WO2022025220A1 - キメラ受容体を発現する細胞を含む医薬組成物 - Google Patents

キメラ受容体を発現する細胞を含む医薬組成物 Download PDF

Info

Publication number
WO2022025220A1
WO2022025220A1 PCT/JP2021/028230 JP2021028230W WO2022025220A1 WO 2022025220 A1 WO2022025220 A1 WO 2022025220A1 JP 2021028230 W JP2021028230 W JP 2021028230W WO 2022025220 A1 WO2022025220 A1 WO 2022025220A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
antibody
amino acid
receptor
binding
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.)
Ceased
Application number
PCT/JP2021/028230
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
智之 井川
実香 櫻井
隆 鈴木
加奈子 辰巳
駿 清水
耕治 玉田
幸美 佐古田
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.)
Chugai Pharmaceutical Co Ltd
Yamaguchi University NUC
Original Assignee
Chugai Pharmaceutical Co Ltd
Yamaguchi University NUC
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 Chugai Pharmaceutical Co Ltd, Yamaguchi University NUC filed Critical Chugai Pharmaceutical Co Ltd
Priority to CN202180058518.9A priority Critical patent/CN116194124A/zh
Priority to KR1020237006010A priority patent/KR20230048059A/ko
Priority to EP21850145.0A priority patent/EP4197545A4/en
Priority to MX2023001120A priority patent/MX2023001120A/es
Priority to CA3190649A priority patent/CA3190649A1/en
Priority to IL300225A priority patent/IL300225A/en
Priority to BR112023000537A priority patent/BR112023000537A2/pt
Priority to AU2021317974A priority patent/AU2021317974A1/en
Priority to JP2022539585A priority patent/JPWO2022025220A1/ja
Priority to US18/018,807 priority patent/US20230322898A1/en
Publication of WO2022025220A1 publication Critical patent/WO2022025220A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] 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
    • 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
    • A61K40/34—Antigenic peptides
    • 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
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • 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
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K16/2818—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 CD28 or CD152
    • 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
    • C07K16/283—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 Fc-receptors, e.g. CD16, CD32, CD64
    • 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
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • 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
    • C07K16/303—Liver or Pancreas
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N5/0636—T lymphocytes
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
    • 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
    • A61K2239/39—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by a specific adjuvant, e.g. cytokines or CpG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • 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
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • 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
    • C12N2740/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/10043—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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—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
    • 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
    • C12N2740/15043—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
    • 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

Definitions

  • the present invention relates to a chimeric receptor, cells expressing the chimeric receptor, and a method for treating a disease using the cells, for example, adoptive cell immunotherapy, particularly adoptive cell immunotherapy using T cells expressing the chimeric receptor. Regarding.
  • Chimeric antigen receptor (hereinafter also referred to as "CAR") artificially fuses an antigen-binding molecule that recognizes an antigen on the cell surface with a signal transduction region that induces activation of immune cells. It is a chimeric protein.
  • CAR Chimeric antigen receptor
  • immune cells expressing CAR are produced.
  • CAR-expressing cells exhibit cytotoxicity to cells expressing the target antigen without depending on the interaction with the major histocompatibility complex (MHC), and for diseases such as cancer caused by adoptive cell immunotherapy. Used for treatment (Non-Patent Document 1).
  • MHC major histocompatibility complex
  • Patent Documents 1 to 5 The establishment of technology for producing immune cells that can universally recognize target cells expressing various antigens has important clinical significance in generalizing adoptive cell immunotherapy. So far, there have been reports on such studies (Patent Documents 1 to 5 and Non-Patent Documents 2 to 5).
  • Non-Patent Documents 6 to 7 the therapeutic effects of antibodies that inhibit the immune checkpoint molecules CTLA-4, PD-1, and PD-L1 have been reported (Non-Patent Documents 6 to 7), and they have been approved as pharmaceuticals.
  • Non-Patent Document 8 it has been demonstrated in a mouse model that an antitumor effect is exhibited by activating a co-stimulator expressed on immune cells with an agonist antibody.
  • a therapeutic method for enhancing the drug efficacy by co-administering a CD137 agonist antibody that activates immune cells and CAR-T has been devised (Patent Document 6 and Non-Patent Document 9).
  • One of the problems in the treatment of cancer by adoptive cell immunotherapy using CAR is that the therapeutic effect on solid cancer is poor. This is due to the fact that the tumor antigens expressed on individual cancer cells in cancer tissue are heterogeneous, so CAR-expressing cells that recognize only a single target antigen cannot recognize all tumor cells, and tumors. An immunosuppressive mechanism in a microenvironment is considered. As a strategy for solving this problem, it is conceivable to activate not only CAR-expressing cells but also endogenous immune cells to develop an antitumor effect in cooperation with the endogenous immune cells, and one of the means for that purpose. As one of them, concomitant therapy with a CD137 agonist antibody that activates immune cells can be considered.
  • CD137 agonist antibody induces activation of immune cells in non-tumor tissues such as liver and develops toxicity.
  • another issue of cancer treatment by adoptive cell immunotherapy using CAR includes the following points. First, since the tumor antigen expressed on the surface of the tumor cell differs for each cancer cell, it is necessary to construct the antigen recognition site of CAR for each target tumor antigen. Second, the tumor antigens targeted by CAR can cause tumor antigen escape by reducing or mutating their expression levels with treatment. Tumor antigen escape reduces the therapeutic effect, as many current CAR-expressing cells recognize only a single target antigen.
  • the present disclosure is a chimera having a multispecific antigen-binding molecule having a site that exerts agonist activity on an immunoreceptor and a site that recognizes a target antigen, and an immunoreceptor recognized by the antigen-binding molecule in an extracellular domain. It provides a therapeutic method in combination with an immune cell into which a receptor has been introduced.
  • the antigen-binding molecule in the present disclosure since the antigen-binding molecule in the present disclosure accumulates at a lesion site expressing a target antigen, its agonist activity can be selectively expressed at the lesion site, and as a result, the development of toxicity can be avoided.
  • the chimeric receptor in the present disclosure does not have the binding property to the target antigen, and therefore has the characteristic of not inhibiting the binding of the antigen-binding molecule in the present disclosure to the target antigen. Furthermore, since the chimeric receptor in the present disclosure has the binding property to the antigen-binding molecule in the present disclosure, the activation of the endogenous immune cell and the activity of the immune cell expressing the chimeric receptor by the antigen-binding molecule alone are used. It is possible to induce the formation at the same time. That is, the present invention discloses the following.
  • a pharmaceutical composition containing cells expressing a chimeric receptor for use in combination with administration of an antigen-binding molecule comprises an extracellular domain, the extracellular domain comprising an extracellular domain of an immune receptor, an extracellular domain variant of an immune receptor, or a fragment thereof.
  • the antigen-binding molecule is a pharmaceutical composition, which is a multispecific antigen-binding molecule having a target antigen recognition site and an immunoreceptor recognition site that recognizes the immunoreceptor.
  • a pharmaceutical composition containing an antigen-binding molecule for use in combination with administration of cells expressing a chimeric receptor is a pharmaceutical composition, which is a multispecific antigen-binding molecule having a target antigen recognition site and an immunoreceptor recognition site that recognizes the immunoreceptor.
  • a chimeric receptor comprises an extracellular domain, the extracellular domain comprising an extracellular domain of an immune receptor, an extracellular domain variant of an immune receptor, or a fragment thereof.
  • the antigen-binding molecule is a pharmaceutical composition, which is a multispecific antigen-binding molecule having a target antigen recognition site and an immunoreceptor recognition site that recognizes the immunoreceptor. [3] The pharmaceutical composition according to [1] or [2], wherein the immune receptor recognition site recognizes the extracellular domain of the chimeric receptor and the endogenous immune receptor.
  • the extracellular domain of the chimeric receptor comprises a human CD137 extracellular domain variant or a fragment thereof in which at least a part or all of the cysteine rich domain 3 and the cysteine rich domain 4 of the human CD137 extracellular domain are deleted. , [1] to [6].
  • the target antigen is a receptor, a tumor antigen, an MHC antigen, or a differentiation antigen.
  • the antigen-binding molecule is a bispecific antibody.
  • a chimeric receptor comprising an extracellular domain of an immune receptor, an extracellular domain variant of an immune receptor, or an extracellular domain comprising a fragment thereof.
  • the extracellular domain of the chimeric receptor comprises a human CD137 variant or fragment thereof in which at least some or all of the cysteine-rich domain 3 and the cysteine-rich domain 4 of the human CD137 extracellular domain have been deleted [13]. ]-[16].
  • the chimeric receptor according to any one of. [18] The chimeric receptor according to any one of [13] to [17], wherein the chimeric receptor comprises an intracellular signal transduction domain and the intracellular signal transduction domain comprises an intracellular signal transduction domain of CD3 zeta.
  • [19] A cell expressing the chimeric receptor according to any one of [13] to [18].
  • [20] A composition comprising the cells of [19].
  • [21] The nucleic acid encoding the chimeric receptor according to any one of [13] to [18].
  • [22] A vector into which the nucleic acid according to [21] is inserted.
  • a pharmaceutical composition containing cells expressing a chimeric receptor for use in combination with administration of an antigen-binding molecule comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain, the extracellular domain comprising an extracellular domain of an immune receptor, an extracellular domain variant of an immune receptor, or a fragment thereof.
  • the antigen-binding molecule is a pharmaceutical composition comprising a target antigen recognition site and a bispecific antigen-binding site having an immunoreceptor recognition site that recognizes the immunoreceptor.
  • a pharmaceutical composition containing an antigen-binding molecule for use in combination with administration of cells expressing a chimeric receptor is a pharmaceutical composition comprising a target antigen recognition site and a bispecific antigen-binding site having an immunoreceptor recognition site that recognizes the immunoreceptor.
  • the chimeric receptor comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain, the extracellular domain comprising an extracellular domain of an immune receptor, an extracellular domain variant of an immune receptor, or a fragment thereof.
  • the antigen-binding molecule is a pharmaceutical composition comprising a target antigen recognition site and a bispecific antigen-binding site having an immunoreceptor recognition site that recognizes the immunoreceptor. [A3] The pharmaceutical composition according to [A1] or [A2], wherein the immune receptor recognition site recognizes the extracellular domain of the chimeric receptor and the endogenous immunoreceptor.
  • [A4] Described in any of [A1] to [A3], wherein the extracellular domain of the chimeric receptor comprises an extracellular domain variant of the immune receptor or a fragment thereof whose binding to an endogenous immune ligand is attenuated.
  • Pharmaceutical composition [A5] The pharmaceutical composition according to any one of [A1] to [A4], wherein the immunoreceptor is a co-stimulating molecule.
  • [A6] The pharmaceutical composition according to any one of [A1] to [A5], wherein the immune receptor is human CD137.
  • the extracellular domain of the chimeric receptor comprises an extracellular domain variant or fragment thereof in which at least a part or all of the cysteine rich domain 3 and the cysteine rich domain 4 of the human CD137 extracellular domain are deleted.
  • [A8] The pharmaceutical composition according to any one of [A1] to [A7], wherein the intracellular signal transduction domain comprises the intracellular signal transduction domain of CD3 zeta.
  • [A9] The pharmaceutical composition according to any one of [A1] to [A8], wherein the target antigen is a receptor, a tumor antigen, an MHC antigen, or a differentiation antigen.
  • the target antigen is a receptor, a tumor antigen, an MHC antigen, or a differentiation antigen.
  • [A11] The pharmaceutical composition according to any one of [A1] to [A10], wherein the antigen-binding molecule is a bispecific antibody.
  • [A12] The pharmaceutical composition according to any one of [A1] to [A11] for use in the treatment or prevention of cancer.
  • [A13] A chimeric receptor comprising an extracellular domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular domain comprises an extracellular domain of an immune receptor, an extracellular domain variant of an immune receptor or a fragment thereof. ..
  • [A14] The chimeric receptor according to [A13], wherein the extracellular domain of the chimeric receptor comprises an extracellular domain variant of the immune receptor or a fragment thereof whose binding to an endogenous immune ligand is attenuated.
  • [A15] The chimeric receptor according to [A13] or [A14], wherein the immunoreceptor is a co-stimulator molecule.
  • [A16] The chimeric receptor according to any one of [A13] to [A15], wherein the immune receptor is human CD137.
  • the extracellular domain of the chimeric receptor comprises an extracellular domain variant or fragment thereof in which at least some or all of the cysteine rich domain 3 and the cysteine rich domain 4 of the human CD137 extracellular domain have been deleted [A17].
  • [A18] The chimeric receptor according to any one of [A13] to [A17], wherein the intracellular signal transduction domain comprises the intracellular signal transduction domain of CD3 zeta.
  • [A19] A cell expressing the chimeric receptor according to any one of [A13] to [A18].
  • a composition comprising the cells of [A20] and [A19].
  • [A21] A nucleic acid encoding the chimeric receptor according to any one of [A13] to [A17].
  • [A22] A vector into which the nucleic acid according to [A21] is inserted.
  • [A23] The method for producing a cell according to [A18], which comprises transfecting or transducing the cell using the nucleic acid according to [A21] or the vector according to [A22].
  • [B1] A method of treating a disease. Administering an effective amount of the antigen-binding molecule to a subject in need of treatment, Administering cells expressing the chimeric receptor to the subject, Including The chimeric receptor comprises an extracellular domain, the extracellular domain comprising an extracellular domain of an immune receptor, an extracellular domain variant of an immune receptor, or a fragment thereof.
  • the method described above, wherein the antigen-binding molecule is a multispecific antigen-binding molecule having a target antigen recognition site and an immunoreceptor recognition site that recognizes the immunoreceptor.
  • the chimeric receptor further comprises a transmembrane domain and an intracellular signal transduction domain.
  • [B3] The method according to [B1] or [B2], wherein the immune receptor recognition site recognizes the extracellular domain of the chimeric receptor and the endogenous immunoreceptor.
  • [B5] The method according to any one of [B1] to [B4], wherein the immune receptor is a co-stimulating molecule.
  • [B6] The method according to any of [B1] to [B5], wherein the immune receptor is human CD137.
  • the extracellular domain of the chimeric receptor comprises a human CD137 extracellular domain variant or a fragment thereof in which at least a part or all of the cysteine rich domain 3 and the cysteine rich domain 4 of the human CD137 extracellular domain are deleted. , [B1] to [B6].
  • [B8] The method according to any one of [B1] to [B7], wherein the chimeric receptor comprises an intracellular signaling domain and the intracellular signaling domain comprises an intracellular signaling domain of CD3 zeta.
  • [B9] The method according to any one of [B1] to [B8], wherein the target antigen is a receptor, a tumor antigen, an MHC antigen, or a differentiation antigen.
  • the target antigen is a tumor antigen.
  • the antigen-binding molecule is a bispecific antibody.
  • [B12] The method according to any one of [B1] to [B11], wherein the disease is cancer.
  • Cancer is primary or metastatic melanoma, thoracic adenoma, lymphoma, sarcoma, lung cancer, colorectal cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer ,
  • the method according to [B12] which is selected from bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer.
  • [C1] A method of treating a disease. Administering an effective amount of the antigen-binding molecule to a subject in need of treatment, Administering cells expressing the chimeric receptor to the subject, Including The chimeric receptor comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain, the extracellular domain comprising an extracellular domain of an immune receptor, an extracellular domain variant of an immune receptor, or a fragment thereof.
  • the antigen binding molecule comprises a bispecific antigen binding site having a target antigen recognition site and an immunoreceptor recognition site that recognizes the immunoreceptor.
  • [C2] The method according to [C1], wherein the chimeric receptor further comprises a transmembrane domain and an intracellular signal transduction domain.
  • [C3] The method according to [C1] or [C2], wherein the immune receptor recognition site recognizes the extracellular domain of the chimeric receptor and the endogenous immunoreceptor.
  • [C4] Described in any of [C1] to [C3], wherein the extracellular domain of the chimeric receptor comprises an extracellular domain variant of the immune receptor or a fragment thereof whose binding to an endogenous immune ligand is attenuated. the method of.
  • [C5] The method according to any one of [C1] to [C4], wherein the immune receptor is a costimulatory molecule.
  • [C6] The method according to any of [C1] to [C5], wherein the immune receptor is human CD137.
  • the extracellular domain of the chimeric receptor comprises an extracellular domain variant or fragment thereof in which at least some or all of the cysteine rich domain 3 and the cysteine rich domain 4 of the human CD137 extracellular domain have been deleted [C7].
  • [C8] The method according to any one of [C1] to [C7], wherein the intracellular signal transduction domain comprises the intracellular signal transduction domain of the CD3 zeta.
  • [C9] The method according to any one of [C1] to [C8], wherein the target antigen is a receptor, a tumor antigen, an MHC antigen, or a differentiation antigen.
  • the target antigen is a tumor antigen.
  • the antigen-binding molecule is a bispecific antibody.
  • Cancer is primary or metastatic melanoma, thoracic adenoma, lymphoma, sarcoma, lung cancer, colorectal cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer ,
  • the method according to [C12] which is selected from bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer.
  • an antigen-binding molecule for example, a bispecific antibody
  • a chimeric receptor having an immunoreceptor recognized by the antigen-binding molecule in the extracellular domain are characterized.
  • FIG. 2 shows the cells of CD137 compared to the chimeric receptors CD137-CD8-CD28-CD137-CD3 Zeta (CD137-CR1), trCD137-CD8-CD28-CD137-CD3 Zeta (trCD137-CR) and CD137-CR1.
  • FIG. 6 is a schematic diagram showing a chimeric receptor CD137-CR2 having I64R and V71R mutations in the outer domain.
  • FIG. 3 is a schematic diagram of the sequence of the lentiviral vector construct expressing pCDH-CD137-CR1 described in Example 3-1 and the frame-by-frame components from the 5'end to the 3'end.
  • FIG. 4 shows CD137-CR1 when co-cultured with cells expressing human GPC3 as a target antigen and a bispecific antibody consisting of an anti-GPC3 antibody and an anti-CD137 antibody according to the reporter assay described in Example 3-4. It is a figure which shows the evaluation result of the activation ability of Jurkat cell.
  • FIG. 5 shows CD137-CR1 when co-cultured with cells expressing human GPRC5D as a target antigen and a bispecific antibody consisting of an anti-GPRC5D antibody and an anti-CD137 antibody according to the reporter assay described in Example 3-5.
  • -It is a figure which shows the evaluation result of the activation ability of copperGFPJurkat cells.
  • FIG. 6 shows cells co-cultured with cells expressing human IL-6R as a target antigen and a bispecific antibody consisting of an anti-IL-6R antibody and an anti-CD137 antibody according to the reporter assay described in Example 3-6. It is a figure which shows the evaluation result of the activation ability of NFAT-RE-luc2 Jurkat cell (copGFP) and CD137-CR1-copGFP Jurkat cell (CD137-CR1) which do not express a chimeric receptor but express only copyGFP.
  • FIG. 7 is a schematic diagram of the sequence sequence of the retroviral vector construct expressing pMSGV1-CD137-CR1 according to Example 4-1 and the frame-by-frame components from the 5'end to the 3'end.
  • a bispecific antibody MRAH.v1-F760mnN17 / MRAL.v1-k0.v1 // hCD137VH-F760mnP17 / hCD137VL-k0
  • FIG. 7 is a schematic diagram of the sequence sequence of the retroviral vector construct expressing pMSGV1-CD137-CR1 according to Example 4-1 and the frame-by-frame components from the 5'end to the 3'end.
  • the human liver cancer cell line (SK-Hep-1) described in Example 4-4 and SK-pca60 cells expressing human GPC3 as a target antigen are used, and three types of bispecific antibodies are used.
  • CD137-CR1-eGFP-expressing T cells using the number of residual tumor cells as an index when co-cultured with consisting of anti-GPC3 antibody and anti-KLH antibody, anti-KLH antibody and anti-CD137 antibody, anti-GPC3 antibody and anti-CD137 antibody
  • the vertical axis shows the number of residual tumor cells, and the horizontal axis shows the presence or absence of three bispecific antibodies.
  • FIG. 8B the human liver cancer cell line (SK-Hep-1) described in Example 4-5 and SK-pca60 cells expressing human GPC3 as target antigens were used, and from anti-GPC3 antibody and anti-CD137 antibody.
  • the ratio of effector cells to target cells (E: T) and the presence or absence of bispecific antibodies are evaluated (indicated as “with antibody” and “without antibody” in FIG. 8B).
  • the vertical axis shows cell proliferation activity (%), and the horizontal axis shows the number of days since the addition of effector cells and antibodies.
  • FIG. 9 is a schematic diagram showing a target antigen-independent activation mechanism of chimeric receptor-expressing cells by binding to cells expressing the ligand.
  • FIG. 10 shows trCD137-CR when co-cultured with cells expressing human GPC3 as a target antigen and a bispecific antibody consisting of an anti-GPC3 antibody and an anti-CD137 antibody according to the reporter assay described in Example 6-4. It is a graph which shows the result of having evaluated the activation ability of -copGFP Jurkat cell (trCD137-CR).
  • FIG. 11 shows CD137-CR2 when co-cultured with cells expressing human GPC3 as a target antigen and a bispecific antibody consisting of an anti-GPC3 antibody and an anti-CD137 antibody according to the reporter assay described in Example 7-4. It is a graph which shows the result of having evaluated the activation ability of -copGFP Jurkat cell (CD137-CR2).
  • FIG. 12A shows the CD137-CR1-copGFP Jurkat cells (CD137-CR1) prepared in Example 3-2 in the presence / absence of the ligand (human CD137L) -expressing cells (Raj) described in Example 8. It is a graph which shows the result of having evaluated the activation ability of the chimeric receptor-expressing cell.
  • the vertical axis shows the luminescence intensity of luciferase.
  • FIG. 12B shows the trCD137-CR-copGFP Jurkat cells (trCD137-CR) prepared in Example 6-2 in the presence / absence of the ligand (human CD137L) -expressing cells (Raj) described in Example 8. It is a graph which shows the result of having evaluated the activation ability of the chimeric receptor-expressing cell. The vertical axis shows the luminescence intensity of luciferase.
  • FIG. 12C shows the CD137-CR2-copGFP Jurkat cells (CD137-CR2) prepared in Example 7-2 in the presence / absence of the ligand (human CD137L) -expressing cells (Raj) described in Example 8. It is a graph which shows the result of having evaluated the activation ability of the chimeric receptor-expressing cell.
  • FIG. 13 is a graph showing the results of measuring the binding activity of BB0000 and BB0077 to the in vivo ligand (human CD137L) described in Example 9-3-1.
  • the horizontal axis shows the concentration of CD137L, and the vertical axis shows the amount of binding of CD137L to the supplementary amount of B0000 and BB0077.
  • FIG. 14 is a graph showing the results of ECM binding evaluation of BB0000 and BB0077 described in Example 9-3-2.
  • the vertical axis shows the emission intensity indicating the ECM bond.
  • FIG. 15 is a graph showing the results of measuring the binding activity to the in vivo ligand (human CD137L) from BB0124 to BB0138 described in Example 9-3-4.
  • the horizontal axis shows the evaluated CD137 variant, and the vertical axis shows the amount of binding of CD137L to the supplementary amount of each variant.
  • FIG. 16A is a graph showing the results of ECM binding evaluation from CD137 variant BB0124 to BB0138 described in Example 9-3-5.
  • the vertical axis shows the emission intensity indicating the ECM bond.
  • FIG. 16B is a graph showing the results of ECM binding evaluation of the CD137 variant BB0139 described in Example 9-3-3.
  • the vertical axis shows the emission intensity indicating the ECM bond.
  • FIG. 17 shows the evaluation of bispecific antibody (denoted as Ab in FIG. 17) -dependent activation ability consisting of anti-GPC3 antibody and anti-CD137 antibody in the presence of target cells by the reporter assay described in Example 10-4. It is a graph which shows the result of performing.
  • the horizontal axis is a chimeric receptor in which the cytoplasmic regions of CD8-CD28-CD137-CD3 zeta are linked with eight CD137 variants of BB0000, BB0077, BB0127, BB0128, BB0131, BB0133, BB0134, and BB0139 as extracellular domains.
  • FIG. 18 is a graph showing the results of evaluation of the activation ability of Jurkat cells having a CD137 variant in the extracellular domain in the presence of ligand-expressing cells by the reporter assay described in Example 10-5.
  • the axis is Jurkat cells expressing a chimeric receptor to which the cytoplasmic region of CD8-CD28-CD137-CD3 zeta is linked, with eight species of BB0000, BB0077, BB0127, BB0128, BB0131, BB0133, BB0134, and BB0139 as extracellular domains.
  • the parent strain (Mock) of NFAT-RE-luc2 Jurkat cells into which no gene has been introduced is shown, and the vertical axis shows the luminescence intensity of luciferase.
  • FIG. 19 is a schematic diagram showing the chimeric receptor CD137-CD8-CD3 zeta encoded by the lentiviral vector described in Example 11-1.
  • FIG. 20 shows the evaluation of CD137 activation ability of three bispecific antibody-dependent Jurkat cells consisting of anti-GPC3 antibody and anti-CD137 antibody in the presence of target cells by the reporter assay described in Example 11-4. It is a graph which shows the result which performed. On the horizontal axis, express the three types of Jurkat cells prepared in Example 11-2, that is, CD137-CR1 Jurkat cells (CD137-CR1), CD137-CR3-copGFP Jurkat cells (CD137-CR3), and chimeric receptors. The presence or absence of NF-kB-Luc2 / 4-1BB Jurkat cells (copGFP) expressing only copyGFP and bispecific antibody is shown. The vertical axis shows the luminescence intensity of luciferase.
  • FIG. 21 is a schematic diagram showing a construct (CD28-CR1) using human CD8 and a construct (CD28-CR2) using human CD28 in the transmembrane domain encoded by the lentiviral vector described in Example 12-1.
  • FIG. 22 is a graph showing the results of evaluation of the activation ability of the CD28-CR1 Jurkat cells and CD28-CR2 Jurkat cells described in Example 12-4 in the presence of target cells.
  • On the horizontal axis three types of Jurkat cells, that is, NFAT-RE-luc2 Jurkat cells (copGFP) that do not express chimeric receptors and express only copGFP, and CD28-CR1-copGFP prepared in Example 12-2 are expressed.
  • the polypeptide in the present invention usually refers to a peptide and a protein having a length of about 4 amino acids or more. Further, the polypeptide in the present invention is usually a polypeptide consisting of an artificially designed sequence, but is not particularly limited, and may be, for example, a polypeptide derived from an organism. Further, it may be either a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide or the like. Further, fragments of the above-mentioned polypeptides are also included in the polypeptides of the present invention.
  • Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q. , Ser / S, Glu / E, Thr / T, Gly / G, Thrp / W, His / H, Tyr / Y, Ile / I, Val / V, amino acids are 1-letter code or 3 It is written in character code or both.
  • amino acid 37V contained in a single domain antibody represents Val at position 37 represented by Kabat numbering.
  • a cell-free translation system (CloverDirect (ProteinExpress)) containing a tRNA in which an unnatural amino acid is bound to a complementary amber suppressor tRNA of a UAG codon (amber codon), which is one of the stop codons, is also suitable.
  • CloverDirect ProteinExpress
  • modifications include, but are not limited to, substitutions.
  • the meaning of the term “and / or” used to describe the modified position of an amino acid includes any combination of "and” and “or” as appropriate.
  • “the amino acids 37, 45, and / or 47 are substituted” includes variations of the following amino acid modification positions; (a) 37, (b) 45. Numbers, (c) 47, (d) 37 and 45, (e) 37 and 47, (f) 45 and 47, (g) 37 and 45 and 47.
  • an expression representing the modification of an amino acid an expression in which the one-character code or the three-character code of the amino acid before and after the modification is written before and after the number indicating a specific position can be appropriately used.
  • the modification F37V or Phe37Val used when substituting an amino acid contained in an antibody variable region or monodomain antibody represents the substitution of Phe at position 37 represented by Kabat numbering with Val. That is, the number represents the position of the amino acid represented by Kabat numbering, the one-letter code or three-letter code of the amino acid described before it is the amino acid before substitution, and the one-letter code or 3 of the amino acid described after that.
  • the character code represents the amino acid after substitution.
  • the modification P238A or Pro238Ala used to add amino acid substitutions to the Fc region contained in the antibody constant region represents the substitution of Pro at position 238 with Ala, represented by EU numbering. That is, the number represents the position of the amino acid represented by the EU numbering, the one-letter code or three-letter code of the amino acid described before it is the amino acid before substitution, and the one-letter code or 3 of the amino acid described after that.
  • the character code represents the amino acid after substitution.
  • antibody is used in the broadest sense and is not limited thereto as long as it exhibits a desired antigen-binding activity, but is not limited to a monoclonal antibody, a polyclonal antibody, and a multispecific antibody (for example, two). Includes a variety of antibody structures, including heavily specific antibodies), monoclonal antibodies, and antibody fragments.
  • the "multispecific antigen-binding molecule” refers to a molecule capable of specifically binding to various different antigens, specifically, various epitopes contained in various antigens. That is, a multispecific antigen-binding molecule is a molecule having specificity for at least two different epitopes, and includes molecules that bind to different antigens as well as molecules that bind to different epitopes on the same antigen. Multispecific antigen-binding molecule is a concept including bispecific antigen-binding molecule, and examples thereof include multispecific antibody and bispecific antibody. Bispecific antibodies are those in which such molecules bind to two antigens, whereas multispecific antibodies are two or more antigens (eg, three). It may have specificity for the antigen. Bispecific and multispecific antibodies can be prepared as full-length antibodies or as molecules containing antibody fragments.
  • Antibody fragment refers to a molecule other than the complete antibody, which comprises a portion of the complete antibody that binds to the antigen to which the complete antibody binds.
  • Examples of antibody fragments are, but are not limited to, Fv, Fab, Fab', Fab'-SH, F (ab') 2 , diabodies, linear antibodies, single chain antibody molecules (eg, scFv). , And multispecific antibodies formed from antibody fragments.
  • full-length antibody refers to an antibody having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein.
  • the term “homoantibody” refers to an antibody that binds to a single antigen and has a four-chain structure in which two polypeptide chains, a light chain and a heavy chain, are present.
  • variable region refers to the heavy or light chain domain of an antibody involved in binding the antibody to an antigen.
  • the variable domains of the heavy and light chains of an antibody typically have similar structures, each domain containing four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • CDRs complementarity determining regions
  • VH H1, H2, H3
  • VL VL1, L2, L3
  • Exemplary CDRs herein include: (A) At amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).
  • the resulting hypervariable loop (Chothia and Lesk, J. Mol. Biol.
  • 262 732-745 (1996)); (D) HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49 A combination of (a), (b), and / or (c), comprising -65 (H2), 93-102 (H3), and 94-102 (H3).
  • CDR residues and other residues in the variable domain are numbered herein according to Kabat et al., Supra. It was
  • “Framework” or “FR” refers to variable domain residues other than complementarity determining region (CDR) residues.
  • the variable domain FR usually consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of CDRs and FRs usually appear in VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4. It was
  • variable region refers to a portion of the antibody other than the variable region.
  • an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical disulfide-bonded light chains and two identical heavy chains, directed from the N-terminal to the C-terminal.
  • Each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by a heavy chain constant region (CH) containing a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.
  • VH variable region
  • CH heavy chain constant region
  • each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, followed by a stationary light chain (CL) domain.
  • VL variable region
  • CL stationary light chain
  • the light chain of a native antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • the term "Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes the Fc region of the native sequence and the mutant Fc region.
  • the heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, follow the EU numbering system (also called EU index) described in MD 1991.
  • the "class" of an antibody refers to the type of constant domain or constant region in the heavy chain of an antibody.
  • Heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as alpha, delta, epsilon, gamma, and mu, respectively. It was
  • the "target antigen recognition site” is a site of an antigen-binding molecule capable of recognizing a target antigen (target antigen) and specifically binding to the target antigen, and is also referred to as a target antigen-binding domain.
  • the target antigen recognition site may be a site having any structure as long as it binds to the target antigen. Examples of such sites include, but are not limited to, heavy chain variable regions (VHs) of antibodies and light chain variable regions (VL) of antibodies, monodomain antibodies (sdAb), which are present in vivo.
  • VHs heavy chain variable regions
  • VL light chain variable regions
  • sdAb monodomain antibodies
  • a module called A domain (Internationally published WO2004 / 040411, WO2005 / 040229) containing about 35 amino acids contained in the cell membrane protein Avimer, and the 10Fn3 domain, which is a domain that binds to a protein in the antibody that is a glycoprotein expressed on the cell membrane.
  • Antibody International Publication WO2002 / 032925
  • Affibody International Publication WO1995 / 001937
  • IgG binding domain constituting a bundle of three helices consisting of 58 amino acids of Protein A as scaffold, Turn containing 33 amino acid residues.
  • DARPins Designed Ankyrin Repeat proteins
  • AR ankyrin repeat
  • NGAL neutrophil gelatinase-associated lipocalin
  • VLR variable lymphocyte receptor
  • An example is a recessed region of a parallel sheet structure inside a horse-shaped structure in which repeat (LEUCINE-rich-repeat (LRR)) modules are repeatedly stacked (International Publication WO2008 / 016854).
  • the target antigen recognition site of the present invention include a heavy chain variable region (VH) of an antibody and a light chain variable region (VL) of an antibody, and a particularly suitable example is a multiplex having an immune receptor recognition site.
  • VH heavy chain variable region
  • VL light chain variable region
  • a particularly suitable example is a multiplex having an immune receptor recognition site.
  • examples thereof include a target antigen recognition site in a specific antibody, for example, a target antigen recognition site in a bispecific antibody.
  • a site capable of recognizing and binding to a specific antigen possessed by the antigen-binding molecule of the present invention may be referred to as an antigen-binding domain.
  • a multispecific antibody has multiple antigen-binding domains, of which the antigen-binding domain capable of recognizing and binding to a target antigen functions as a target antigen-binding site and can recognize and bind to an immune receptor.
  • the resulting antigen-binding domain functions as an immune receptor recognition site. Therefore, both the target antigen-binding site and the immune receptor recognition site of the multispecific antibody correspond to the antigen-binding domain of the multispecific antibody.
  • Including a bispecific antigen-binding site means having two types of antigen-binding domains.
  • target antigen means a substance recognized as a target by a molecule such as an antibody
  • target antigen refers to an antigen to which an antigen-binding molecule can bind via a target antigen recognition site. means.
  • the target antigen contains an epitope to which the target antigen recognition site can bind.
  • the target antigen is an antigen used for treating a disease caused by a target tissue, and a suitable example thereof is, but is not limited to, the surface of a target cell (eg, cancer cell, inflammatory cell).
  • a molecule expressed in a cell a molecule expressed on the surface of another cell in a tissue containing a target cell, a molecule expressed on the surface of a cell having an immunological role for the target cell and the tissue containing the target cell, and a target cell. Examples thereof include large molecules existing in the interstitial space of the containing tissue.
  • the antigens shown below can be mentioned as examples of target antigens.
  • the target antigen in the present invention is preferably, for example, a receptor, a tumor antigen, an MHC antigen, a differentiation antigen, or the like.
  • Target antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2 , Actibin, Actibin A, Actibin AB, Actibin B, Actibin C, Actibin RIA, Actibin RIA ALK-2, Actibin RIB ALK-4, Actibin RIIA, Actibin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC,
  • Receptors are also described in the above-mentioned examples of target antigens, but even when these receptors are present in a soluble form in a biological fluid, they can be used as an antigen to which the target antigen recognition site of the present invention binds. ..
  • soluble receptors for example, soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968).
  • a protein that is can be exemplified.
  • receptors include, for example, hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase type receptor family, serine / threonine kinase type receptor family, TNF receptor family, G protein conjugated receptor family, GPI.
  • receptors belonging to a receptor family such as an anchor-type receptor family, a tyrosine phosphatase-type receptor family, an adhesion factor family, and a hormone receptor family.
  • many documents such as Cooke BA., King RJB., Van der Molen HJ. Ed.
  • Specific receptors belonging to the above receptor family include, for example, human or mouse erythropoietin (EPO) receptors (Blood (1990) 76 (1), 31-35, Cell (1989) 57 (2), 277-. 285), Human or Mouse Granulocyte Colony Stimulating Factor (G-CSF) Receptor (Proc. Natl. Acad. Sci. USA. (1990) 87 (22), 8702-8706, mG-CSFR, Cell (1990) 61 (2), 341-350), Human or Mouse Thrombopoietin (TPO) Receptor (Proc Natl Acad Sci U S A. (1992) 89 (12), 5640-5644, EMBO J.
  • EPO erythropoietin
  • human or mouse leptin receptor human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL) -10 receptor, human or mouse insulin-like growth factor (IGF) -I receptor, human or mouse leukemia inhibitor (LIF) receptor, human or mouse hair Erythropoievous factor (CNTF) receptors and the like are preferably exemplified.
  • GH growth hormone
  • IL interleukin
  • IGF insulin-like growth factor
  • LIF human or mouse leukemia inhibitor
  • CNTF hair Erythropoievous factor
  • target antigens examples include membrane-type molecules expressed on the cell membrane and soluble-type molecules secreted extracellularly from the cell.
  • the antigen-binding domain of the present invention binds to a soluble molecule secreted from a cell, it is preferable that the antigen-binding domain has neutralizing activity.
  • the solution in which the soluble molecule is present is not limited, and the soluble molecule can be present in a biological fluid, that is, any liquid that fills between vessels or tissues / cells in the living body.
  • the soluble molecule to which the target antigen recognition site of the present invention binds can be present in extracellular fluid.
  • extracellular fluid is a component in bone and cartilage such as plasma, interstitial fluid, lymph, tight connective tissue, cerebrospinal fluid, cerebrospinal fluid, puncture fluid, or joint fluid, and alveolar fluid (bronchial lung).
  • Cell permeate such as plasma fluid), ascites, pleural fluid, cerebrospinal fluid, cyst fluid, or atrioventricular fluid (interstitial fluid) (fluids in various gland cavities resulting from active cell transport / secretion activity, and gastrointestinal tract) It is a general term for other cerebrospinal fluids).
  • tumor antigen refers to an antigen expressed on a cancer cell, and means an antigenic biomolecule whose expression becomes recognized in relation to malignant changes in the cell.
  • Tumor antigens include tumor-specific antigens (antigens that are present only in tumor cells and not found in other normal cells) and tumor-related antigens (other organs and tissues or heterologous and allogeneic normal cells). Includes antigens that are present or that are expressed during development and / or differentiation).
  • abnormal sugar chains that appear on the cell surface or protein molecules when cells become cancerous are also tumor antigens and are also called cancer sugar chain antigens.
  • the target antigen is a tumor antigen.
  • tumor antigens examples include GPC3 (Int J Cancer. (2003) 103 (4), which belongs to the GPI-anchored receptor family as the above-mentioned receptor but is expressed in some cancers including liver cancer. , 455-65), EpCAM (Proc Natl Acad Sci U S A. (1989) 86 (1), 27-31) expressed in multiple cancers including lung cancer (its polynucleotide sequence is RefSeq registration number NM_002354. 2. Polypeptide sequences are described in RefSeq registration number NP_002345.2), EGFR, CA19-9, CA15-3, serial SSEA-1 (SLX), Her2, Her3, prostate stem cell antigen (PSCA).
  • GPC3 Int J Cancer. (2003) 103 (4), which belongs to the GPI-anchored receptor family as the above-mentioned receptor but is expressed in some cancers including liver cancer. , 455-65
  • EpCAM Proc Natl Acad Sci U S A. (1989) 86 (1), 27-31 expressed
  • Alpha-Fet Protein AFP
  • CEA Cancer Fetal Antigen
  • CA-125 Tumor Antigen-125
  • Carretinin MUC-1, MUC-16, Epithelial Membrane Protein (EMA), Epithelial Tumor Antigen (ETA) ), Tyrosinase, melanoma-related antigen (MAGE), chromogranin, cytokeratin, desmin, glial fibrous acid protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen , Protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, nerve-specific enolase (NSE), placenta alkaline phosphatase, synaptopha Isis, tyroglobulin, thyroid transcription factor-1, pyruvate kinase isoenzyme type M2 dimer form (tumor M2-PK), GD2 (gan
  • tumor antigens include, for example, thyroid stimulating hormone receptor (TSHR); CD171; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule-1 (CLL-1).
  • TSHR thyroid stimulating hormone receptor
  • CD171 CD171
  • CS-1 CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24
  • CLL-1 C-type lectin-like molecule-1
  • MHC antigen is a gene product of major histocompatibility complex (MHC), and among them, glycoproteins expressed on cell membranes are mainly classified into MHC class I antigen and MHC class II antigen.
  • MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, -H, and MHC class II antigens include HLA-DR, -DQ, -DP. Is done. It also includes tumor antigen-derived peptides presented to these MHC antigens.
  • Tumor antigens such as GP100, MART-1, MAGE-1, MAGE-A4, NY-ESO-1 or complexes with MHC presenting mutants such as RAS and p53 are also considered as one of the tumor antigens. Can be caught.
  • Differentiation antigen is a general term for cell surface molecules in which macrophages, T cells, B cells, etc. are dissipated as they differentiate from bone marrow stem cells.
  • Differentiation antigens include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27.
  • tumor generally refers to a tissue mass formed by the autonomous overgrowth of tissues and cells against the control in the living body. Tumors are malignant, which has three characteristics of autonomous growth, infiltration and metastasis, and malaise, and benign, which is characterized only by autonomous growth.
  • the malignant tumor controls abnormal cells. Refers to a disease characterized by impossibility of growth. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described in this disclosure and are not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney. These include cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.
  • tumor and cancer are used interchangeably in the present disclosure, eg, both terms include solid and humoral, eg, diffuse or circulating tumors.
  • cancer or “tumor” as used herein includes precancerous, as well as malignant cancers and tumors.
  • cancers in the anticancer agents disclosed in the present disclosure and the cancer treatment methods described below include adenocarcinoma, squamous epithelial cancer, glandular squamous epithelial cancer, undifferentiated cancer, large cell cancer, and small cells.
  • sarcomas such as chondrosarcoma, Ewing sarcoma, malignant vascular endothelial tumor, malignant Schwan tumor, osteosarcoma, and soft tissue sarcoma, hepatoblastoma, myelblastoma, renal blastoma, and neuroblast.
  • blastoma such as tumor, pancreatic blastoma, pleural lung blastoma, and retinal blastoma, embryonic cell
  • the tumor antigen in the context of a cancer type is a marker expressed by both normal cells and cancer cells, eg, a lineage marker, eg, CD19 on B cells.
  • the tumor antigens of the present disclosure include primary or metastatic melanoma, thoracic adenoma, lymphoma, sarcoma, lung cancer, colorectal cancer, liver cancer, non-hodgkin lymphoma, hodgkin lymphoma, leukemia, uterus.
  • Cancer derived from cancer including but not limited to cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer and the like. do.
  • the tumor antigen is an antigen that is common to certain proliferative disorders.
  • the cancer-related antigen is overexpressed in cancer cells as compared to normal cells, eg, 1-fold expression, 2-fold overexpression, 3-fold overexpression as compared to normal cells.
  • the cancer-related antigen contains a deletion, addition or mutation as compared to a cell surface molecule that is improperly synthesized in cancer cells, eg, a molecule expressed in normal cells. It is a molecule.
  • the cancer-related antigen is expressed exclusively on the cell surface of cancer cells, either in whole or as a fragment (eg, MHC / peptide), and is neither synthesized nor expressed on the surface of normal cells.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • the MHC class I complex is constitutively expressed by all nucleated cells.
  • virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy.
  • TCR-like antibodies targeting peptides derived from viruses or tumor antigens in the context of human leukocyte antigen (HLA) -A1 or HLA-A2 have been described [eg, Sastry et al., J Virol. 2011 85 (5).
  • TCR-like antibodies can be identified by screening libraries such as the human scFv phage presentation library.
  • antigens to which the antigen-binding molecules of the present disclosure bind include viral antigens, bacterial (particularly infectious bacterial) antigens, parasite antigens, and cell surface markers on target cells associated with a particular pathology (eg, tumor antigens). ), And surface molecules of immune cells that give rise to autoimmunity.
  • the present disclosure in one aspect, is a retroviral family (eg, human immunodeficiency virus, eg, HIV-1 and HIV-LP), a picorna virus family (eg, poliovirus, hepatitis A virus, enterovirus, human coxsackie virus, etc.).
  • a retroviral family eg, human immunodeficiency virus, eg, HIV-1 and HIV-LP
  • a picorna virus family eg, poliovirus, hepatitis A virus, enterovirus, human coxsackie virus, etc.
  • Rhinovirus and Echovirus ruin virus, coronavirus, bullous stomatitis virus, mad dog disease virus, Ebola virus, parainfluenza virus, epidemic parotitis virus, measles virus, respiratory follicles virus, influenza virus, B Hepatitis virus, parvovirus, adenovirus family, herpesvirus family [eg, type 1 and type 2 simple herpesvirus (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpesvirus], poxvirus family (Eg, acne virus, vaccinia virus, and pox virus), or a chimeric receptor capable of binding via an antigen-binding molecule to an antigen derived from hepatitis C virus.
  • herpesvirus family eg, type 1 and type 2 simple herpesvirus (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpesvirus
  • poxvirus family Eg, acne virus, vaccinia virus
  • the present disclosure is an antigen-binding molecule derived from a strain of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella. Provides a chimeric receptor that can bind via.
  • infectious bacteria such as Helicobacter pyroris, Legionella pneumophilia, Mycobacterium tuberculosis strains (eg, M. tuberculosis), M. tuberculosis (M. tuberculosis). avium), M. intracellulare, M. kansaii, or M.
  • a chimeric receptor capable of binding to an antigen derived from Mycobacterium tuberculosis (Clostridium tetani) via an antigen-binding molecule.
  • the antigen-binding molecules are 5T4, alpha 5 beta1-integrin, 707-AP, AFP, ART-4, B7H4, B7H3, BAGE, beta-catenin / m, Bcr-abl, MN.
  • the "immune receptor recognition site” is a site of an antigen-binding molecule capable of recognizing a target immune receptor and specifically binding to the immune receptor, and is referred to as an immunoreceptor binding domain. You can also.
  • the immune receptor recognition site may be a site of any structure as long as it binds to the target immune receptor.
  • VHs heavy chain variable regions
  • VL light chain variable regions
  • sdAb monodomain antibodies
  • a domain of about 35 amino acids contained in Avimer which is a cell membrane protein existing in the living body (international release WO2004 / 044011, WO2005 / 040229), and protein in fibronectin, which is a glycoprotein expressed on cell membrane.
  • Adnectin containing the 10Fn3 domain which is the binding domain (Internationally published WO2002 / 032925), and Affibody (Internationally published WO1995 / 001937), whose IgG binding domain is scaffold, which constitutes a bundle of three helices consisting of 58 amino acids of Protein A.
  • DARPins Designed Ankyrin Repeat proteins
  • ARs ankyrin repeats
  • LRR Leucine-rich-repeat
  • the immune receptor recognition site of the present invention include a heavy chain variable region (VH) of an antibody that recognizes an immune receptor as an antigen and a light chain variable region (VL) of an antibody, and particularly suitable examples thereof.
  • VH heavy chain variable region
  • VL light chain variable region
  • An immune receptor recognition site in a multispecific antibody having a target antigen recognition site for example, an immunoreceptor recognition site in a bispecific antibody.
  • the site capable of recognizing and binding to a specific immunoreceptor possessed by the antigen-binding molecule of the present invention is a part of an antibody that recognizes the immunoreceptor as an antigen
  • the site for recognizing the immunoreceptor is referred to as an antigen-binding domain. May be called.
  • a multispecific antibody has multiple antigen-binding domains, of which the antigen-binding domain capable of recognizing and binding to a target antigen functions as a target antigen-binding site and can recognize and bind to an immune receptor.
  • the resulting antigen-binding domain functions as an immune receptor recognition site. Therefore, both the target antigen-binding site and the immune receptor recognition site of the multispecific antibody correspond to the antigen-binding domain of the multispecific antibody.
  • immunoreceptors recognized by antigen-binding molecules include immunoreceptors on T cells and immune receptors on natural killer (NK) cells.
  • NK natural killer
  • Specific examples include TIGIT, PD-1, PD-L1, TIM-3, LAG-3, BTLA, CD268, CD267, CD266, CD226, CD160, CD137, CD96, CD70, CD47, CD40, CD30, CD28, CD27, CD18, NKG2D, VISTA, ICOS, B7-HE, GITR, OX40, KIR, SLAM7, CTLA-4, RANK, Osteoprotegerin, BCMA, TNFR1, TNFR2, Fas, DR1, DR2, DR3, DR4, Examples include DR5, DR6, HVEM, NGFR, EDA2R, TR6, TROY, etc.
  • the immunoreceptor recognition site of the antigen-binding molecule uses an agonist antibody against a costimulatory molecule belonging to the tumor necrosis factor receptor superfamily (TNFRSF) (hereinafter referred to as an agonist antibody of TNFRSF) or an antigen-binding fragment thereof.
  • TNFRSF tumor necrosis factor receptor superfamily
  • An agonist antibody of TNFRSF is used on cells, tissues or living organisms expressing the TNF receptor superfamily when 100% activation is at an activation level achieved under physiological conditions by an equimolar amount of binding partner. When added, it means an antibody that activates cells expressing the TNF receptor superfamily by at least about 5%, specifically at least about 10%, more specifically at least about 15%.
  • the TNFRSF agonist antibodies used as the pharmaceutical compositions of the invention have at least about 20%, 30%, 40%, 50%, 60%, 70%, 80% of the activity of the cells. Can be activated 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 750%, 1000% or more. ..
  • the immune receptor recognition site of the antigen binding molecule binds as an agonist to the endogenous immunoreceptor and also as an agonist to the chimeric receptor of the invention.
  • the target molecule of the agonist antibody of TNFRSF is not particularly limited as long as it is a factor that activates cells expressing the TNF receptor superfamily (for example, T cells and NK cells).
  • Preferred factors include, for example, CD137 and CD40. Further preferred factors include, for example, CD137.
  • examples of the CD137 agonist antibody include Urrelumab (CAS Registry Number: 934823-49-1) and various known CD137 agonist antibodies.
  • CD137 agonist antibodies include the following antibodies, such as those set forth in WO2015 / 156268, such as SEQ ID NO: [1] An antibody having the amino acid sequence set forth in SEQ ID NO: 66 as a heavy chain variable region and the amino acid sequence set forth in SEQ ID NO: 85 as a light chain variable region; [2] An antibody having the amino acid sequence set forth in SEQ ID NO: 67 as a heavy chain variable region and the amino acid sequence set forth in SEQ ID NO: 86 as a light chain variable region; [3] An antibody having the amino acid sequence set forth in SEQ ID NO: 70 as a heavy chain variable region and the amino acid sequence set forth in SEQ ID NO: 89 as a light chain variable region; [4] An antibody having the amino acid sequence set forth in SEQ ID NO: 76 as a heavy chain variable region and the amino acid sequence set forth in SEQ ID NO: 95 as a light chain variable region; [5] An antibody having the amino acid sequence set forth in SEQ ID NO: 77 as a heavy antibody
  • equivalent activity means that the agonist activity on CD137 is 70% or more, preferably 80% of the binding activity of the antibody described in any of the above [1] to [7]. The above is more preferably 90% or more.
  • the present invention also provides an antibody that binds to the same epitope as the epitope to which the anti-CD137 antibody disclosed in the present invention described in the above [9] binds.
  • an antibody can be obtained, for example, by the following method.
  • an antibody that binds to the same epitope as the epitope to which the antibody described in any of the above [1] to [7] binds for example, the sequence of SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 15 described in WO2015 / 156268).
  • an antibody that recognizes a region having can be mentioned.
  • an antibody that recognizes a region having the sequence of DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 16 in WO2015 / 156268) in the CD137 protein can be mentioned.
  • the desired effect can be obtained.
  • a tumor-specific antigen antibody for example, anti-human GPC3 antibody
  • An antitumor antigen / anti-human CD137 bispecific antibody that exerts can be provided, and the antibody can be used as the antigen-binding molecule of the present invention.
  • an epitope meaning an antigenic determinant present in an antigen means a site on an antigen (target antigen or immunoreceptor) to which an antigen-binding molecule disclosed herein binds.
  • an epitope can be defined by its structure.
  • the epitope can also be defined by the antigen-binding activity of the antigen-binding molecule that recognizes the epitope.
  • the antigen is a peptide or polypeptide, it is also possible to identify the epitope by the amino acid residues constituting the epitope.
  • the epitope is a sugar chain, it is also possible to specify the epitope by a specific sugar chain structure.
  • a linear epitope is an epitope containing an epitope in which a primary amino acid sequence is recognized.
  • Linear epitopes typically contain at least three, and most usually at least five, such as about 8 to about 10, 6 to 20 amino acids in a unique sequence.
  • a steric epitope in contrast to a linear epitope, is an epitope in which the primary sequence of the amino acid containing the epitope is not a single defining component of the recognized epitope (eg, the primary sequence of the amino acid does not necessarily define the epitope. It is an epitope that is not recognized by.
  • the steric epitope may contain an increased number of amino acids relative to the linear epitope.
  • the target antigen recognition site or immunoreceptor recognition site of the antigen-binding molecule recognizes the three-dimensional structure of the peptide or protein.
  • a protein molecule when a protein molecule is folded to form three-dimensional structure, certain amino acid and / or polypeptide backbones that form the steric epitope are parallel, allowing the antibody to recognize the epitope.
  • Methods for determining the conformation of an epitope include, but are not limited to, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy and site-specific spin labeling and electromagnetic paramagnetic resonance spectroscopy. For example, see Epitope Mapping Protocols in Methods in Molecular Biology (1996), Volume 66, Morris (eds.). The
  • the structure of the target antigen recognition site or immunoreceptor recognition site of the antigen-binding molecule that binds to the epitope of the target antigen or immunoreceptor is called a paratope.
  • the epitope and paratope are stably bound by hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc. that act between the epitope and the paratope.
  • the binding force between this epitope and the paratope is called affinity.
  • the sum of the binding forces when multiple antigens and multiple antigen-binding domains bind is called avidity.
  • an antibody or the like containing a plurality of antigen-binding domains that is, polyvalent
  • the antigen-binding molecules provided herein are ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10nM, ⁇ 1nM, ⁇ 0.1nM, ⁇ 0.01nM or ⁇ 0.001nM (eg, 10 -8 (1E-). 08) Dissociation constant of M or less, for example 10 -8 (1E-08) M to 10 -13 (1E-13) M, for example 10 -9 (1E-09) M to 10 -13 (1E-13) M) Has (Kd).
  • the method for confirming the binding to the epitope by the antigen-binding molecule containing the antigen-binding domain for the antigen or the antigen-binding domain as the target antigen recognition site or the immunoreceptor recognition site can be appropriately carried out according to the following examples.
  • an antigen when an antigen is a receptor expressed on the cell surface, it can be confirmed that the antigen-binding domain for the antigen recognizes a linear epitope present in a certain antigen molecule, for example, as follows.
  • a linear peptide consisting of amino acid sequences constituting the extracellular domain of an antigen is synthesized.
  • the peptide can be chemically synthesized.
  • it can be obtained by a genetic engineering technique using a region encoding an amino acid sequence corresponding to an extracellular domain in the cDNA of a certain antigen.
  • the binding activity between the linear peptide consisting of the amino acid sequence constituting the extracellular domain and the antigen-binding domain for a certain antigen is evaluated.
  • an ELISA using an immobilized linear peptide as an antigen can evaluate the binding activity of the antigen-binding domain to the peptide.
  • the binding activity to the linear peptide can be clarified based on the level of inhibition by the linear peptide in the binding of the antigen binding domain to a certain antigen-expressing cell. These tests can reveal the binding activity of the antigen-binding domain to the linear peptide.
  • the target antigen recognition site or the immune receptor recognition site recognizes the steric epitope as follows.
  • Cells expressing the target antigen or immune receptor are prepared.
  • a target antigen recognition site or immunoreceptor recognition site for a target antigen or immunoreceptor binds strongly to the cell when it comes into contact with the cell, while the recognition site is an immobilized target antigen or immunoreceptor.
  • substantially non-binding means that the binding activity of the latter is 80% or less, usually 50% or less, preferably 30% or less, particularly preferably 15% or less of the binding activity to the target antigen or immunoreceptor expressing cells. Refers to the case of.
  • a method for confirming the binding activity of the antigen-binding molecule to the target antigen or immune receptor for example, there is a method of measuring the Kd value by a radiolabeled antigen binding assay (RIA).
  • the RIA is performed with an antigen binding molecule and a target antigen or immune receptor or a fragment thereof.
  • in-solution binding affinity of an antigen-binding molecule equilibrates the antigen-binding domain with the lowest concentration of (125I) -labeled antigen in the presence of an increasing series of unlabeled antigens, and then coats the bound antigen with the antigen-binding domain. Measured by capture with a plate (see, eg, Chen et al., J. Mol. Biol. 293: 865-881 (1999)).
  • Kd is measured by a surface plasmon resonance method using BIACORE®.
  • a measurement method using BIACORE®-2000 or BIACORE®-3000 is a CM5 with approximately 10 response unit (RU) antigens immobilized. It is carried out at 25 ° C using a chip.
  • the carboxymethylated dextran biosensor chip CM5, BIACORE, Inc.
  • EDC N-ethyl-N'-(3-dimethylaminopropyl) -carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Antigen to 5 ⁇ g / ml (approximately 0.2 ⁇ M) using 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 ⁇ l / min to achieve approximately 10 reaction unit (RU) protein binding. It is diluted. After injection of the antigen, 1M ethanolamine is injected to block unreacted groups.
  • Antigen-binding molecules (target antigen recognition site or target antigen recognition site) in a 0.05% polysorbate 20 (TWEEN-20®) detergent-containing PBS (PBST) at 25 ° C., approximately 25 ⁇ l / min for kinetic measurements. A 2-fold serial dilution (0.78nM-500nM) of the immune receptor recognition site) is infused.
  • Binding rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting sensorgrams of binding and dissociation using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). To. The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. Furthermore, it is also possible to obtain the apparent dissociation constant (Kd) by using the equilibrium method analysis. These methods refer to the protocol attached to BIACORE®. See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999) and Methods Enzymol. 2000; 323: 325-40.
  • a spectrometer eg, Aviv Instruments or a stirring cuvette. Fluorescence intensity at 25 ° C. of 20 nM antigen binding domain in PBS, pH 7.2 in the presence of increasing concentrations of
  • the binding activity of the antigen-binding molecule to the target antigen or immune receptor can also be measured by a known method for measuring intermolecular interaction such as an electrochemical luminescence method.
  • a fragment corresponding to a target antigen recognition site or an immune receptor recognition site can be used, and a target antigen recognition site or an immunoreceptor recognition site can be used.
  • a fragment corresponding to a target antigen recognition site or an immune receptor recognition site, or an antigen-binding molecule can be used.
  • the binding activity of the antigen-binding molecule is quantitatively evaluated by comparing the signal levels generated by the enzymatic reaction. That is, a test antigen-binding molecule is added to an ELISA plate on which a cell expressing a certain antigen is immobilized, and the test antigen-binding molecule bound to the cell is detected using an enzyme-labeled antibody that recognizes the test antigen-binding domain.
  • a test antigen-binding molecule is added to an ELISA plate on which a cell expressing a certain antigen is immobilized, and the test antigen-binding molecule bound to the cell is detected using an enzyme-labeled antibody that recognizes the test antigen-binding domain.
  • FACS by creating a diluted series of test antigen-binding molecules and determining the antibody binding titer (titer) for cells expressing a certain antigen, the binding activity of the test antigen-binding molecule to cells expressing a certain antigen can be compared. Can be done.
  • the binding of the test antigen-binding molecule to the target antigen or immune receptor expressed on the cell surface suspended in a buffer solution or the like can be detected by a flow cytometer.
  • a flow cytometer for example, the following devices are known.
  • FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM both are product names of BD Biosciences
  • EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are product names of Beckman Coulter).
  • an antigen-binding molecule As an example of a suitable method for measuring the binding activity of an antigen-binding molecule to a target antigen or immune receptor, the following method can be mentioned. First, it is stained with a FITC-labeled secondary antibody that recognizes a test antigen-binding molecule reacted with a cell expressing a certain antigen.
  • the antigen-binding molecule is prepared and used at a desired concentration by diluting the test antigen-binding molecule with a suitable buffer solution as appropriate. For example, it can be used at any concentration between 10 ⁇ g / ml and 10 ng / ml.
  • the fluorescence intensity and cell number are measured by FACSCalibur TM (BD).
  • the amount of antigen-binding molecule bound to the cell is reflected in the fluorescence intensity obtained by analysis using CELL QUEST Software (BD), that is, the value of Geometric Mean. That is, by obtaining the value of the Geometric Mean, the binding activity of the test antigen-binding molecule represented by the binding amount of the test antigen-binding molecule can be measured.
  • BD CELL QUEST Software
  • the sharing of an epitope with another antigen-binding molecule by one antigen-binding molecule can be confirmed by competition for the same epitope by both. Competition between antigen-binding molecules is detected by cross-blocking assays and the like. For example, a competitive ELISA assay is a preferred cross-blocking assay.
  • an antigenic protein from a target antigen or immune receptor coated on a well of a microtiter plate is present or absent from a candidate competing antigen-binding molecule.
  • the test antigen binding molecule is added.
  • the amount of test antigen-binding molecule bound to a certain antigen protein in the well indirectly correlates with the binding ability of a competing antigen-binding molecule that is a competing candidate for binding to the same epitope. That is, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to the well coated with the antigen protein.
  • the amount of the test antigen-binding molecule bound to the well via the antigen protein can be easily measured by labeling the antigen-binding molecule in advance.
  • a biotin-labeled antigen-binding molecule is measured by using an avidin peroxidase conjugate and a suitable substrate.
  • a cross-blocking assay that utilizes an enzyme label such as peroxidase is particularly referred to as a competitive ELISA assay.
  • the antigen-binding molecule can be labeled with other labeling substances that can be detected or measured. Specifically, a radial label, a fluorescent label, or the like is known.
  • the competitive antigen-binding molecule binds the antigen-binding molecule to an antigen by at least 20%, preferably at least, as compared to the binding activity obtained in a control test performed in the absence of a candidate competing antigen-binding molecule aggregate. If 20-50%, more preferably at least 50%, can be blocked, the test antigen-binding molecule binds to substantially the same epitope as the competing antigen-binding molecule, or competes for binding to the same epitope. It is a molecule.
  • the sharing of the epitope between the test antigen-binding molecule and the control antigen-binding molecule means that the peptide in which an amino acid mutation is introduced into the peptide constituting the epitope is introduced. It can be evaluated by comparing the binding activity of both antigen-binding molecules to the polypeptide.
  • binding activity for example, it can be measured by comparing the binding activity of the test antigen-binding molecule and the control antigen-binding molecule to the linear peptide into which the mutation has been introduced in the above-mentioned ELISA format.
  • the binding activity to the mutant peptide bound to the column is quantified as the antigen-binding molecule eluted in the eluent after the test antigen-binding molecule and the control antigen-binding molecule are allowed to flow down the column. It can also be measured by.
  • a method of adsorbing a mutant peptide on a column as, for example, a fusion peptide with GST is known.
  • the sharing of the epitope between the test antigen-binding molecule and the control antigen-binding molecule can be evaluated by the following method.
  • cells expressing a certain antigen and cells expressing a certain antigen having a mutation introduced into the epitope are prepared.
  • a test antigen-binding molecule and a control antigen-binding molecule are added to the cell suspension in which these cells are suspended in a suitable buffer such as PBS.
  • a FITC-labeled antibody capable of recognizing the test antigen-binding molecule and the control antigen-binding molecule is added to the cell suspension washed with a buffer solution as appropriate.
  • the fluorescence intensity and cell number of cells stained with the labeled antibody are measured by FACSCalibur TM (BD).
  • the concentration of the test antigen-binding molecule and the control antigen-binding molecule is adjusted to a desired concentration and used by appropriately diluting with a suitable buffer solution. For example, it is used at any concentration between 10 ⁇ g / ml and 10 ng / ml.
  • the amount of labeled antibody bound to the cells is reflected in the fluorescence intensity obtained by analysis using CELL QUEST TM Software (BD), that is, the value of Geometric Mean. That is, by obtaining the value of the Geometric Mean, the binding activity of the test antigen-binding molecule and the control antigen-binding molecule represented by the binding amount of the labeled antibody can be measured.
  • radiolabeled antigen binding assay RIA
  • BIACORE registered trademark
  • the Geometric Mean comparison value (molecular ⁇ Geo-Mean value of the mutated antigen) that reflects the amount of the mutated antigen-binding molecule bound to the expressing cells of the test antigen-binding molecule obtained by the analysis is the expression of the antigen of the test antigen-binding molecule. Compare with the ⁇ Geo-Mean comparison value that reflects the amount of binding to cells.
  • the concentrations of the test antigen-binding molecules used to obtain the ⁇ Geo-Mean comparison values for the mutated cells expressing a certain antigen and the cells expressing a certain antigen shall be prepared to be the same or substantially the same concentration as each other. Is particularly preferable.
  • An antigen-binding molecule confirmed to recognize an epitope among certain antigens in advance is used as a control antigen-binding molecule.
  • the ⁇ Geo-Mean comparison value for cells expressing a certain antigen in which the test antigen-binding molecule is mutated is at least 80%, preferably 50%, more preferably more preferably the ⁇ Geo-Mean comparison value for cells expressing a certain antigen of the test antigen-binding molecule. If it is less than 30%, particularly preferably 15%, it is considered to "substantially do not bind to cells expressing a mutated antigen".
  • the calculation formula for obtaining the Geo-Mean value (Geometric Mean) is described in the CELLQUEST Software User's Guide (BD biosciences).
  • the epitopes of the test antigen-binding molecule and the control antigen-binding molecule can be evaluated to be the same to the extent that they can be substantially equated by comparing the comparative values.
  • the term "carrying portion” refers to a portion of the antigen-binding molecule other than the target antigen recognition site and the immune receptor recognition site.
  • the transport portion of the present invention is usually a peptide or polypeptide composed of amino acids, and as a specific embodiment, the transport portion in the antigen-binding molecule is linked to a target antigen recognition site and an immune receptor recognition site. There is.
  • the transport portion of the present invention may be a series of peptides or polypeptides linked by amide bonds, and a plurality of peptides or polypeptides are covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds and hydrophobic interactions. It may be a complex formed by binding.
  • the antigen-binding molecule is an antibody
  • one embodiment of extending the half-life in blood is to impart FcRn-binding property to the antigen-binding molecule.
  • FcRn binding property there is usually a method of providing an FcRn binding region in the antigen-binding molecule.
  • the FcRn binding region refers to a region having binding property to FcRn, and any structure can be used as long as it has binding property to FcRn.
  • IgG molecules incorporated into endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions in endosomes. IgG molecules that could not bind to FcRn proceed to lysosomes and are degraded there, but IgG molecules that bind to FcRn migrate to the cell surface and dissociate from FcRn under neutral plasma conditions and return to plasma again. ..
  • the FcRn binding region is preferably a region that directly binds to FcRn.
  • a preferred example of an FcRn binding region is the Fc region of an antibody.
  • a region capable of binding to a polypeptide having FcRn-binding ability such as albumin or IgG can indirectly bind to FcRn via albumin or IgG, and thus the FcRn-binding region in the present invention. May be a region that binds to a polypeptide capable of binding to such FcRn.
  • the binding activity of the FcRn binding region in the present invention to FcRn, particularly human FcRn, can be measured by a method known to those skilled in the art as described in the section of binding activity, and the conditions can be measured by those skilled in the art. Can be determined as appropriate.
  • the binding activity to human FcRn is KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Dissociation rate), or apparent kd (Apparent). It can be evaluated as dissociation (apparent dissociation rate) and the like. These can be measured by methods known to those skilled in the art. For example, Biacore (GE healthcare), scatchard plot, flow cytometer, etc. can be used.
  • the binding activity to human FcRn is KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Dissociation rate), or apparent kd (Apparent). It can be evaluated as dissociation
  • the conditions for measuring the FcRn-binding activity of the FcRn-binding region can be appropriately selected by those skilled in the art, and are not particularly limited. For example, it can be measured in MES buffer, 37 ° C. conditions as described in WO2009 / 125825. Further, the measurement of the binding activity of the FcRn binding region of the present invention to FcRn can be performed by a method known to those skilled in the art, and can be measured using, for example, Biacore (GE Healthcare).
  • the binding affinity between the FcRn binding region and FcRn may be evaluated at any pH of pH 4.0 to pH 6.5.
  • a pH of pH 5.8 to pH 6.0 which is close to the pH in the early endosomes in vivo, is used to determine the binding affinity between the FcRn binding region and human FcRn.
  • the binding affinity between the FcRn binding region and FcRn may be evaluated at any temperature of 10 ° C to 50 ° C.
  • a temperature of 15 ° C to 40 ° C is used to determine the binding affinity between the FcRn binding region and human FcRn.
  • any temperature of is also used to determine the binding affinity between the FcRn binding region and the FcRn.
  • a temperature of 25 ° C. is a non-limiting example of an embodiment of the present invention.
  • FcRn binding region is, but is not limited to, the IgG antibody Fc region.
  • the type is not limited, and it is possible to use an Fc region such as IgG1, IgG2, IgG3, or IgG4.
  • Fc region of the natural IgG antibody not only the Fc region of the natural IgG antibody but also the modified Fc region in which one or more amino acids are substituted can be used as long as it has FcRn binding property.
  • EU numbering 237th, 238th, 239th, 248th, 250th, 252nd, 254th, 255th, 256th, 257th, 258th, 265th, 270th, 286th in the IgG antibody Fc region EU numbering 237th, 238th, 239th, 248th, 250th, 252nd, 254th, 255th, 256th, 257th, 258th, 265th, 270th, 286th in the IgG antibody Fc region.
  • EU numbering in the IgG antibody Fc region Amino acid substitution that replaces Gly at position 237 with Met, Amino acid substitution that replaces Pro at position 238 with Ala, Amino acid substitution that replaces Ser at position 239 with Lys, Amino acid substitution that replaces the 248th Lys with Ile, Amino acid substitution that replaces the 250th Thr with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr, Amino acid substitution that replaces the 252nd Met with Phe, Trp, or Tyr, Amino acid substitution that replaces the 254th Ser with Thr, Amino acid substitution that replaces the 255th Arg with Glu, Amino acid substitution that replaces the 256th Thr with Asp, Glu, or Gln, Amino acid substitution that replaces the 257th Pro with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val, Amino acid substitution that replaces the 257
  • Amino acid substitution that replaces His at position 433 with Lys Amino acid substitutions that replace Asn at position 434 with Ala, Phe, His, Ser, Trp, or Tyr, and amino acid substitutions that replace Tyr or Phe at position 436 with His. It is possible to use a modified Fc region containing at least one amino acid substitution selected from.
  • albumin is not excreted by the kidneys and has FcRn binding, it has a long half-life in blood of 17 to 19 days (J Clin Invest. 1953 Aug; 32 (8): 746-768.). Therefore, it has been reported that the protein bound to albumin becomes bulky and can indirectly bind to FcRn, resulting in an increase in blood half-life (Antibodies 2015, 4 (3), 141-156).
  • the antigen binding molecule comprises an antibody Fc region.
  • the antigen-binding molecule comprises the CH2 and CH3 domains of a human IgG antibody.
  • the antigen-binding molecule comprises a portion extending from the human IgG1 antibody heavy chain Cys226 or from Pro230 to the carboxyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) in the Fc region may or may not be present.
  • the antigen binding molecule comprises an antibody constant region. In a more preferred embodiment, the antigen binding molecule comprises an IgG antibody constant region. In a more preferred embodiment, the antigen binding molecule comprises a human IgG antibody constant region. In a more preferred embodiment, the antigen binding molecule is a bispecific antibody and comprises a human IgG antibody constant region.
  • the antigen-binding molecule has a region having a structure substantially similar to that of the antibody heavy chain constant region, and a covalent bond such as a disulfide bond, a hydrogen bond, or a hydrophobic interaction with the region. It contains a region having a structure similar to that of an antibody light chain, which is bound by a non-covalent bond such as.
  • IgG antibody-like molecule refers to a moiety that is substantially similar to the structure of a constant domain or constant region, such as an IgG antibody, and the structure and substance of a variable domain or variable region, such as an IgG antibody. It is used to define a molecule that has a similar moiety and a three-dimensional structure that is substantially similar to an IgG antibody.
  • the "IgG antibody-like molecule” in the present specification is not limited to exhibiting antigen-binding activity while maintaining a structure similar to that of an IgG antibody.
  • the antigen-binding molecule is an IgG antibody-like molecule
  • the term "specificity" refers to the property that the target antigen recognition site or immunoreceptor recognition site of an antigen-binding molecule does not substantially bind to a molecule other than a specific target antigen or immunoreceptor, respectively. Say. It is also used when the antigen-binding molecule has specificity for an epitope contained in a specific target antigen or immune receptor. Substantially non-binding is determined according to the above method described for the measurement of binding activity, and the binding activity of the special binding molecule to a molecule other than the specific target antigen or immunoreceptor is the binding activity to the other molecule. 80% or less, usually 50% or less, preferably 30% or less, particularly preferably 15% or less. The
  • the antigen-binding molecule in the present invention can usually be prepared by carrying (inserting) a nucleic acid encoding it into an appropriate vector, introducing it into a host cell, and using a usual method.
  • the vector is not particularly limited as long as it stably retains the inserted nucleic acid.
  • Escherichia coli is used as a host
  • a pBluescript vector manufactured by Stratagene
  • Various vectors of the above can be used.
  • Expression vectors are particularly useful when vectors are used to produce the polypeptides used in the practice of the invention (eg, chimeric receptors, IgG antibodies, bispecific antibodies, antigen binding molecules, etc.).
  • the expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in vitro, in Escherichia coli, in cultured cells, or in an individual organism.
  • pBEST vector manufactured by Promega
  • Escherichia coli If it is a pET vector (manufactured by Invitrogen), if it is a cultured cell, pME18S-FL3 vector (GenBank Accession No.
  • DNA of the present invention can be inserted into a vector by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley &amp.; Sons. Section 11.4-11.11).
  • the host cell is not particularly limited, and various host cells are used depending on the purpose.
  • Examples of cells for expressing antigen-binding molecules include bacterial cells (eg, Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), and insect cells (eg, drosophila).
  • S2, Spodoptera SF9 animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells can be exemplified.
  • Vector introduction into host cells is, for example, calcium phosphate precipitation method, electric pulse perforation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofectamine method ( It can be performed by a known method such as (manufactured by GIBCO-BRL) and the microinjection method.
  • calcium phosphate precipitation method Electric pulse perforation method
  • electric pulse perforation method Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9
  • lipofectamine method It can be performed by a known method such as (manufactured by GIBCO-BRL) and the microinjection method.
  • an appropriate secretory signal can be incorporated into the nucleic acid encoding the antigen-binding molecule of interest. can. These signals may be endogenous or heterologous to the polypeptide of interest.
  • the medium is recovered.
  • the antigen-binding molecule of the present invention is produced intracellularly, the cell is first lysed and then the antigen-binding molecule is recovered.
  • ammonium sulfate or ethanol precipitation acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography.
  • Known methods including hydroxylapatite chromatography and lectin chromatography can be used.
  • amino acids contained in the amino acid sequence described in the present invention may undergo post-translational modifications (eg, modification of N-terminal glutamine to pyroglutamylation by pyroglutamylation is a modification well known to those skilled in the art). , Even when the amino acid is modified after translation as such, it is naturally included in the amino acid sequence described in the present invention.
  • post-translational modifications eg, modification of N-terminal glutamine to pyroglutamylation by pyroglutamylation is a modification well known to those skilled in the art.
  • a method for producing an antibody having a desired binding activity is known to those skilled in the art.
  • an antigen-binding molecule having a molecule expressed on the surface of a target cell (lesion cell) as an antigen (target antigen) can be used.
  • the target cell is a tumor cell or a cancer cell
  • the antigen is exemplified here as a tumor antigen.
  • the following is an example of a method for producing an antibody that binds to a tumor antigen.
  • Antibodies that bind to tumor antigens can be obtained as polyclonal or monoclonal antibodies using known means.
  • a monoclonal antibody derived from a mammal can be preferably produced.
  • Monoclonal antibodies derived from mammals include those produced by hybridomas, those produced by host cells transformed with an expression vector containing an antibody gene by genetic engineering techniques, and the like.
  • the monoclonal antibody-producing hybridoma can be produced, for example, as follows by using a known technique. That is, the tumor antigen protein is used as a sensitizing antigen and the mammal is immunized according to the usual immunization method. The resulting immune cells are fused with known parent cells by conventional cell fusion methods. Hybridomas that produce anti-tumor antigen antibodies can then be selected by screening monoclonal antibody-producing cells by conventional screening methods.
  • a tumor antigen protein used as a sensitizing antigen for antibody acquisition can be obtained. That is, a suitable host cell is transformed by inserting a gene sequence encoding a tumor antigen into a known expression vector.
  • the desired human tumor antigen protein is purified from the host cell or the culture supernatant by a known method.
  • a protein in which the portion constituting the hydrophobic region of the tumor antigen polypeptide sequence is deleted can be used.
  • Purified natural GPC3 protein can also be used as a sensitizing antigen as well.
  • the purified tumor antigen protein can be used as a sensitizing antigen used for immunization against mammals. Partial peptides of tumor antigen can also be used as sensitizing antigens. At this time, the partial peptide can also be obtained by chemical synthesis from the amino acid sequence of human tumor antigen. It can also be obtained by incorporating a part of the tumor antigen gene into an expression vector and expressing it. Further, it can be obtained by degrading the tumor antigen protein using a proteolytic enzyme, but the region and size of the tumor antigen peptide used as the partial peptide are not particularly limited to special embodiments.
  • the number of amino acids constituting the peptide to be the sensitizing antigen is preferably at least 5, for example, 6 or more, or 7 or more. More specifically, peptides with 8 to 50, preferably 10 to 30 residues can be used as sensitizing antigens.
  • a desired partial polypeptide of the tumor antigen protein or a fusion protein obtained by fusing a peptide with a different polypeptide can be used as a sensitizing antigen.
  • fusion proteins used as sensitizing antigens for example, Fc fragments of antibodies, peptide tags and the like can be suitably utilized.
  • a vector expressing a fusion protein can be prepared by in-frame fusion of genes encoding two or more desired polypeptide fragments and inserting the fusion gene into the expression vector as described above. The method for producing the fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, Jet al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab.
  • GPC3 used as a sensitizing antigen and an immunization method using the same are specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693 and the like.
  • the mammal immunized with the sensitizing antigen is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Generally, rodent animals such as mice, rats, hamsters, or rabbits, monkeys, etc. are preferably used.
  • the above animals are immunized with the sensitizing antigen according to a known method.
  • immunization is performed by injecting a sensitizing antigen intraperitoneally or subcutaneously into a mammal.
  • a sensitizing antigen diluted with PBS (Phosphate-Buffered Saline), physiological saline or the like at an appropriate dilution ratio is optionally mixed with a usual adjuvant, for example, Freund's complete adjuvant, and then emulsified.
  • the sensitizing antigen is administered to the mammal several times every 4 to 21 days.
  • suitable carriers may be used during immunization of the sensitizing antigen.
  • a partial peptide having a small molecular weight when used as a sensitizing antigen, it may be desirable to immunize the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocianine.
  • a carrier protein such as albumin or keyhole limpet hemocianine.
  • Hybridomas that produce the desired antibody can also be produced using DNA immunity as follows.
  • DNA immunity means that a sensitized antigen is expressed in the living body of the immune animal to which the vector DNA constructed in such a manner that the gene encoding the antigen protein can be expressed in the immune animal is administered. It is an immune method in which an immune stimulus is given by being performed. Compared with the general immunization method in which a protein antigen is administered to an immunized animal, DNA immunization is expected to have the following advantages. -Immune stimulation can be given by maintaining the structure of membrane proteins. -No need to purify immune antigens.
  • a DNA expressing a tumor antigen protein is administered to an immune animal.
  • the DNA encoding the tumor antigen can be synthesized by a known method such as PCR.
  • the obtained DNA is inserted into a suitable expression vector and administered to an immune animal.
  • a commercially available expression vector such as pcDNA3.1 can be preferably used.
  • a method for administering the vector to a living body a generally used method can be used.
  • DNA immunization is performed by introducing gold particles adsorbed by an expression vector into cells of an immune animal individual by a gene gun.
  • the production of an antibody that recognizes a tumor antigen can also be produced by using the method described in International Publication WO2003 / 104453.
  • immune cells are collected from the animal and subjected to cell fusion.
  • Particularly preferred immune cells may be splenocytes.
  • Mammalian myeloma cells are used as cells fused with the immune cells.
  • Myeloma cells are preferably equipped with a suitable selectable marker for screening.
  • Selectable markers refer to traits that can (or cannot) survive under specific culture conditions.
  • Known selectable markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency).
  • HGPRT deficiency hypoxanthine-guanine-phosphoribosyltransferase deficiency
  • TK deficiency thymidine kinase deficiency
  • Cells with HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity).
  • HGPRT-deficient and TK-deficient cells can be selected in a medium containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5'bromodeoxyuridine, respectively.
  • 8AG 8-azaguanine
  • 5'bromodeoxyuridine normal cells that incorporate these pyrimidine analogs into their DNA die.
  • cells deficient in these enzymes that cannot uptake these pyrimidine analogs can survive in selective media.
  • G418 resistance confer resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) by means of the neomycin resistance gene.
  • gentamicin analogs gentamicin analogs
  • myeloma cells suitable for cell fusion are known.
  • Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-. 7), NS-1 (C.Eur.J.Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 ( Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133) and the like can be preferably used.
  • cell fusion between the immune cells and myeloma cells is performed according to a known method, for example, the method of Koehler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).
  • the cell fusion can be carried out, for example, in the presence of a cell fusion promoter in a normal nutrient culture medium.
  • a cell fusion promoter for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and an auxiliary agent such as dimethyl sulfoxide is optionally added and used in order to further increase the fusion efficiency.
  • the usage ratio between immune cells and myeloma cells can be set arbitrarily. For example, it is preferable to increase the number of immune cells by 1 to 10 times that of myeloma cells.
  • the culture medium used for the cell fusion for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, and other ordinary culture mediums used for this type of cell culture are used, and further, cattle.
  • a serum supplement such as fetal serum (FCS) can be suitably added.
  • a predetermined amount of the immune cells and myeloma cells are well mixed in the culture medium, and a PEG solution preheated to about 37 ° C. (for example, an average molecular weight of about 1000 to 6000) is usually 30 to 60%. It is added at a concentration of (w / v).
  • the desired fusion cells are formed by gently mixing the mixed solution. Then, the appropriate culture solution mentioned above is sequentially added, and by repeating the operation of centrifuging and removing the supernatant, the cell fusion agent or the like which is not preferable for the growth of the hybridoma can be removed.
  • the hybridoma thus obtained can be selected by culturing in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Culture using the above HAT culture medium can be continued for a sufficient time (usually, such sufficient time is several days to several weeks) for cells other than the desired hybridoma (non-fused cells) to die.
  • a HAT culture medium a culture medium containing hypoxanthine, aminopterin and thymidine.
  • Culture using the above HAT culture medium can be continued for a sufficient time (usually, such sufficient time is several days to several weeks) for cells other than the desired hybridoma (non-fused cells) to die.
  • the hybridomas that produce the desired antibody are then screened and single cloned by conventional limiting dilution methods.
  • the hybridoma thus obtained can be selected by using a selective culture medium according to the selectable marker possessed by the myeloma used for cell fusion.
  • a selective culture medium for example, cells with deficiency of HGPRT or TK can be selected by culturing in HAT culture medium (culture medium containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have succeeded in cell fusion with normal cells can selectively proliferate in the HAT culture medium. Culture using the above HAT culture medium is continued for a sufficient time for cells other than the desired hybridoma (non-fused cells) to die.
  • the desired hybridoma can be selected by culturing for several days to several weeks. Screening and single cloning of hybridomas producing the desired antibody can then be performed by conventional limiting dilution methods.
  • Screening and single cloning of the desired antibody can be suitably performed by a screening method based on a known antigen-antibody reaction.
  • a monoclonal antibody that binds to GPC3 can bind to GPC3 expressed on the cell surface.
  • Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting).
  • FACS fluorescence activated cell sorting
  • FACS is a system that makes it possible to measure the binding of an antibody to the cell surface by analyzing the cells in contact with the fluorescent antibody with laser light and measuring the fluorescence emitted by each cell.
  • cells expressing GPC3 are prepared.
  • Preferred cells for screening are mammalian cells that have been forcibly expressed with the tumor antigen used.
  • the binding activity of the antibody to the tumor antigen on the cell surface can be selectively detected. That is, a hybridoma that produces a tumor antigen monoclonal antibody can be obtained by selecting a hybridoma that produces an antibody that does not bind to a host cell but binds to a GPC3 forced expression cell.
  • the antibody binding activity to immobilized tumor antigen-expressing cells can be evaluated based on the principle of ELISA.
  • GPC3-expressing cells are immobilized in wells of an ELISA plate.
  • the hybridoma culture supernatant is contacted with the immobilized cells in the well, and the antibody that binds to the immobilized cells is detected.
  • the monoclonal antibody is of mouse origin, the antibody bound to the cell can be detected by an anti-mouse immunoglobulin antibody.
  • the hybridomas selected by these screenings that produce the desired antibody capable of binding to an antigen can be cloned by a limiting dilution method or the like.
  • the hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture medium.
  • the hybridoma can be stored in liquid nitrogen for a long period of time.
  • the hybridoma can be cultured according to a usual method, and a desired monoclonal antibody can be obtained from the culture supernatant.
  • hybridomas can be administered to compatible mammals for growth and monoclonal antibodies can be obtained from the ascites.
  • the former method is suitable for obtaining a high-purity antibody.
  • An antibody encoded by an antibody gene cloned from an antibody-producing cell such as the hybridoma can also be preferably used.
  • the cloned antibody gene By incorporating the cloned antibody gene into an appropriate vector and introducing it into a host, the antibody encoded by the gene is expressed.
  • Methods for the isolation of antibody genes, introduction into vectors, and transformation of host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767- 775). Methods for producing recombinant antibodies are also known, as described below.
  • cDNA encoding the variable region (V region) of the antibody is obtained from a hybridoma cell that produces an antibody that binds to a tumor antigen.
  • the total RNA is usually first extracted from the hybridoma.
  • the following methods can be used: -guanidine ultracentrifugation method (Biochemistry (1979) 18 (24), 5294-5299) -AGPC method (Anal. Biochem). . (1987) 162 (1), 156-159).
  • the extracted mRNA can be purified using mRNA Purification Kit (manufactured by GE Healthcare Bioscience) or the like.
  • kits for extracting total mRNA directly from cells such as QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Bioscience) are also commercially available.
  • mRNA can be obtained from hybridomas.
  • a cDNA encoding the antibody V region can be synthesized from the obtained mRNA using reverse transcriptase.
  • the cDNA can be synthesized by AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation) or the like.
  • the 5'-RACE method Provided. Natl. Acad.
  • the desired cDNA fragment is purified from the obtained PCR product and then ligated with the vector DNA.
  • a desired recombinant vector can be prepared from the Escherichia coli that formed the colony. Then, whether or not the recombinant vector has the base sequence of the target cDNA is confirmed by a known method, for example, the dideoxynucleotide chain lamination method or the like.
  • cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained.
  • kits such as SMART RACE cDNA amplification kits are appropriately used for the synthesis of the 5'-RACE cDNA library.
  • the antibody gene is amplified by the PCR method.
  • Primers for mouse antibody gene amplification can be designed based on known antibody gene sequences. These primers have different base sequences for each subclass of immunoglobulin. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as the IsoStrip mouse monoclonal antibody isotyping kit (Roche Diagnostics).
  • a primer capable of amplifying a gene encoding ⁇ 1, ⁇ 2a, ⁇ 2b, ⁇ 3 as a heavy chain and a gene encoding a ⁇ chain and a ⁇ chain as a light chain is used.
  • a primer that anneals to a portion corresponding to a constant region close to the variable region is generally used as a primer on the 3'side.
  • the primer attached to the 5'RACE cDNA library preparation kit is used as the primer on the 5'RACE cDNA library preparation kit.
  • the PCR product thus amplified can be used to reconstitute an immunoglobulin consisting of a combination of heavy and light chains.
  • the desired antibody can be screened using the binding activity of the reconstituted immunoglobulin to the antigen as an index. For example, when the purpose is to obtain an antibody against GPC3, it is more preferable that the binding of the antibody to GPC3 is specific.
  • Antibodies used in the present invention can be screened, for example, as follows; (1) A step of contacting an antibody containing a V region encoded by cDNA obtained from a hybridoma with an antigen-expressing cell. (2) A step of detecting the binding between the antigen-expressing cell and the antibody, and (3) A step of selecting an antibody that binds to an antigen-expressing cell.
  • a method for detecting the binding between an antibody and a tumor antigen-expressing cell is known. Specifically, the binding between the antibody and the tumor antigen-expressing cell can be detected by a method such as FACS described above. Fixed specimens of tumor antigen-expressing cells can be appropriately used to evaluate the binding activity of the antibody.
  • a panning method using phage is also preferably used.
  • an antibody gene is obtained as a library of heavy chain and light chain subclasses from a polyclonal antibody-expressing cell group
  • a screening method using phage is advantageous.
  • Genes encoding the variable regions of the heavy and light chains can be linked with the appropriate linker sequence to form a single chain Fv (scFv).
  • scFv single chain Fv
  • the DNA encoding scFv having the desired binding activity can be recovered. By repeating this operation as necessary, scFv having the desired binding activity can be concentrated.
  • the cDNA is digested by a restriction enzyme that recognizes the restriction enzyme sites inserted at both ends of the cDNA.
  • Preferred restriction enzymes recognize and digest base sequences that rarely appear in the base sequences that make up an antibody gene. Further, in order to insert one copy of the digested fragment into the vector in the correct direction, it is preferable to insert a restriction enzyme that gives an attachment end.
  • An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-GPC3 antibody digested as described above into an appropriate expression vector.
  • a chimeric antibody is obtained.
  • the chimeric antibody means that the origins of the constant region and the variable region are different. Therefore, in addition to heterologous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention.
  • a chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector having a constant region in advance.
  • a restriction enzyme recognition sequence of a restriction enzyme that digests the V region gene can be appropriately arranged on the 5'side of an expression vector holding a DNA encoding a desired antibody constant region (C region). .. Chimeric antibody expression vectors are constructed by in-frame fusion of both digested with the same combination of restriction enzymes.
  • the antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression control region.
  • the expression control region for expressing an antibody includes, for example, an enhancer or a promoter.
  • an appropriate signal sequence can be added to the amino terminus so that the expressed antibody is secreted extracellularly.
  • a peptide having the amino acid sequence MGWSCIILFLVATATGVHS is used as the signal sequence, but a suitable signal sequence is added in addition to this.
  • the expressed polypeptide is cleaved at the carboxyl-terminal portion of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Then, by transforming a suitable host cell with this expression vector, recombinant cells expressing DNA encoding an antibody that binds to a target tumor antigen can be obtained.
  • the DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is integrated into different expression vectors.
  • An antibody molecule having an H chain and an L chain can be expressed by co-transfecting the same host cell with a vector incorporating the H chain and the L chain.
  • the host cell can be transformed by integrating the DNA encoding the H and L chains into a single expression vector (see WO 94/11523).
  • host cells and expression vectors for producing antibodies by introducing the isolated antibody gene into a suitable host are known. Any of these expression systems can be applied to isolate the domain containing the antibody variable region of the present invention.
  • animal cells, plant cells, or fungal cells may be used as appropriate. Specifically, the following cells can be exemplified as animal cells.
  • Mammalian cells CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc.
  • Amphibian cells Xenopus oocytes, etc.
  • Insect cells sf9, sf21, Tn5, etc.
  • an expression system of an antibody gene by a cell derived from the genus Nicotiana such as Nicotiana tabacum is known.
  • Callus-cultured cells can be appropriately used for transformation of plant cells.
  • -Yeast Saccharomyces such as Saccharomyces serevisiae
  • Pichia such as Pichia pastoris
  • -Filamentous fungi Aspergillus genus such as Aspergillus niger.
  • an expression system for antibody genes using prokaryotic cells is also known.
  • bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be appropriately used.
  • An expression vector containing the antibody gene of interest is introduced into these cells by transformation. By culturing the transformed cells in vitro, the desired antibody can be obtained from the culture of the transformed cells.
  • transgenic animals can also be used for the production of recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding a desired antibody has been introduced.
  • an antibody gene can be constructed as a fusion gene by in-frame insertion into a gene encoding a protein that is uniquely produced in milk.
  • a protein secreted into milk for example, goat ⁇ casein and the like can be utilized.
  • the DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat's embryo, and the injected embryo is introduced into a female goat.
  • the desired antibody can be obtained as a fusion protein with a milk protein from the milk produced by a transgenic goat (or its progeny) born from the goat that received the embryo. Hormones can also be administered to transgenic goats to increase the amount of milk containing the desired antibody produced by transgenic goats (Bio / Technology (1994), 12 (7), 699-702). ..
  • the antigen-binding molecule described in the present specification When the antigen-binding molecule described in the present specification is administered to a human, a gene artificially modified for the purpose of reducing heterologous antigenicity to humans as a domain containing an antibody variable region in the antigen-binding molecule.
  • the domain derived from the recombinant antibody can be appropriately adopted.
  • Genetically modified antibodies include, for example, Humanized antibodies and the like. These modified antibodies are appropriately produced using known methods.
  • variable regions of an antibody used to create the domain comprising the antibody variable regions in the antigen-binding molecules described herein are usually three complementarity determining sandwiched between four framework regions (FRs). It is composed of regions (complementarity-determining regions; CDRs).
  • the CDR is essentially the region that determines the binding specificity of the antibody.
  • the amino acid sequence of the CDR is rich in variety. On the other hand, the amino acid sequences constituting FR often show high identity even among antibodies having different binding specificities. Therefore, it is generally said that the binding specificity of one antibody can be transplanted to another antibody by transplanting CDR.
  • Humanized antibodies are also referred to as reshaped human antibodies.
  • animals other than humans for example, humanized antibodies obtained by transplanting CDRs of mouse antibodies into human antibodies are known.
  • Common genetic recombination techniques for obtaining humanized antibodies are also known.
  • Overlap Extension PCR is known as a method for transplanting the CDR of a mouse antibody into a human FR.
  • Overlap Extension PCR a base sequence encoding the CDR of a mouse antibody to be transplanted is added to a primer for synthesizing FR of a human antibody. Primers are prepared for each of the four FRs.
  • human FRs having high identity with mouse FRs in maintaining the function of CDRs. That is, in general, it is preferable to use human FR having an amino acid sequence having a high identity with the amino acid sequence of FR adjacent to the mouse CDR to be transplanted.
  • the base sequences to be linked are designed to be linked to each other in frame.
  • Human FR is individually synthesized by each primer. The result is a product in which the DNA encoding the mouse CDR is added to each FR.
  • the nucleotide sequences encoding the mouse CDRs of each product are designed to overlap each other. Subsequently, the overlapping CDR moieties of the products synthesized using the human antibody gene as a template are annealed to each other to carry out a complementary strand synthesis reaction. By this reaction, human FR is linked via the sequence of mouse CDR.
  • a vector for human antibody expression can be prepared by inserting the DNA obtained as described above and the DNA encoding the human antibody C region into an expression vector so as to be fused in frame. After introducing the integrated vector into a host to establish recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed so that the humanized antibody is a culture of the cultured cells. Produced in (see European Patent Publication EP 239400, International Publication WO 1996/002576).
  • the CDR forms a good antigen-binding site when linked via the CDR.
  • the FR of a human antibody such as that can be preferably selected.
  • the amino acid residues of FR can be substituted so that the CDRs of the reconstituted human antibody form the appropriate antigen binding site.
  • the PCR method used for transplanting mouse CDRs into human FR can be applied to introduce mutations in the amino acid sequence into FR.
  • a partial base sequence mutation can be introduced into the primer annealed to FR.
  • a mutation in the base sequence is introduced into the FR synthesized by such a primer.
  • a mutant FR sequence having desired properties can be selected by measuring and evaluating the binding activity of the amino acid-substituted mutant antibody to the antigen by the above method (Sato, K. et al., Cancer Res, 1993, 53, 851-856).
  • transgenic animals having the entire repertoire of human antibody genes were used as immune animals, and DNA immunization was performed.
  • the desired human antibody can be obtained.
  • the V region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method.
  • Phage expressing scFv that binds to the antigen can be selected.
  • the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined.
  • An expression vector can be prepared by determining the DNA sequence of scFv that binds to the antigen, fusing the V region sequence with the sequence of the desired human antibody C region in-frame, and then inserting it into an appropriate expression vector.
  • the human antibody is obtained by introducing the expression vector into suitable expressing cells as listed above and expressing the gene encoding the human antibody. These methods are already known (see International Publication WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).
  • Fv variable fragment
  • VL light chain variable region
  • VH heavy chain variable region
  • Fv is, for example, the following antigen-binding molecule;
  • the monovalent scFv is one polypeptide that constitutes the Fc region via the heavy chain Fv fragment that constitutes the CD3 binding domain, and the other monovalent scFv is the light chain that constitutes the CD3 binding domain.
  • the bivalent antigen-binding domain linked to the other polypeptide constituting the Fc region via the Fv fragment is a bivalent scFv (1) bivalent antigen-binding domain, (2) IgG1, IgG2a, A light chain in an antigen-binding molecule containing an Fc region that does not have Fc gamma receptor-binding activity among the amino acids constituting the Fc region of IgG3 or IgG4, and (3) at least a monovalent CD3 binding domain.
  • single chain antibody includes variable regions derived from both heavy and light chains within a single polypeptide chain. Means an antibody fragment lacking a constant region.
  • single chain antibodies further comprise a polypeptide linker between the VH and VL domains that allows them to form the desired structure that appears to allow antigen binding.
  • Single-chain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg, and Moore, Springer-Verlag, New York, 269-315 (1994). Similarly, see International Patent Application Publication WO 1988/001649 and US Pat. Nos. 4,946,778 and 5,260,203.
  • the single chain antibody can also be bispecific and / or humanized.
  • ScFv is an antigen-binding domain in which VH and VL constituting Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. U.S.A. (1988) 85 (16), 5879-5883).
  • the peptide linker can keep VH and VL in close proximity.
  • sc (Fv) 2 is a single-chain antibody in which four variable regions of two VL and two VHs are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-). 2), 177-189).
  • the two VHs and VLs may be derived from different monoclonal antibodies.
  • bispecific sc (Fv) 2 ) that recognize two epitopes present in the same antigen as disclosed in the Journal of Immunology (1994) 152 (11), 5368-5374 are also preferred.
  • sc (Fv) 2 can be produced by a method known to those skilled in the art. For example, it can be prepared by linking scFv with a linker such as a peptide linker.
  • two VHs and two VLs are VH, VL, VH, VL ([ Examples of the antibody are characterized in that they are arranged in the order of VH] linker [VL] linker [VH] linker [VL]), but the order of the two VHs and the two VLs is not particularly limited to the above configuration. It may be arranged in any order. For example, the following order structure can be mentioned.
  • the antigen-binding molecule of the present invention may be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent.
  • a glycosylated sequence can be inserted, and the glycosylated chain can be suitably added for the purpose of obtaining a desired effect.
  • linker that binds the variable region of the antibody examples include any peptide linker that can be introduced by genetic engineering, or a linker disclosed in a synthetic compound linker (see, for example, Protein Engineering, 9 (3), 299-305, 1996).
  • a peptide linker is preferable.
  • the length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the intended purpose, but the preferred length is 5 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably 30 amino acids or less). Is 20 amino acids or less), particularly preferably 15 amino acids.
  • sc (Fv) 2 contains three peptide linkers, peptide linkers of the same length may be used, or peptide linkers of different lengths may be used.
  • Synthetic chemical linkers are commonly used cross-linking agents for cross-linking peptides such as N-hydroxysuccinimide (NHS), dissuccinimidylsvelate (DSS), bis (sulfosuccinimidyl).
  • NHS N-hydroxysuccinimide
  • DSS dissuccinimidylsvelate
  • bis sulfosuccinimidyl
  • BS3 Dithiobis (Sulfine Imidyl Propionate) (DSP), Dithiobis (Sulfone Imidyl Propionate) (DTSSP), Ethylene Glycolbis (Sulfine Imidyl Succinate) (EGS), Ethylene Glycolbis (Sulfone Sulfine Imidyl Succinate) (Sulfone-EGS), Dissuccini Imidyl Tartrate (DST), Disulfosuccinimidyl Tartrate (Sulfone-DST), Bis [2- (Sulfone Imidoxycarbonyloxy) ) Ethyl] sulfone (BSOCOES), bis [2- (sulfosuccinimideoxycarbonyloxy) ethyl] sulfone (sulfo-BSOCOES), etc., and these cross-linking agents are commercially available.
  • linkers When binding four antibody variable regions, three linkers are usually required, but the same linker may be used or different linkers may be used.
  • Fab is composed of one light chain and one heavy chain CH1 region and variable region.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • F (ab') 2 " and “Fab'” are produced by treating an immunoglobulin (monoclonal antibody) with a proteolytic enzyme such as pepsin or papain, and are produced between two H chains in a hinge region. Means an antibody fragment produced by digestion before and after the disulfide bond present in. For example, by treating IgG with papain, L is cleaved upstream of the disulfide bond existing between two H chains in the hinge region and consists of VL (L chain variable region) and CL (L chain constant region).
  • F (ab') 2 contains two light chains and two constant regions of the CH1 domain and a portion of the CH2 domain such that disulfide bonds between the chains are formed between the two heavy chains. Includes heavy chains.
  • F (ab') 2 constituting the antigen-binding molecule disclosed in the present specification is obtained by partially digesting a full-length monoclonal antibody or the like having a desired antigen-binding domain with a proteolytic enzyme such as pepsin, and then proteinizing the Fc fragment. It can be suitably obtained by adsorbing it on the A column and removing it.
  • the proteolytic enzyme is not particularly limited as long as it can digest the full-length antibody so as to produce F (ab') 2 in a limited manner by appropriately setting the reaction conditions of the enzyme such as pH.
  • pepsin, ficin, and the like can be exemplified.
  • the Fc region constituting the antigen-binding molecule disclosed herein is obtained after partially digesting an antibody such as a monoclonal antibody with a proteolytic enzyme such as pepsin and then adsorbing the fragment on a protein A column or a protein G column.
  • a proteolytic enzyme such as pepsin
  • the proteolytic enzyme is not particularly limited as long as it can digest an antibody such as a monoclonal antibody by appropriately setting the reaction conditions of the enzyme such as pH, and examples thereof include pepsin and ficin.
  • the antigen-binding molecule described in the present specification includes an Fc region in which the binding activity to the Fc gamma receptor is reduced among the amino acids constituting the Fc region of IgG1, IgG2, IgG3 or IgG4.
  • the antibody isotype is determined by the structure of the constant region.
  • the constant regions of the IgG1, IgG2, IgG3, and IgG4 isotypes are called C gamma 1, C gamma 2, C gamma 3, and C gamma 4, respectively.
  • the Fc region contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains such that disulfide bonds between the chains are formed between the two heavy chains.
  • (ab') Refers to the area excluding 2 .
  • the Fc region constituting the antigen-binding molecule disclosed in the present specification is a fraction adsorbed on a protein A column after partial digestion of IgG1, IgG2, IgG3, IgG4 monoclonal antibody and the like with a proteolytic enzyme such as pepsin. Can be suitably obtained by re-eluting.
  • the proteolytic enzyme is not particularly limited as long as it can digest the full-length antibody so as to produce F (ab') 2 in a limited manner by appropriately setting the reaction conditions of the enzyme such as pH.
  • pepsin, ficin, and the like can be exemplified.
  • Fc ⁇ receptor means a receptor that can bind to the Fc region of an IgG1, IgG2, IgG3, IgG4 monoclonal antibody, and means substantially any member of the family of proteins encoded by the Fc ⁇ receptor gene. In humans, this family includes Fc ⁇ RI (CD64) containing isoforms Fc ⁇ RIa, Fc ⁇ RIb and Fc ⁇ RIc; isoforms Fc ⁇ RIIa (including allotypes H131 and R131), Fc ⁇ RIIb (including Fc ⁇ RIIb-1 and Fc ⁇ RIIb-2) and Fc ⁇ RIIc.
  • Fc ⁇ RI CD64
  • Fc ⁇ RII CD32
  • Fc ⁇ RIII CD16
  • Fc ⁇ RIIIa including allotypes V158 and F158
  • Fc ⁇ RIIIb including allotypes Fc ⁇ RIIIb-NA1 and Fc ⁇ RIIIb-NA2
  • Fc ⁇ Rs may be of any organism, including but not limited to humans, mice, rats, rabbits and monkeys.
  • Mouse Fc ⁇ Rs include Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), Fc ⁇ RIII (CD16) and Fc ⁇ RIII-2 (CD16-2), as well as any undiscovered mouse Fc ⁇ Rs or Fc ⁇ R isoforms or allotypes. Not limited to. Suitable examples of such Fc ⁇ receptors include human Fc ⁇ RI (CD64), Fc ⁇ RIIA (CD32), Fc ⁇ RIIB (CD32), Fc ⁇ RIIIA (CD16) and / or Fc ⁇ RIIIB (CD16).
  • the Nucleotide sequence and amino acid sequence of Fc ⁇ RI are RefSeq registration numbers in NM_000566.3 and NP_000557.1, respectively, and the polynucleotide sequence and amino acid sequence of Fc ⁇ RIIA are RefSeq registration numbers in BC020823.1 and 30AAH20823.1, respectively.
  • the polynucleotide and amino acid sequences are RefSeq registration numbers, BC146678.1 and AAI46679.1, respectively, and the polynucleotide and amino acid sequences of Fc ⁇ RIIIA are RefSeq registration numbers, BC033678.1 and AAH33678.1, respectively, and the poly of Fc ⁇ RIIIB.
  • Nucleotide and amino acid sequences are described in BC128562.1 and AAI28563.1 with RefSeq registration numbers, respectively. Whether or not the Fc ⁇ receptor has binding activity to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies can be determined by the FACS and ELISA formats described above, as well as the ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and surface. It can be confirmed by the BIACORE method using the plasmon resonance (SPR) phenomenon (Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010).
  • SPR plasmon resonance
  • Fc ligand or "effector ligand” means a molecule derived from any organism, preferably a polypeptide, which binds to the Fc region of an antibody to form an Fc / Fc ligand complex. Binding of the Fc ligand to Fc preferably induces one or more effector functions.
  • Fc ligands include Fc receptors, Fc ⁇ R, Fc ⁇ R, Fc ⁇ R, FcRn, C1q, C3, mannan-binding lectin, mannose receptors, staphylococcus protein A, staphylococcus protein G and viral Fc ⁇ R. , Not limited to these.
  • Fc ligands also include Fc receptor homologues (FcRH) (Davis et al., (2002) Immunological Reviews 190, 123-136), a family of Fc receptors homologous to Fc ⁇ R. Fc ligands can also include undiscovered molecules that bind to Fc
  • Fc region has reduced binding activity to the Fc ⁇ receptor of any of Fc ⁇ I, Fc ⁇ IIA, Fc ⁇ IIB, Fc ⁇ IIIA and / or Fc ⁇ IIIB is described in the FACS and ELISA formats described above, as well as the ALPHA screen (Amplified Luminescent Proximity). It can be confirmed by the BIACORE method using Homogeneous Assay) or surface plasmon resonance (SPR) phenomenon (Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010).
  • SPR surface plasmon resonance
  • the ALPHA screen is implemented based on the following principle by ALPHA technology using two beads, donor and acceptor.
  • the luminescent signal is detected only when the molecule bound to the donor bead biologically interacts with the molecule bound to the acceptor bead and the two beads are in close proximity.
  • the photosensitizer in the donor beads excited by the laser converts the surrounding oxygen into excited singlet oxygen. Singlet oxygen diffuses around the donor beads, and when it reaches the adjacent acceptor beads, it triggers a chemiluminescent reaction inside the beads, eventually emitting light.
  • the chemiluminescent reaction does not occur because the singlet oxygen produced by the donor bead does not reach the acceptor bead.
  • a biotin-labeled antigen-binding molecule is bound to the donor beads, and a glutathione S transferase (GST) -tagged Fc ⁇ receptor is bound to the acceptor beads.
  • GST glutathione S transferase
  • the antigen-binding molecule with a wild-type Fc region interacts with the Fc ⁇ receptor to produce a signal of 520-620 nm.
  • Antigen-binding molecules with untagged mutant Fc regions compete with interactions between Fc ⁇ receptors and antigen-binding molecules with wild-type Fc regions. Relative binding affinity can be determined by quantifying the decrease in fluorescence that results from competition.
  • an antigen-binding molecule such as an antibody is biotinylated using Sulfo-NHS-biotin or the like.
  • a method of tagging the Fc ⁇ receptor with GST it is expressed in a cell or the like in which a fusion gene in which a polynucleotide encoding an Fc ⁇ receptor and a polynucleotide encoding GST are fused in frame is held in an expressible vector.
  • a method of purifying using a glutathione column or the like can be appropriately adopted.
  • the obtained signal is suitably analyzed by fitting it into a one-site competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).
  • the Biacore system takes the above-mentioned shift amount, that is, the change in mass on the surface of the sensor chip on the vertical axis, and displays the change in mass over time as measurement data (sensorgram).
  • Kinetics Bonding rate constant (ka) and dissociation rate constant (kd) are obtained from the curve of the sensorgram, and affinity (KD) is obtained from the ratio of the constants.
  • the inhibition measurement method is also preferably used. Examples of inhibition measures are described in Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010.
  • ADCC Antibody Dependent Cell-mediated Cytotoxicity
  • CDC Complement
  • Dependent Cytotoxicity exerts an antitumor effect on cancer cells.
  • ADCC is the cytotoxicity exerted by these effector cells against the target cancer cells to which the antibody is bound by binding to the Fc receptors present on effector cells such as NK cells and macrophages in the Fc region of the antibody. ..
  • the complement complex binds to the complement binding site present in the structure of the antibody.
  • the Fc ⁇ receptor is a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and when the binding activity to the Fc ⁇ receptor is low, T is independent of the cancer antigen. Receptors expressed in cells and NK cells, macrophages, etc. are not cross-linked. Therefore, cancer antigen-independent cytokine induction does not occur.
  • Antibodies whose Fc region has reduced binding activity to any Fc ⁇ receptor of Fc ⁇ I, Fc ⁇ IIA, Fc ⁇ IIB, Fc ⁇ IIIA and / or Fc ⁇ IIIB are desirable as antigen-binding molecules.
  • the fact that the Fc ⁇ receptor-binding activity is reduced means that, for example, based on the above analysis method, the competitive activity of the test antigen-binding molecule is compared with the competitive activity of the control antigen-binding molecule.
  • an antigen-binding molecule having an Fc region of an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be appropriately used.
  • the structure of the Fc region the sequence in which A is added to the N end of RefSeq registration number AAC82527.1, the sequence in which A is added to the N end of RefSeq registration number AAB59393.1, and the N end of RefSeq registration number CAA27268.1 are A.
  • An example is a sequence in which A is added to the N-terminal of RefSeq registration number AAB59 394.1.
  • an antigen-binding molecule having a variant of the Fc region of a specific isotype antibody is used as a test substance
  • an antigen-binding molecule having an Fc region of the specific isotype antibody is used as a control.
  • the effect of the mutation possessed by the mutant on the binding activity to the Fc ⁇ receptor will be verified.
  • an antigen-binding molecule having a mutant of the Fc region whose binding activity to the Fc ⁇ receptor has been verified to be reduced is appropriately prepared.
  • mutants are deletions of the amino acid 231A-238S identified according to EU numbering (WO 2009/011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum.Antibod.Hybridomas (1990) 1 (1), 47-54), C226S, C229S, E233P, L234V, L235A (Blood (2007) 109, 1185-1192) It is known.
  • any of the following amino acids identified according to EU numbering at positions 220, 226, 229, 231 and 232, 233, and 234.
  • Preferred examples thereof include antigen-binding molecules having an Fc region in which positions, 300, 325, 327, 328, 329, 330, 331, and 332 are substituted.
  • the isotype of the antibody originating from the Fc region is not particularly limited, and an Fc region originating from an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be appropriately used, but an Fc region originating from an IgG1 antibody is preferably used. Will be done.
  • amino acids constituting the Fc region of the IgG1 antibody one of the following substitutions specified according to the EU numbering (the number is the position of the amino acid residue specified according to the EU numbering, the one-letter amino acid located before the number) The symbol represents the amino acid residue before replacement, and the one-letter amino acid symbol located after the number represents the amino acid residue before replacement); (A) L234F, L235E, P331S, (B) C226S, C229S, P238S, (C) C226S, C229S, (D) C226S, C229S, E233P, L234V, L235A (E) L234A, L235A or L235R, N297A (F) An antigen-binding molecule having an Fc region with L235A or L235R, S239K, N297A or an Fc region lacking the amino acid sequence at positions 231 to 238 can also be used as appropriate.
  • any of the following substitutions specified according to the EU numbering (the number is the position of the amino acid residue specified according to the EU numbering, the one-letter amino acid located before the number) The symbol represents the amino acid residue before replacement, and the one-letter amino acid symbol located after the number represents the amino acid residue before replacement); (G) H268Q, V309L, A330S, P331S (H) V234A (I) G237A (J) V234A, G237A (K) A235E, G237A (L) Antigen-binding molecules having an Fc region coated with V234A, A235E, G237A can also be used as appropriate.
  • any of the following substitutions specified according to the EU numbering (the number is the position of the amino acid residue specified according to the EU numbering, the one-letter amino acid located before the number) The symbol represents the amino acid residue before replacement, and the one-letter amino acid symbol located after the number represents the amino acid residue before replacement); (M) F241A (N) D265A (O) Antigen-binding molecules having an Fc region coated with V264A can also be used as appropriate.
  • any of the following substitutions specified according to the EU numbering (the number is the position of the amino acid residue specified according to the EU numbering, the one-letter amino acid located before the number) The symbol represents the amino acid residue before replacement, and the one-letter amino acid symbol located after the number represents the amino acid residue before replacement); (P) L235A, G237A, E318A (Q) L235E (R) Antigen-binding molecules having an Fc region coated with F234A and L235A can also be used as appropriate.
  • any of the following amino acids identified according to EU numbering 233, 234, 235, 236, 237, 327, 330.
  • examples thereof include antigen-binding molecules having an Fc region in which the EU numbering is substituted with the corresponding amino acid in the corresponding IgG2 or IgG4 at positions 331.
  • amino acids constituting the Fc region of the IgG1 antibody one or more of the following amino acids specified according to EU numbering; the 234th, 235th, and 297th positions are replaced by other amino acids.
  • Preferred examples thereof include antigen-binding molecules having an Fc region.
  • the type of amino acid present after the substitution is not particularly limited, but an antigen-binding molecule having an Fc region in which one or more amino acids at the 234th, 235th, and 297th positions are replaced with alanine is particularly preferable.
  • amino acids constituting the Fc region of the IgG1 antibody one of the following amino acids identified according to EU numbering; an antigen-binding molecule having an Fc region in which the 265th position is replaced by another amino acid is used.
  • the type of amino acid present after the substitution is not particularly limited, but an antigen-binding molecule having an Fc region in which the amino acid at position 265 is substituted with alanine is particularly preferable.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cellular cytotoxicity
  • the Fc of the IgG antibody is used. Binding of the region to the Fc ⁇ receptor (Fc ⁇ R) present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages is required.
  • Enhancement of cytotoxic effector functions such as ADCC and ADCP is a promising means for enhancing the antitumor effect of anticancer antibodies. It is suggested that an antibody having an Fc region with optimized binding to the Fc ⁇ receptor mediates a stronger effector function, thereby exerting an effective antitumor effect. Therefore, various reports (for example, WO2013047752) have been made so far as an antibody engineering method for enhancing or improving the antitumor activity of an antibody drug against a cancer antigen.
  • the binding activity to the Fc ⁇ receptor plays an important role in the cytotoxic activity. Therefore, when the cytotoxic activity is required, the human IgG1 having a high binding activity to Fc ⁇ R is required. Istotype is used, and it is a widely used technique that the cytotoxic activity can be enhanced by enhancing the binding activity to the Fc ⁇ receptor. In addition, it has been attempted to enhance the binding activity to the Fc ⁇ receptor even in an antibody targeting a soluble antigen (WO2013047752).
  • the amino acid at position 221 is either Lys or Tyr
  • the amino acid at position 222 is either Phe, Trp, Glu or Tyr
  • the amino acid at position 223 is either Phe, Trp, Glu or Lys
  • the amino acid at position 224 is either Phe, Trp, Glu or Tyr
  • the amino acid at position 225 is either Glu, Lys or Trp
  • the amino acid at position 227 is either Glu, Gly, Lys or Tyr
  • the amino acid at position 228 is either Glu, Gly, Lys or Tyr
  • the amino acid at position 230 is either Ala, Glu, Gly or Tyr
  • the amino acid at position 231 is either Glu, Gly, Lys, Pro or Tyr
  • the amino acid at position 232 is either Glu, Gly, Lys or Tyr
  • the amino acid at position 233 is either Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gl
  • the amino acid at position 234 is either Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 235 is either Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 236 is either Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 237 is either Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 238 is either Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 239 is either Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr.
  • the amino acid at position 240 is either Ala, Ile, Met or Thr
  • the amino acid at position 241 is either Asp, Glu, Leu, Arg, Trp or Tyr
  • the amino acid at position 243 is either Leu, Glu, Leu, Gln, Arg, Trp or Tyr
  • the amino acid at position 244 is His
  • the amino acid at position 245 is Ala
  • the amino acid at position 246 is either Asp, Glu, His or Tyr
  • the amino acid at position 247 is either Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr.
  • the amino acid at position 249 is either Glu, His, Gln or Tyr
  • the amino acid at position 250 is either Glu or Gln
  • the amino acid at position 251 is Phe
  • the amino acid at position 254 is either Phe, Met or Tyr
  • the amino acid at position 255 is either Glu, Leu or Tyr
  • the amino acid at position 256 is either Ala, Met or Pro
  • the amino acid at position 258 is either Asp, Glu, His, Ser or Tyr
  • the amino acid at position 260 is either Asp, Glu, His or Tyr
  • the amino acid at position 262 is either Ala, Glu, Phe, Ile or Thr
  • the amino acid at position 263 is either Ala, Ile, Met or Thr
  • the amino acid at position 264 is either Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr.
  • the amino acid at position 265 is either Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 266 is either Ala, Ile, Met or Thr.
  • the amino acid at position 267 is either Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr.
  • the amino acid at position 268 is either Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp.
  • the amino acid at position 269 is either Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 270 is either Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr.
  • the amino acid at position 271 is either Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 272 is either Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 273 is either Phe or Ile.
  • the amino acid at position 274 is either Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 275 is either Leu or Trp,
  • the amino acid at position 276 is either Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 278 is either Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp.
  • the amino acid at position 279 is Ala
  • the amino acid at position 280 is either Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr.
  • the amino acid at position 281 is either Asp, Lys, Pro or Tyr
  • the amino acid at position 282 is either Glu, Gly, Lys, Pro or Tyr
  • the amino acid at position 283 is either Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr.
  • the amino acid at position 284 is either Asp, Glu, Leu, Asn, Thr or Tyr
  • the amino acid at position 285 is either Asp, Glu, Lys, Gln, Trp or Tyr
  • the amino acid at position 286 is either Glu, Gly, Pro or Tyr
  • the amino acid at position 288 is either Asn, Asp, Glu or Tyr
  • the amino acid at position 290 is either Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr.
  • the amino acid at position 291 is either Asp, Glu, Gly, His, Ile, Gln or Thr.
  • the amino acid at position 292 is either Ala, Asp, Glu, Pro, Thr or Tyr
  • the amino acid at position 293 is either Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 294 is either Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 295 is either Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 296 is either Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val.
  • the amino acid at position 297 is either Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 298 is either Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr.
  • the amino acid at position 299 is either Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr.
  • the amino acid at position 300 is either Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp.
  • the amino acid at position 301 is either Asp, Glu, His or Tyr
  • the amino acid at position 302 is Ile
  • the amino acid at position 303 is either Asp, Gly or Tyr
  • the amino acid at position 304 is either Asp, His, Leu, Asn or Thr
  • the amino acid at position 305 is either Glu, Ile, Thr or Tyr
  • the amino acid at position 311 is either Ala, Asp, Asn, Thr, Val or Tyr
  • the amino acid at position 313 is Phe
  • the amino acid at position 315 is Leu
  • the amino acid at position 317 is Glu or Gln
  • the amino acid at position 318 is either His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr.
  • the amino acid at position 320 is either Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 322 is either Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 323 is Ile,
  • the amino acid at position 324 is either Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr.
  • the amino acid at position 325 is either Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 326 is either Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 327 is either Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr.
  • the amino acid at position 328 is either Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 329 is either Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 330 is Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 331 is either Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr.
  • the amino acid at position 332 is either Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr.
  • the amino acid at position 333 is either Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr.
  • the amino acid at position 334 is either Ala, Glu, Phe, Ile, Leu, Pro or Thr.
  • the amino acid at position 335 is either Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr.
  • the amino acid at position 336 is either Glu, Lys or Tyr.
  • the amino acid at position 337 is either Glu, His or Asn,
  • the amino acid at position 339 is either Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr.
  • the amino acid at position 376 is either Ala or Val, The amino acid at position 377 is either Gly or Lys, The amino acid at position 378 is Asp, The amino acid at position 379 is Asn, The amino acid at position 380 is either Ala, Asn or Ser, The amino acid at position 382 is either Ala or Ile, The amino acid at position 385 is Glu, The amino acid at position 392 is Thr, The amino acid at position 396 is Leu, The amino acid at position 421 is Lys, The amino acid at position 427 is Asn, The amino acid at position 428 is either Phe or Leu, The amino acid at position 429 is Met, The amino acid at position 434 is Trp, The amino acid at position 436 is Ile, and An IgG antibody having an Fc region in which the amino acid at position 440 contains at least one amino acid selected from the group of Gly, His, Ile, Leu or Tyr, and ADCC in which an IgG antibody-like molecule is a secondary molecule. It is preferable
  • a multispecific antibody As the antigen-binding molecule of the present invention, a multispecific antibody can be mentioned.
  • the multispecific antibody is an antibody having a plurality of different specificities.
  • multispecific antibodies include bispecific antibodies. IgG-type bispecific antibodies can be secreted by hybridomas (quadromas) produced by fusion of two IgG antibody-producing hybridomas (Milstein C et al. Nature (1983) 305, 537-540). ..
  • an Fc region having a reduced binding activity to an Fc ⁇ receptor is used as the Fc region of a bispecific antibody, an Fc region originating from the bispecific antibody is also appropriately used.
  • Multispecific antibodies manipulate electrostatic steering effects to create Fc heterodimer molecules (WO2009 / 089004A1); crosslinks of two or more antibodies or fragments (US patent). See Nos.
  • Modified antibodies with three or more functional antigen binding sites are also included herein (see, eg, US Patent Application Publication No. 2006/0025576 A1).
  • the antigen binding molecule comprises a bispecific antigen binding site having a target antigen recognition site and an immune receptor recognition site.
  • the antigen-binding molecule may have an additional antigen-binding site in addition to the bispecific antigen-binding site, and examples of the antigen-binding molecule of this embodiment include a bispecific antigen-binding molecule and three types. Examples thereof include a multispecific antigen-binding molecule having the above-mentioned antigen-binding site, and more specifically, a bispecific antibody and a multi-specific antibody having three or more kinds of antigen-binding sites.
  • IgG-type bispecific antibodies are secreted by introducing into cells a total of four genes, the L-chain and H-chain genes that make up the two target IgGs, and co-expressing them.
  • L-chain and H-chain genes that make up the two target IgGs, and co-expressing them.
  • H chain and L chain of IgG produced by these methods. It is difficult to purify an IgG consisting of H chains and L chains of the desired combination from 10 types of IgG.
  • the amount of secretion of the desired combination is theoretically significantly reduced, which requires a large culture scale and further increases the manufacturing cost.
  • the association of multispecific antibodies is not intended by introducing chargeable repulsion at the interface of the second constant region (CH2) of the antibody H chain or the third constant region (CH3) of the H chain.
  • Techniques for suppressing association between H chains can be applied (WO2006 / 106905).
  • the amino acid residue that contacts at the interface of other constant regions of the H chain is, for example, EU in the CH3 region. Regions facing the 356th residue of numbering, the 439th residue of EU numbering, the 357th residue of EU numbering, the 370th residue of EU numbering, the 399th residue of EU numbering, and the 409th residue of EU numbering. Can be mentioned.
  • a set or 3 sets of amino acid residues can be used as an antibody having the same kind of charge; (1) Amino acid residues contained in the CH3 region of the H chain, which are amino acid residues at EU numbering positions 356 and 439, (2) Amino acid residues contained in the H chain CH3 region, which are amino acid residues at positions 357 and 370 of the EU numbering, and (3) Amino acid residues contained in the H chain CH3 region, which are located at the EU numbering positions 399. And the amino acid residue at position 409.
  • the set of amino acid residues corresponding to the set of amino acid residues shown in (1) to (3) having the same kind of charge in the first H chain CH3 region is the first H chain CH3 region. It can be an antibody having a charge opposite to that of the corresponding amino acid residue in.
  • amino acid residues described in (1) to (3) above are close to each other when associated.
  • a person skilled in the art will find a site corresponding to the amino acid residue described in (1) to (3) above by homology modeling or the like using commercially available software for a desired H chain CH3 region or H chain constant region. It is possible to modify the amino acid residue of the site as appropriate.
  • the "charged amino acid residue” is preferably selected from, for example, amino acid residues included in any of the following groups (a) or (b); (A) Glutamic acid (E), aspartic acid (D), (B) Lysine (K), arginine (R), histidine (H).
  • “having the same charge” means that, for example, any of two or more amino acid residues has an amino acid residue contained in any one group of (a) or (b) above.
  • Means that. “Having opposite charges” means, for example, that at least one amino acid residue among two or more amino acid residues is contained in the group of any one of (a) and (b) above. If, it means that the remaining amino acid residues have amino acid residues contained in different groups.
  • the first H chain CH3 region and the second H chain CH3 region may be crosslinked by a disulfide bond.
  • amino acid residue to be modified in the present invention is not limited to the amino acid residue in the variable region of the antibody or the constant region of the antibody described above. Those skilled in the art can find amino acid residues forming an interface on a polypeptide variant or heterologous multimer by homology modeling using commercially available software, and control the association of the site. Amino acid residues can be used for modification.
  • bispecific antibody of the present invention A portion of CH3 of one H chain of the antibody was made into an IgA-derived sequence corresponding to that portion, and an IgA-derived sequence corresponding to that portion was introduced into a complementary portion of CH3 of the other H chain.
  • exchange engineered domain CH3 By using exchange engineered domain CH3, the association of polypeptides having different sequences can be efficiently triggered by the complementary association of CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). .. Even if this known technique is used, the desired bispecific antibody can be efficiently formed.
  • bispecific antibodies For the formation of bispecific antibodies, the association of CH1 and CL of the antibodies described in WO2011 / 028952, WO2014 / 018572 and Nat Biotechnol. 2014 Feb; 32 (2): 191-8., VH, Antibody production technology using VL association, technology for producing bispecific antibodies using separately prepared monoclonal antibodies described in WO2008 / 119353 and WO2011 / 131746 (Fab Arm Exchange), WO2012 / 058768 And WO2013 / 063702, a technique for controlling the association of antibody heavy chains between CH3, and WO2012 / 023053 to prepare bispecific antibodies composed of two types of light chains and one type of heavy chain.
  • the present invention can also be obtained by separating and purifying the desired bispecific antibody from the produced antibody. It is possible to obtain bispecific antibodies. For example, by introducing an amino acid substitution into the variable region of two types of H chains and imparting a difference in isoelectric point (pI), it becomes possible to purify the two types of homozygous substances and the desired hetero-antibody by ion exchange chromatography. The method has been reported (WO2007114325).
  • a method for purifying a heterozygous substance a method for purifying a heterodimerized antibody consisting of an H chain of mouse IgG2a that binds to protein A and an H chain of rat IgG2b that does not bind to protein A using protein A has been used so far. Has been reported (WO98050431, WO95033844).
  • an H chain in which the amino acid residues 435 and 436 of the EU numbering, which are the binding sites of IgG and Protein A, are replaced with amino acids having different binding strengths to Protein A such as Tyr and His is used, or Reference Example 5
  • H chains having different binding force to Protein A obtained according to the method described in the above the interaction between each H chain and Protein A is changed, and by using the Protein A column, only the heterodimerized antibody can be obtained. It can also be efficiently purified.
  • a common L chain capable of imparting a binding ability to a plurality of different H chains may be obtained and used as a common L chain of a bispecific antibody.
  • Efficient bispecific IgG can be expressed by introducing IgG into cells by introducing multiple H-chain genes different from the common L-chain (Nature Biotechnology (1998) 16, 677-). 681).
  • a method of selecting a common L chain corresponding to any different H chain and exhibiting high binding ability can also be used (WO2004 / 065611).
  • an Fc region in which the heterogeneity of the C-terminal of the Fc region is improved can be appropriately used. More specifically, of the amino acid sequences of the two polypeptides constituting the Fc region originating from IgG1, IgG2, IgG3 or IgG4, the glycine at position 446 and the lysine at position 447 specified according to EU numbering are deleted. The Fc region is provided.
  • the antigen-binding molecule of the present invention may be a separately prepared antigen-binding molecule having the same amino acid sequence based on the modified molecule described above.
  • the "functionally equivalent" antibody variable region is not particularly limited as long as it is an antibody H chain variable region and / or an antibody L chain variable region that satisfies the above conditions.
  • antibody variable regions for example, one or more amino acids (eg, 1, 2, 3, 4, 5 or 10 amino acids) are substituted or deleted in the amino acid sequences of the variable regions shown in Tables 1 to 3 above. , Addition and / or may be inserted.
  • a method well known to those skilled in the art for substituting, deleting, adding and / or inserting one or more amino acids in an amino acid sequence is a method of introducing a mutation into a protein.
  • amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and aromatic-containing sides Amino acids having chains (H, F, Y, W) can be mentioned (all in parentheses represent the one-letter notation of amino acids).
  • substitutions of amino acids within each of these groups are referred to as conservative substitutions. It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution with another amino acid of one or more amino acid residues to an amino acid sequence maintains its biological activity. Yes (Mark, D. F. et al., Proc.Natl.Acad.Sci.USA (1984) 81: 5662-6; Zoller, M.J. and Smith, M., Nucleic Acids Res. (1982) 10 : 6487-500; Wang, A. et al., Science (1984) 224: 1431-3; Dalbadie-McFarland, G.
  • variable region of the invention containing such amino acid modifications is at least 70%, more preferably at least 75%, more preferably at least 80% of the amino acid sequence of the CDR sequence, FR sequence or the entire variable region of the variable region before modification. , More preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% amino acid sequence identity.
  • sequence identity is the amino acid sequence of the original H chain variable region or L chain variable region after the sequences are arranged as necessary so as to maximize the sequence identity and a gap is introduced as appropriate. It is defined as the proportion of residues that are identical to the residues in.
  • the identity of the amino acid sequence can be determined by the method described below.
  • the "functionally equivalent antibody variable region” is, for example, a nucleic acid that hybridizes to a nucleic acid consisting of a base sequence encoding the amino acid sequence of the variable region shown in Tables 1 to 3 above under stringent conditions. It is also possible to obtain from.
  • the stringent hybridization conditions for isolating a nucleic acid that hybridizes to a nucleic acid consisting of a base sequence encoding a variable region amino acid sequence under stringent conditions include 6M urea, 0.4% SDS, and 0.5x. SSC, 37 ° C. conditions or equivalent stringency hybridization conditions can be exemplified.
  • Isolation of nucleic acids with higher homology can be expected by using conditions with higher stringency, for example, 6M urea, 0.4% SDS, 0.1xSSC, and 42 ° C.
  • the washing conditions after hybridization are, for example, 0.5xSSC (1xSSC is 0.15M NaCL, 0.015M sodium citrate, pH 7.0) and 0.1% SDS, washing at 60 ° C, more preferably 0.2xSSC, and 0.1% SDS, Cleaning at 60 ° C, more preferably 0.2xSSC and 0.1% SDS, cleaning at 62 ° C, more preferably 0.2xSSC and 0.1% SDS, cleaning at 65 ° C, more preferably 0.1xSSC and 0.1% SDS, 65 ° C. Cleaning in.
  • the sequence of the isolated nucleic acid can be determined by a known method described later.
  • the homology of the isolated nucleic acid is at least 50% or more, more preferably 70% or more, still more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99%) in the entire base sequence. It has the same sequence of the above).
  • a gene amplification method using a primer synthesized based on the base sequence information encoding the amino acid sequence of the variable region for example, a polymerase chain reaction (PCR) method. It is also possible to isolate a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence encoding the amino acid sequence of the region.
  • PCR polymerase chain reaction
  • the Fc region contained in the bispecific antibody of the present invention is not particularly limited as long as it is an Fc region in which the binding activity to the Fc ⁇ receptor is reduced.
  • a preferred Fc region of the present invention is, for example, WO2016 / 047722A1.
  • the combination of the Fc region portion of E22Hh and the Fc region portion of E22Hk, the combination of the Fc region portion of E2702GsKsc and the Fc region portion of E2704sEpsc, and the combination of the Fc region portion of E2702sKsc and the Fc region portion of E2704sEpsc can be mentioned.
  • the gene encoding the antigen-binding molecule of the present invention into a gene therapy vector to perform gene therapy.
  • a gene therapy vector in addition to direct administration with a naked plasmid, it can be packaged in liposomes or the like, or as various viral vectors such as retrovirus vector, adenovirus vector, vaccinia virus vector, poxvirus vector, adenovirus-related vector, and HVJ vector. It can be formed (see Adolph "Viral Genome Method", CRC Press, Florid (1996)) or coated on a bead carrier such as colloidal gold particles (WO93 / 17706, etc.) and administered.
  • the nucleic acid encoding the antigen-binding molecule of the present invention may be directly administered to a living body, or may be directly administered to a living body by an electroporation method.
  • the mRNA encoding the antigen-binding molecule of the present invention is chemically modified to enhance the stability of the mRNA in vivo, and the mRNA is directly administered to humans to express the antigen-binding molecule of the present invention in vivo.
  • the antigen-binding molecule of the present invention can be administered by the method of causing (see EP2101823B, WO2013 / 120629).
  • the antigen-binding molecule may be administered by any method.
  • injection, infusion, or gas-induced particle impact (electron) is performed via a suitable parenteral route (intravenous, intraperitoneal, subcutaneous, intracutaneous, adipose tissue, mammary gland tissue, inhalation or intramuscular route).
  • a sufficient amount is administered by (using a gun, etc.), a method via a mucosal route such as a nasal spray, etc.).
  • the present invention is produced by administering to blood cells, bone marrow-derived cells, etc. using liposome transfection, particle impact method (US Patent No. 4,945,050), or virus infection in ex vivo, and reintroducing the cells into animals.
  • a gene encoding an antigen-binding molecule may be administered.
  • treatment and its grammatical derivatives such as “treat”, “treat”, etc.
  • treatment is clinically intended to alter the natural course of the individual being treated. It means intervention and can be performed both for prevention and during the course of clinical pathology.
  • the desired effects of treatment are, but are not limited to, prevention of the onset or recurrence of the disease, relief of symptoms, diminishing any direct or indirect pathological effects of the disease, prevention of metastasis, progression of the disease. Includes slowing, recovery or alleviation of disease state, and ameliorated or improved prognosis.
  • the pharmaceutical compositions of the invention are used to delay the onset of a disease or slow the progression of a disease.
  • the pharmaceutical composition usually refers to a drug for treating or preventing a disease, or for testing / diagnosis.
  • the pharmaceutical composition when used in combination with the administration of other ingredients, the pharmaceutical composition can be administered simultaneously, separately or continuously with the administration of the other ingredients.
  • the pharmaceutical composition of the present invention may contain other ingredients as ingredients.
  • the pharmaceutical composition containing the antigen-binding molecule of the present invention can be formulated using a method known to those skilled in the art.
  • it can be used parenterally in the form of aseptic solutions with water or other pharmaceutically acceptable liquids, or injectable suspensions.
  • pharmacologically acceptable carriers or media specifically sterile water, saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives.
  • the sterile composition for injection can be formulated according to the usual formulation practice using a vehicle such as distilled water for injection.
  • Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol, sodium chloride).
  • Suitable solubilizing agents such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), nonionic surfactants (polysorbate 80 (TM), HCO-50, etc.) can be used in combination.
  • TM polysorbate 80
  • oily liquid examples include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol can also be used in combination as a solubilizing agent. It may also be formulated with buffers (eg, phosphate buffers and sodium acetate buffers), soothing agents (eg, procaine hydrochloride), stabilizers (eg, benzyl alcohol and phenols), antioxidants.
  • buffers eg, phosphate buffers and sodium acetate buffers
  • soothing agents eg, procaine hydrochloride
  • stabilizers eg, benzyl alcohol and phenols
  • antioxidants antioxidants.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the pharmaceutical composition containing the antigen-binding molecule of the present invention is preferably administered by parenteral administration.
  • parenteral administration for example, an injection type, a nasal administration type, a pulmonary administration type, and a transdermal administration type composition are administered.
  • intracardiac injection intralesional injection such as tumor, or a method using a catheter.
  • the administration method can be appropriately selected depending on the patient's age and symptoms.
  • the dose of the pharmaceutical composition of the present invention can be set, for example, in the range of 0.0001 mg to 1000 mg per 1 kg of body weight at a time. Alternatively, for example, a dose of 0.001 to 100,000 mg per patient can be set, but the present invention is not necessarily limited to these values.
  • the dose and administration method vary depending on the weight, age, symptoms and the like of the patient, but those skilled in the art can set an appropriate dose and administration method in consideration of these conditions.
  • the pharmaceutical composition of the present invention is encapsulated in microcapsules (microcapsules of hydroxymethyl cellulose, gelatin, poly [methyl methacrylate], etc.) and colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, etc.). Nanoparticles and nanocapsules, etc.) (see Remington's Pharmaceutical Science 16th edition, Oslo Ed. (1980), etc.). Furthermore, a method of using a drug as a sustained release drug is also known, and the method can be applied to the bispecific antigen-binding molecule of the present invention (J.Biomed.Mater.Res. (1981) 15, 267- 277, Chemtech. (1982) 12, 98-105, US Patent No. 3773719, European Patent Publication Publications EP58481 and EP133988, Biopolymers (1983) 22, 547-556).
  • microcapsules microcapsules of hydroxymethyl cellulose, gelatin, poly [methyl methacrylate], etc.
  • chimeric receptor causes specificity and intracellular signal production for target cells, such as cancer cells, when expressed on immune effector cells, including at least extracellular domains, transmembrane domains and intracellular signaling domains. , Refers to a recombinant polypeptide.
  • the extracellular domain of a chimeric receptor means any proteinaceous molecule or a part thereof that can specifically bind to a predetermined molecule.
  • the extracellular domain of a chimeric receptor is a polypeptide comprising the sequence of the extracellular domain of an immune receptor or a variant thereof.
  • the extracellular domain of the chimeric receptor may be a polypeptide consisting only of the sequence of the extracellular domain of the immune receptor or a variant thereof, the extracellular domain of the immunoreceptor or the same thereof.
  • the sequence of variants may contain additional polypeptide or amino acid residues.
  • the extracellular domain of the chimeric receptor may comprise a sequence of fragments of the extracellular domain of the immune receptor or a variant thereof.
  • the extracellular domain of an immune receptor in the present invention refers to an extracellular portion of an immune receptor that can bind to an endogenous immune ligand.
  • CD137 it refers to SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132.
  • the extracellular domain variant of an immune receptor means a polypeptide in which a part of the extracellular domain of an immune receptor is added, deleted, or substituted.
  • An extracellular domain variant of an immune receptor is preferably one that attenuates binding to an endogenous immune ligand, eg, an immune receptor identified by X-ray crystal structure analysis described in the literature and its endogenous.
  • a modification of the binding site of the immune receptor to the endogenous ligand of the extracellular domain based on the binding site to the ligand.
  • examples of extracellular domain variants of immune receptors that have diminished binding to endogenous immune ligands include cysteine-rich domain 3, cysteine-rich domain 4, and transmembrane domains of the immune receptor of CD137.
  • Examples include extracellular domain variants lacking the stalk (Genbank NM001561.6, Cys88-Gln186) that connects to the region.
  • the fragment of the extracellular domain of the immune receptor may be a fragment of a polypeptide constituting the extracellular domain of the immune receptor, and if the antigen-binding molecule is recognizable, the number of constituent amino acids. Etc. are not particularly limited. Examples of such fragments include polypeptides or fragments thereof that constitute an epitope recognized by an antibody having agonist activity on an immune receptor.
  • the extracellular domain of the chimeric receptor may contain a variant of the epitope, and the fragment of the polypeptide constituting the extracellular domain of the immunoreceptor contained in the epitope may also contain "an immunoreceptor. Corresponds to "fragment of extracellular domain".
  • transmembrane domain includes a polypeptide that is located between the extracellular domain and the intracellular signaling domain and has the function of penetrating the cell membrane.
  • intracellular signaling domain is known to serve to transmit signals that trigger the activation or inhibition of intracellular biological processes, such as the activation of immune cells such as T cells or NK cells.
  • domain means, for example, a region of a polypeptide that is folded into a specific structure and / or has a specific function independently of other regions.
  • the domain can be, for example, the cytoplasmic portion of the molecule or part thereof.
  • the "cytoplasmic domain” of a molecule means the full-length cytoplasmic domain, or a portion thereof that transmits intracellular signals when activated.
  • a chimeric receptor is a molecule comprising a domain as defined below.
  • the chimeric receptor comprises a chimeric fusion protein comprising an extracellular domain, an extracellular hinge domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory molecule signaling domain derived from a stimulating molecule. ..
  • the chimeric receptor comprises an extracellular domain, an extracellular hinge domain, a transmembrane domain, a co-stimulatory molecule signaling domain derived from a co-stimulating molecule, and a functional signaling domain derived from a stimulating molecule.
  • a chimeric fusion protein containing an intracellular signaling domain.
  • the chimeric receptor comprises an extracellular domain, a transmembrane domain, and two functional signaling domains from one or more costimulatory molecules and a functional signaling domain from a stimulatory molecule.
  • a chimeric fusion protein comprising an intracellular signaling domain.
  • the chimeric receptor is an extracellular domain, a transmembrane domain, and at least two costimulatory molecule signaling domains derived from one or more costimulatory molecules, a stimulatory molecule signaling domain derived from a stimulatory molecule.
  • chimeric fusion proteins including and other functional domains and / or intracellular signaling domains containing motifs.
  • i) select from the extracellular domain capable of binding to the immunoreceptor recognition site of the antigen-binding molecule, ii) the transmembrane domain, and ii) the cytoplasmic costimulatory domain and / or the cytoplasmic domain of the interleukin receptor chain.
  • An intracellular segment comprising one or more intracellular signaling domains and a CD3 zeta intracellular signaling domain comprising an extrinsic STAT3-related motif (where the intracellular segment is endogenous or extrinsic).
  • a chimeric receptor comprising a sex JAK binding motif and a STAT5-related motif is disclosed.
  • these domains are optionally fused directly or indirectly, starting at the N-terminus and in the above order.
  • these domains within the intracellular segment are fused in reverse order.
  • an extracellular hinge domain and a transmembrane domain may be included between the extracellular domain and the intracellular signaling domain.
  • the term "extracellular hinge domain” means a domain that connects an extracellular domain with a transmembrane domain.
  • the extracellular hinge domain is not particularly limited as long as it can connect the extracellular domain and the transmembrane domain. It may be derived from a natural protein or it may be artificially designed.
  • the extracellular hinge domain can be composed of, for example, about 10 to 300 amino acids, preferably about 20 to 100 amino acids. It is preferable that the extracellular hinge domain does not interfere with the ability of the extracellular domain to bind to the antigen-binding molecule of the present disclosure and does not interfere with signal transduction via the intracellular signal transduction domain.
  • transmembrane domain is not particularly limited as long as it is a polypeptide that is located between the extracellular domain and the intracellular signal transduction domain and has a function of penetrating the cell membrane.
  • the transmembrane domain may be derived from a natural protein or may be artificially designed.
  • Transmembrane domains derived from native proteins can be obtained from any membrane binding protein or transmembrane protein.
  • the chimeric receptor comprises an additional leader sequence at the amino terminus (N-terminus) of the chimeric receptor fusion protein. In one embodiment, the chimeric receptor further comprises a leader sequence at the N-end of the extracellular domain, where the leader sequence is extracellular during cell processing and localization of the chimeric receptor to the cell membrane. You may be disconnected from the domain.
  • immunocytes are a receptor expressed on immune cells and means a receptor involved in the activation or suppression of immune cells.
  • immunocytes include T cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, NK cells or hematopoietic cells (neutrophils, basophils) and the like.
  • the chimeric receptor has an extracellular domain of an immune receptor or a variant thereof as an extracellular domain.
  • the immunoreceptor is preferably a receptor involved in the activation of immune cells, for example, a costimulatory molecule belonging to the tumor necrosis factor receptor superfamily (TNFRSF), specifically, CD137, CD40, OX40, RANK, and the like. GITR and the like can be mentioned.
  • the immune receptor may be a stimulating molecule or a co-stimulating molecule, and specific examples thereof will be described later.
  • the immunoreceptor recognition site of the antigen-binding molecule in the present invention can recognize the extracellular domain of the immunoreceptor or a fragment thereof contained in the extracellular binding domain of the chimeric receptor. More preferably, the immunoreceptor recognition site of the antigen-binding molecule recognizes a portion different from the binding site of the endogenous ligand.
  • the endogenous ligand binding site on the immune receptor is identified, for example, by structural biology analysis described in the literature. For example, in CD137, the X-ray crystal structure of the complex with CD137L has been clarified (Chin SM et al (2016) Nat Communi. 9, 4679), F36 on CRD1, P49, S52, Q59 on CRD2.
  • the extracellular domain of the chimeric receptor comprises the extracellular domain of a co-stimulator molecule belonging to TNFRSF or a fragment thereof, and the immunoreceptor recognition site of the antigen-binding molecule is an agonist antibody against the co-stimulator molecule belonging to TNFRSF. (Hereinafter referred to as an agonist antibody of TNFRSF) or an antigen-binding fragment thereof can be used.
  • the antigen-binding molecule used as the pharmaceutical composition of the present invention has at least about 20%, 30%, 40%, 50%, 60%, 70% of the activity of the chimeric receptor expressing cells. Activate 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 750%, 1000% or more be able to.
  • the extracellular domain of the chimeric receptor comprises a target molecule or fragment thereof of an agonist antibody of TNFRSF.
  • the target molecule of the agonist antibody of TNFRSF is not particularly limited as long as it is a factor that activates cells expressing the TNF receptor superfamily (for example, T cells and NK cells).
  • Preferred factors include, for example, CD137 and CD40. Further preferred factors include, for example, CD137.
  • examples of the CD137 agonist antibody include Urrelumab (CAS Registry Number: 934823-49-1) and various known CD137 agonist antibodies.
  • the extracellular domain of the chimeric receptor comprises an epitope of an antibody having agonist activity against the immune receptor as a fragment of the immune receptor.
  • immune receptors are as described above, preferably CD137.
  • the extracellular domain of the chimeric receptor comprises a target molecule or fragment thereof of a CD137 agonist antibody.
  • CD137 agonist antibodies include, for example, the following antibodies set forth in WO2015 / 156268, such as by SEQ ID NO:: [1] An antibody having the amino acid sequence set forth in SEQ ID NO: 66 as a heavy chain variable region and the amino acid sequence set forth in SEQ ID NO: 85 as a light chain variable region; [2] An antibody having the amino acid sequence set forth in SEQ ID NO: 67 as a heavy chain variable region and the amino acid sequence set forth in SEQ ID NO: 86 as a light chain variable region; [3] An antibody having the amino acid sequence set forth in SEQ ID NO: 70 as a heavy chain variable region and the amino acid sequence set forth in SEQ ID NO: 89 as a light chain variable region; [4] An antibody having the amino acid sequence set forth in SEQ ID NO: 76 as a heavy chain variable region and the amino acid sequence set forth in SEQ ID NO: 85 as
  • equivalent activity means that the agonist activity on CD137 is 70% or more, preferably 80% of the binding activity of the antibody described in any of the above [1] to [7]. The above is more preferably 90% or more.
  • an antibody that binds to the same epitope as the epitope to which the antibody described in any of the above [1] to [7] binds for example, an antibody that recognizes a region having a sequence of SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC in the CD137 protein can be mentioned. can. Furthermore, an antibody that recognizes a region having a sequence of DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC in the CD137 protein can be mentioned.
  • examples of the extracellular hinge domain include the extracellular hinge domains of CD8 alpha, CD8 beta, CD28, CD4, NKp30, NKp44, and NKp46. You may also use the hinge region of an immunoglobulin (eg, IgG4, etc.).
  • an immunoglobulin eg, IgG4, etc.
  • the proteins from which the transmembrane domain is derived include, for example, alpha and beta chains of T cell receptors, CD3 zetas, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 alpha, CD8 beta, CD9, etc.
  • CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, NKp30, NKp44, NKp46 and the like can be mentioned.
  • the protein from which the transmembrane domain is derived is CD8alpha or CD28.
  • signal transduction domain is used to regulate cell activity via defined signaling pathways by producing a second messenger or by functioning as an effector in response to such a messenger.
  • signal transduction domain refers to the functional part of a protein that acts by transmitting information within.
  • Intracellular signaling domain refers to the intracellular portion of a molecule.
  • the intracellular signal transduction domain can generate a signal that promotes the immune effector function of a cell containing a chimeric receptor, for example, a chimeric receptor-expressing T cell.
  • immune effector functions in chimeric receptor-expressing T cells include cell lytic activity and helper activity, such as cytokine secretion.
  • an intracellular signaling domain is a portion of a protein that transmits an effector function signal and causes the cell to perform a specialized function. The entire intracellular signaling domain may be employed, but in many cases it is not always necessary to use the entire chain.
  • intracellular signaling domain As long as the truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of an intact chain as long as it transmits an effector function signal.
  • intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit effector function signals.
  • the intracellular signaling domain may include a primary intracellular signaling domain.
  • Typical primary intracellular signaling domains include those derived from molecules involved in primary stimulation or antigen-dependent simulation.
  • the intracellular signaling domain may comprise a co-stimulating intracellular domain.
  • Typical intracellular signal transduction domains include co-stimulation signals or those derived from molecules involved in antigen-independent stimulation.
  • the primary intracellular signaling domain may comprise the intracellular sequence of the T cell receptor, and the co-stimulating intracellular signaling domain may be a co-receptor or co-receptor. It may contain intracellular sequences from stimulatory molecules.
  • CD3 zeta means all mammalian species, preferably human differentiation antigen group 3 (CD3) T cell co-receptors.
  • CD3 comprises a CD3 zeta chain, a CD3 delta chain and two CD3 epsilon chains.
  • the CD3 zeta chain (eg, NCBI RefSeq: NP_932170.1) comprises an intracellular signaling domain that can be used to manipulate the chimeric receptor.
  • the primary signaling sequence of the CD3-zeta is the full length or part of the cytoplasmic region sequence (nucleotide sequence 299-634) of Genbank NM000734.3, or a non-human species, eg. Equivalent residues from mice, rodents, monkeys, apes and the like.
  • the intracellular signaling domain may include the cytoplasmic domain of the interleukin receptor chain.
  • the intracellular signaling domain may be CD28, 4-1BB, ICOS, or CD3 zeta-CD28-4-1BB or CD3 zeta-CD28-OX40 in which multiple signaling domains are linked.
  • the protein from which the transmembrane domain is derived is CD8alpha or CD28
  • the intracellular signaling domain is CD28, 4-1BB, ICOS, or a CD3 zeta linking multiple signaling domains. It may be CD28-4-1BB or CD3 zeta-CD28-OX40.
  • i) select from the extracellular domain capable of binding to the immunoreceptor recognition site of the antigen-binding molecule, ii) the transmembrane domain, and ii) the cytoplasmic costimulatory domain and / or the cytoplasmic domain of the interleukin receptor chain.
  • An intracellular segment comprising one or more intracellular signaling domains and a CD3 zeta intracellular signaling domain comprising an extrinsic STAT3-related motif (where the intracellular segment is endogenous or extrinsic).
  • a chimeric receptor comprising a sex JAK binding motif and a STAT5-related motif is disclosed.
  • these domains are optionally fused directly or indirectly, starting at the N-terminus and in the above order.
  • these domains within the intracellular segment are fused in reverse order.
  • stimulation is a primary induced by binding of a stimulating molecule (eg, a TCR / CD3 complex or chimeric receptor) to its ligand (or, in the case of a chimeric receptor, the immunoreceptor recognition site of an antigen-binding molecule).
  • a stimulating molecule eg, a TCR / CD3 complex or chimeric receptor
  • ligand or, in the case of a chimeric receptor, the immunoreceptor recognition site of an antigen-binding molecule.
  • signal transduction events such as, but not limited to, signal transduction via the TCR / CD3 complex, or signal transduction via the appropriate NK receptor or signaling domain of the chimeric receptor. Be mediated. Stimulation may also mediate altered expression of certain molecules.
  • the term "stimulatory molecule” refers to an immune cell, such as a T cell, NK cell or Refers to molecules expressed by B cells.
  • the signal is, for example, a primary signal initiated by the binding of the TCR / CD3 complex to the MHC molecule presenting the peptide, whereby, but not limited to, proliferation, activation, differentiation, and homologous. Mediation of T cell responses such as those occurs.
  • Primary cytoplasmic signaling sequences that act in the form of stimuli may contain signaling motifs, which are immunoreceptor tyrosine-based activations. Known as a motif or ITAM.
  • ITAMs containing intracellular signaling sequences having specific uses in the present disclosure include, but are not limited to, CD3 zetas, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), and the like. Derived from CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 (“ICOS”), Fc epsilon RI, CD66d, CD32, DAP10, DAP12, CLEC2, CLEC7A (Detectin1), CLEC9A, EZRIN, RADIXIN and MOESIN. Things can be mentioned.
  • the intracellular signaling domain in any one or more chimera receptors of the present disclosure comprises an intracellular signaling sequence, eg, a CD3-zeta primary signaling sequence.
  • the extracellular domain of the chimeric receptor of the present invention may be the extracellular domain of the above-mentioned stimulating molecule.
  • co-stimulatory molecule mediates a co-stimulatory response by T cells, such as, but not limited to, by specifically binding to a co-stimulatory ligand (secondary signal) of the same origin on T cells.
  • co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response.
  • co-stimulatory intracellular signaling domain refers to the intracellular portion of a co-stimulatory molecule.
  • the intracellular signaling domain may include the entire intracellular portion, or the entire native intracellular signaling domain of the molecule from which it is obtained, or a functional fragment or derivative thereof.
  • the costimulatory molecule is not limited to these, but is limited to MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), and activated NK cell receptor.
  • BTLA Toll ligand receptor
  • OX40 CD134
  • the co-stimulator molecule is selected from, for example, 4-1BB (ie CD137), CD27, CD28 and / or OX40 to enhance T cell receptor stimulation.
  • 4-1BB or "CD137” is a member of the TNFR superfamily having the sequence amino acid sequence provided as GenBank Accession No. AAA6268.2, or non-human species such as mice, rodents, monkeys, apes and allogeneic.
  • the "4-1BB co-stimulating domain" is the full length, or part or non-human species of the Genbank NM001561.5 nucleotide sequence 886-1026, such as mice, rodents, monkeys, apes and the like. Equivalent residue from.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and their polymers in either single- or double-stranded form.
  • nucleic acid includes a gene, cDNA or mRNA.
  • the nucleic acid molecule is a synthetic (eg, chemically synthesized) nucleic acid molecule or a recombinant nucleic acid molecule.
  • the term includes nucleic acids containing analogs or derivatives of natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequences are also defined as their conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences.
  • degenerate codon substitutions are sequences in which the third position of one or more selected (or all) codons is replaced with a hybrid base and / or deoxyinosine residue. May be achieved by generating [Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)].
  • nucleic acid sequence means a sequence of a nucleoside or nucleotide monomer consisting of a natural base, sugar and sugar-to-sugar (main chain) bond.
  • the term also includes modified or substituted sequences comprising unnatural monomers or parts thereof.
  • the nucleic acid sequence of the present application can be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA) and includes natural bases including adenine, guanine, cytosine, thymine and uracil. These sequences may also contain modified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymine and uracil; as well as xanthines and hypoxanthines.
  • isolated nucleic acid is a cellular material or culture medium when produced by recombinant DNA technology, or a chemical precursor or other chemistry when chemically synthesized. Means a nucleic acid that is substantially free of substances. The isolated nucleic acid is also substantially free of sequences that are naturally adjacent to the nucleic acid from which it is derived (ie, sequences located at the 5'and 3'ends of the nucleic acid).
  • nucleic acid is intended to include DNA and RNA, which may be double-stranded or single-stranded and corresponds to the sense or antisense strand.
  • nucleic acid includes complementary nucleic acid sequences, such as cDNA.
  • coding refers to the synthesis of other polymers and polymers in biological processes that have either a defined sequence of nucleotides (eg, rRNA, tRNA and mRNA) or a defined sequence of amino acids.
  • a defined sequence of nucleotides eg, rRNA, tRNA and mRNA
  • a defined sequence of nucleotides eg, rRNA, tRNA and mRNA
  • a defined sequence of amino acids e.g, rRNA, tRNA and mRNA
  • the nucleotide sequence is identical to the mRNA sequence, and both the coding strand normally shown in the sequence listing and the non-coding strand used as a template for transcription of the gene or cDNA are proteins or other of the gene or cDNA. It can be referred to as encoding the product.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate from each other or that encode the same amino acid sequence. Also, the phrase nucleotide sequence encoding a protein or RNA may include introns to the extent that the nucleotide sequence encoding the protein may contain introns in some cases. Also, the nucleic acid molecule can be operably linked to at least one regulatory element for expression of the chimeric receptor.
  • peptide refers to a compound composed of amino acid residues covalently linked by a peptide bond.
  • the protein or peptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can make up the sequence of the protein or peptide.
  • Polypeptides include any peptide or protein containing two or more amino acids combined with each other in a peptide bond.
  • short chains commonly referred to in the art as, for example, peptides, oligopeptides and oligomers, and proteins in which many types generally exist in the art. Refers to both with longer chains.
  • polypeptide examples include biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, and modified polys. Examples include peptides, derivatives, analogs and fusion proteins. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.
  • the polypeptide in the present disclosure usually refers to a peptide and a protein having a length of about 10 amino acids or more.
  • a chain of amino acids connected by a peptide bond from the N-terminal to the C-terminal is a series of peptide chains
  • a plurality of series of peptide chains interact with each other such as SS bond, hydrophobic interaction, and ionic bond. It may be a complex protein formed by.
  • isolated polypeptide also referred to as “isolated protein” is a cellular material or culture medium when produced by recombinant DNA technology, or a chemical precursor when chemically synthesized. Means a polypeptide that is substantially free of the body or other chemicals.
  • amino acid includes all natural and modified amino acids.
  • a “conservative amino acid variation”, when used in the present disclosure, is one in which one amino acid residue is replaced with another amino acid residue without compromising the desired properties of the protein.
  • conservative sequence modification refers to an amino acid mutation that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present disclosure by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • a conservative amino acid substitution is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • a family of amino acid residues with similar side chains is defined in the art.
  • amino acids with basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine, asparagine, glutamine, serine.
  • Threonine, tyrosine, cysteine, tryptophan non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains.
  • Includes eg, tyrosine, phenylalanine, tryptophan, histidine).
  • one or more amino acid residues within the chimeric receptor of the present disclosure may be replaced with other amino acid residues of the same side chain family, and the modified chimeric receptor is described herein. Can be tested using a functional assay.
  • expression refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.
  • host cell used interchangeably and have foreign nucleic acid-introduced cells (including progeny of such cells).
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived from those cells regardless of passage number.
  • the progeny do not have to be exactly the same in the content of the parent cell and nucleic acid and may contain mutations.
  • mutant offspring having the same function or biological activity as those used when the original transformed cells were screened or selected.
  • vector in the present disclosure refers to a nucleic acid molecule capable of increasing another nucleic acid to which it is linked.
  • the term includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. Certain vectors can result in the expression of nucleic acids to which they are operably linked. Such vectors are also referred to herein as "expression vectors.”
  • Transfection means the uptake of an expression vector by a host cell in which it is not known whether any coding sequence is actually expressed. Many methods of transfection are known to technicians with conventional skills, such as the CaPO 4 sedimentation method and the electroporation method. Successful transfections are generally recognized when signs of action of the vector appear in the host cell.
  • promoter refers to a DNA sequence recognized by a cellular synthetic mechanism or an introduced synthetic mechanism required to initiate specific transcription of a polynucleotide sequence.
  • lentivirus refers to one of the genus of the retrovirus family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, which are gene delivery vectors because they can deliver a significant amount of genetic information to the DNA of the host cell. Is one of the most efficient methods of. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of the lentiviral genome, in particular the self as shown in Milone et al., Mol. Ther. 17 (8): 1453-1464 (2009). Examples include inactivated lentiviral vectors. Other examples of lentiviral vectors that may be used in clinical facilities are, but are not limited to, for example, Oxford BioMedica's LENTIVECTOR® gene delivery technology, Lentien's LENTIMAX TM vector system. And similar ones. Non-clinical type lentiviral vectors are also available and the selection and preparation of the vector can be performed by those of skill in the art as appropriate.
  • the term "pharmaceutically acceptable” is such a composition that, when administered to a mammal (eg, human), is physiologically tolerant and typically does not produce an undesired reaction.
  • a mammal eg, human
  • pharmaceutically acceptable refers to the molecular entity of an object and other components.
  • "pharmaceutically acceptable” in the present disclosure has been approved by federal or state regulatory authorities or is recognized by the United States Pharmacopeia or other generally for use in mammals, more specifically in humans. It means that it is listed in the Pharmacopoeia.
  • the chimeric receptor of the present disclosure comprises the two signaling domains described herein, namely the CD3 zeta and 4-1BB / CD137 or CD3 zeta plus one or more signaling domains.
  • several signaling domains are fused together for additive or synergistic action.
  • useful additional signaling domains are TCR zeta chains, CD27, CD28, OX40 / CD134, 4-1BB / CD137, Fc epsilon RIy, ICOS / CD278, ILRB / CD122, IL-2RG / CD132, Includes one or more parts or all of DAP-10 and CD40.
  • the extracellular domain capable of binding to a predetermined antigen via an antigen-binding molecule
  • ii) the transmembrane domain and iii) the intracellular syntactic domain and / or the intracellular domain of the interleukin receptor chain.
  • An intracellular segment comprising one or more intracellular signaling domains and a CD3 zeta intracellular signaling domain comprising an exogenous STAT3-related motif (where, said intracellular segment is intracellular or extrinsic).
  • a chimeric receptor comprising (contