medium (day 14), or the CAR-iNK third medium (day 24) simultaneously with the reprogramming factor (day 0) or after introduction of the reprogramming factor, as shown in FIG. 2 , and thus were transformed into the cells during the reprograming process by which PBMC were converted into NK cells.
  • NK cells introduced with the CD19-CAR1 gene, CD19-CAR2 gene, MSLN-CAR gene, or HER2-CAR gene were prepared through the direct reprogramming, the cells were stained with the CD56 antibody, CD3 antibody, CD19 antigen, MSLN antigen, and HER2 antigen, and then iNK cell group (CD56+ and CD3 ⁇ ), CD19 CAR-iNK cell group (CD56+ and CD19+), MSLN CAR-iNK cell group (CD56+and MSLN+), and HER2 CAR-iNK cell group (CD56+and HER2+) were analyzed using flow cytometry.
  • NK (iNK) cells induced through direct reprogramming were added to a phosphate buffer (FACS buffer) containing 1% BSA (bovine serum albumin) and 2 mM EDTA (ethylenediaminetetraacetic acid), supplemented with fluorescent-labeled antibodies and antigens, and reacted at room temperature for 20 minutes, and subsequently, the cells were washed and recovered using a centrifuge, and then analyzed by FACS (BD Bioscience).
  • FACS buffer phosphate buffer
  • BSA bovine serum albumin
  • EDTA ethylenediaminetetraacetic acid
  • each CAR gene was inserted into the genome of iNK cells
  • genomic DNA of each cell was analyzed with primers (SEQ ID NOS: 12 and 13) for CD19 CAR, primers (SEQ ID NOS: 14 and 15) for MSLN CAR, and primers (SEQ ID NOS: 16 and 17) for HER2 CAR through PCR gene analysis.
  • CD56+CD3 ⁇ CD19-CAR-iNK cells were produced with a maximum efficiency of 45.9% ( FIG. 3 and FIG. 6A ), that CD56+CD3 ⁇ MSLN-CAR-iNK were produced with a maximum efficiency of 43.7% ( FIG. 4 ), and that CD56+CD3 ⁇ HER2-CAR-iNK cells were produced with a maximum efficiency of 47.8% ( FIG. 5 ).
  • the cells were cultured in the CAR-iNK first medium for different incubation periods (3, 5, 7, and 9 days), and then cultured in the CAR-iNK second medium and the third medium under the same conditions as in Example 1, and the CAR-iNK production yield was analyzed by comparison.
  • the CAR-iNK yield efficiency was increased when cultured in the CAR-iNK third medium containing 20 ng/mL human IL-21, 2 ⁇ M CHIR99021, 2 ⁇ M StemRegenin 1 (SR1), 50 ng/mL human IL-12, and 100 ng/mL human IL-18 ( FIG. 7B ).
  • the CAR-iNK yield efficiency was increased up to 15 times when cultured for 14 days ( FIG. 7B ).
  • the CAR-iNK cells were cultured in the CAR-iNK first medium for 5 days, in the CAR-iNK second medium for 12 days, and in the CAR-iNK third medium for 2 to 3 weeks.
  • the frequency of CD107a+ cells was confirmed through flow cytometry. Specifically, the frequency of CD107a+ cells was determined through FACS analysis after reacting the CAR-iNK cells in FACS buffer supplemented with fluorescent-labeled antibodies against CD56 and CD107a at room temperature for 20 minutes, and then washing and recovering the cells using a centrifuge.
  • the frequency of IFN-gamma cells was confirmed through flow cytometry. Specifically, the frequency of IFN-gamma cells was determined after reacting the CAR-iNK cells in a phosphate buffer containing 0.5% Tween 20 and 0.5% BSA at room temperature for 20 minutes, and then washing and recovering the cells using a centrifuge. The recovered cells were reacted at room temperature for 20 minutes in FACS buffer supplemented with fluorescent-labeled antibodies against CD56 and IFN-gamma, and then the cells were washed and recovered using a centrifuge and subjected to FACS analysis.
  • the cell killing ability of the CAR-iNK cells produced in Example 1 was measured using calcein-AM.
  • the iNK cells were prepared by diluting the cells to a density of 0.25 ⁇ 10 5 cells/mL and 1 ⁇ 10 5 cells/mL using a culture solution, and then dispended in a 96-well plate in an amount of 100 mL.
  • the thus-prepared calcein-labeled target cancer cells (1 ⁇ 10 5 cells/mL) were added to a 96-well plate in an amount of 100 pUwell, centrifuged at 400 g for 1 minute, and then cultured in a cell incubator at 37° C. for 4 hours in the presence of 5% CO 2 , and subsequently, 100 ⁇ L of the supernatant was taken from each well and measured with a fluorescence plater reader (485 nm/535 nm).
  • the cell killing ability (%) was calculated according to the following formula.
  • Cancer Cell Killing Ability(%) ⁇ (Measured Value ⁇ Minimum Value)/(Measured Value ⁇ Minimum Value) ⁇ 100
  • the minimum value is the measured value of a well in which only calcein-labeled target cancer cells exist
  • the maximum value is the measured value of a well in which cells are completely lysed by adding 0.1% TritonX-100 to the calcein-labeled target cancer cells.
  • CD19-CAR-iNK cells FIG. 16
  • MSLN-CAR-iNK cells FIG. 17
  • HER2-CAR-iNK cells FIG. 18
  • the tumor size formed in the iNK (5.35 ⁇ 10 9 radiance) and CAR-NK (3.83 ⁇ 10 9 radiance) cell experimental groups was significantly reduced compared to the tumor size (1.21 ⁇ 10 10 radiance) of the control group formed under the condition of PBS injection on the 14th day, thereby confirming that the CAR-iNK cells exhibited an excellent anticancer effect compared to iNK cells ( FIG. 19 ).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
US17/598,916 2019-03-28 2020-03-27 Method for producing car gene-introduced nk cells and use thereof Pending US20220184128A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
KR10-2019-0036265 2019-03-28
KR20190036265 2019-03-28
KR20190036264 2019-03-28
KR10-2019-0036264 2019-03-28
PCT/KR2020/004197 WO2020197318A1 (fr) 2019-03-28 2020-03-27 Procédé de production de cellules nk avec un gène car introduit dans ces dernières, et son utilisation

Publications (1)

Publication Number Publication Date
US20220184128A1 true US20220184128A1 (en) 2022-06-16

Family

ID=72610717

Family Applications (2)

Application Number Title Priority Date Filing Date
US17/598,916 Pending US20220184128A1 (en) 2019-03-28 2020-03-27 Method for producing car gene-introduced nk cells and use thereof
US17/598,917 Active 2042-11-28 US12435117B2 (en) 2019-03-28 2020-03-27 Method for producing immune cells and use thereof

Family Applications After (1)

Application Number Title Priority Date Filing Date
US17/598,917 Active 2042-11-28 US12435117B2 (en) 2019-03-28 2020-03-27 Method for producing immune cells and use thereof

Country Status (6)

Country Link
US (2) US20220184128A1 (fr)
EP (2) EP3950935A4 (fr)
JP (3) JP2022527932A (fr)
KR (2) KR102298639B1 (fr)
CN (2) CN113874493B (fr)
WO (2) WO2020197318A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024102860A1 (fr) * 2022-11-09 2024-05-16 Shoreline Biosciences, Inc. Cellules ingéniérisées pour une thérapie
CN118240758A (zh) * 2024-04-12 2024-06-25 深圳泽医细胞治疗集团有限公司 一种免疫细胞的培养基及其应用

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112656789A (zh) * 2020-12-31 2021-04-16 中国科学院生态环境研究中心 Ficz在抑制肿瘤细胞迁移中的应用
US20240240148A1 (en) * 2021-05-14 2024-07-18 Appia Bio, Inc. Engineering stem cells for allogenic car t cell therapies
WO2023038476A1 (fr) * 2021-09-10 2023-03-16 한국생명공학연구원 Procédé de production d'une cellule tueuse naturelle et son utilisation pour la prévention ou le traitement de maladies infectieuses
JP2024534981A (ja) * 2021-09-10 2024-09-26 コリア リサーチ インスティテュート オブ バイオサイエンス アンド バイオテクノロジー 誘導ナチュラルキラー細胞の製造方法及びその使用
US20250009879A1 (en) * 2021-11-22 2025-01-09 The Asan Foundation Natural killer cell-specific chimeric antigen receptor and use thereof
CN114350608B (zh) * 2022-01-27 2024-05-28 昭泰英基生物医药（香港）有限公司 一种诱导t细胞重编程为类nk细胞的组合物及其应用
US20250228943A1 (en) * 2022-04-08 2025-07-17 Fate Therapeutics, Inc. Cells having solid tumor targeting backbone and use thereof
CN114958771B (zh) * 2022-06-27 2023-12-05 广东康盾创新产业集团股份公司 一种ipsc-car-nk细胞的制备方法
KR20240066123A (ko) 2022-11-07 2024-05-14 마루테라퓨틱스 주식회사 Nk 세포의 분화 효율 및 기능성 증가를 위한 배양 방법
CN115804776A (zh) * 2022-12-16 2023-03-17 国纳之星(上海)纳米科技发展有限公司 一种芳香烃受体抑制剂和5-氟尿嘧啶合用在治疗抑制肿瘤药物中的应用
WO2024153172A1 (fr) * 2023-01-19 2024-07-25 中国科学院动物研究所 Cellule art-nk ciblant msln et procédé de préparation associé
WO2025033948A1 (fr) * 2023-08-07 2025-02-13 마루테라퓨틱스 주식회사 Vecteur plasmidique double non viral à expression génique hautement efficace, cellules souches l'exprimant et cellule du système hématopoïétique différenciée
AU2024201132A1 (en) * 2023-09-20 2025-04-10 Uci Therapeutics Inc. Anti-mesothelin antibodies and uses thereof
CN117660335A (zh) * 2023-12-14 2024-03-08 广州百康细胞生命科技有限公司 一种使用化学重编辑法将体细胞转化成免疫细胞的方法
WO2025130895A1 (fr) * 2023-12-19 2025-06-26 苏州沙砾生物科技有限公司 Procédé de préparation et utilisation de cellules immunitaires
CN118186007B (zh) * 2024-03-18 2025-02-25 广州市新一代生物工程有限公司 一种基于prf1基因提高car-nk细胞治疗效果的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016123333A1 (fr) * 2015-01-29 2016-08-04 Regents Of The University Of Minnesota Récepteurs d'antigène chimère, compositions, et procédés
US20160257939A1 (en) * 2009-06-05 2016-09-08 Cellular Dynamics International, Inc. Reprogramming t cells and hematopoietic cells

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2602743T3 (es) * 2010-09-08 2017-02-22 Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus Receptores de antígenos quiméricos con una región bisagra optimizada
WO2013163171A1 (fr) * 2012-04-24 2013-10-31 Kaufman Dan S Procédé de développement de cellules tueuses naturelles à partir de cellules souches
KR101550269B1 (ko) * 2014-01-22 2015-09-07 한국생명공학연구원 섬유아세포로부터 조혈줄기세포로의 직접 역분화 유도 방법
KR20200138445A (ko) * 2014-04-24 2020-12-09 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 입양 세포 요법 생성물을 생성하기 위한 유도 만능 줄기 세포의 응용
BR112017014269A2 (pt) * 2014-12-31 2018-03-27 Anthrogenesis Corp células exterminadoras naturais e usos das mesmas
CN106349389B (zh) * 2015-07-21 2019-11-15 科济生物医药（上海）有限公司 肿瘤特异性抗egfr抗体及其应用
US10947502B2 (en) * 2015-10-20 2021-03-16 FUJIFILM Cellular Dynamics, Inc. Methods for directed differentiation of pluripotent stem cells to immune cells
CN109562126A (zh) * 2016-06-24 2019-04-02 美商生物细胞基因治疗有限公司 嵌合抗原受体(car)、组合物及其使用方法
SG11201909331UA (en) * 2017-04-18 2019-11-28 Fujifilm Cellular Dynamics Inc Antigen-specific immune effector cells
EP3630956A4 (fr) * 2017-05-22 2021-03-31 Transfusion Health, LLC Compositions et méthodes de production de cellules souches hématopoïétiques expansées à l'aide d'inhibiteurs de pten
CN107384949B (zh) * 2017-06-27 2019-01-15 闾民 表达GPC3嵌合抗原受体的iNKT细胞及制备、应用
WO2019059713A2 (fr) 2017-09-21 2019-03-28 한국생명공학연구원 Procédé pour la production de cellules tueuses naturelles et son utilisation
KR102167548B1 (ko) * 2017-09-21 2020-10-19 한국생명공학연구원 자연살해세포의 제조방법 및 그의 용도

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160257939A1 (en) * 2009-06-05 2016-09-08 Cellular Dynamics International, Inc. Reprogramming t cells and hematopoietic cells
WO2016123333A1 (fr) * 2015-01-29 2016-08-04 Regents Of The University Of Minnesota Récepteurs d'antigène chimère, compositions, et procédés

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Freud et al., A Human CD34(+) Subset Resides in Lymph Nodes and Differentiates into CD56bright Natural Killer Cells. Immunity. 2005; 22: 295-304. (Year: 2005) *
Kim et al., The Generation of Human Induced Pluripotent Stem Cells from Blood Cells: An Efficient Protocol Using Serial Plating of Reprogrammed Cells by Centrifugation. Stem Cells International. 2016, Article ID 1329459, p. 1-9. (Year: 2016) *
Qi et al., The magic of four: induction of pluripotent stem cells from somatic cells by Oct4, Sox2, Myc and Klf4. Cell Research. 2007; 17:578-580. (Year: 2007) *
Roeven et al. The Aryl Hydrocarbon Receptor Antagonist StemRegenin1 Improves In Vitro Generation of Highly Functional Natural Killer Cells from CD34+ Hematopoietic Stem and Progenitor Cells. Stem Cells and Development. 2015; 24(24): 2886-2898. (Year: 2015) *
Son et al., Interleukin-18 (IL-18) Synergizes with IL-2 to Enhance Cytotoxicity, Interferon-g Production, and Expansion of Natural Killer Cells. Cancer Research. 2001; 61: 884-888. (Year: 2001) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024102860A1 (fr) * 2022-11-09 2024-05-16 Shoreline Biosciences, Inc. Cellules ingéniérisées pour une thérapie
CN118240758A (zh) * 2024-04-12 2024-06-25 深圳泽医细胞治疗集团有限公司 一种免疫细胞的培养基及其应用

Also Published As

Publication number Publication date
JP7429713B2 (ja) 2024-02-08
EP3950935A4 (fr) 2023-06-07
JP2022528086A (ja) 2022-06-08
EP3954762A4 (fr) 2023-01-18
CN113874492B (zh) 2024-03-19
WO2020197318A1 (fr) 2020-10-01
JP2022527932A (ja) 2022-06-07
US12435117B2 (en) 2025-10-07
KR20200115357A (ko) 2020-10-07
KR102298639B1 (ko) 2021-09-07
JP2023179554A (ja) 2023-12-19
EP3950935A1 (fr) 2022-02-09
CN113874493B (zh) 2024-05-10
KR102298640B1 (ko) 2021-09-07
CN113874492A (zh) 2021-12-31
EP3954762A1 (fr) 2022-02-16
US20220160769A1 (en) 2022-05-26
KR20200115358A (ko) 2020-10-07
WO2020197319A1 (fr) 2020-10-01
CN113874493A (zh) 2021-12-31

Similar Documents

Publication Publication Date Title
US20220184128A1 (en) Method for producing car gene-introduced nk cells and use thereof
JP7058728B2 (ja) ナチュラルキラー細胞の製造方法及びその用途
CN116514998B (zh) 一种嵌合抗原受体、嵌合抗原受体-自然杀伤细胞及其在制备抗肿瘤药物中的应用
US20240156963A1 (en) Combinational immunotherapies using car-m, car-nk, car-eos, and car-n cells
CN116286660B (zh) 一种ipsc-（car）自然杀伤细胞、制备方法及其在肿瘤治疗中的应用
US20240318139A1 (en) Method for producing directly reprogrammed natural killer cells and uses thereof
WO2025055493A1 (fr) Lymphocytes infiltrant les tumeurs édités par un gène et leur utilisation dans une immunothérapie cellulaire
JP2023504075A (ja) Ｃａｒ－ｎｋ細胞を得る方法
JP2021069322A (ja) Ｃｄ４陽性制御性ｔ細胞の製造方法
KR20230039789A (ko) 자연살해세포의 제조방법 및 이의 감염성 질환 예방 또는 치료 용도
US20260071181A1 (en) Universal natural killer cells derived from human pluripotent stem cells and method of use
KR20230153301A (ko) 이종 면역세포에 대한 화학주성을 유도하는 전능성 줄기세포 유래 면역세포
CN120272430A (zh) 巨噬细胞的制备及其应用

Legal Events

Date Code Title Description
AS Assignment

Owner name: KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, KOREA, REPUBLIC OF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:CHO, YEE SOOK;KIM, HAN-SEOP;KIM, JAE YUN;AND OTHERS;REEL/FRAME:057655/0255

Effective date: 20210930

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

AS Assignment

Owner name: CEFO CO., LTD., KOREA, REPUBLIC OF

Free format text: LICENSE;ASSIGNOR:KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY;REEL/FRAME:061624/0364

Effective date: 20221019

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION COUNTED, NOT YET MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION COUNTED, NOT YET MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED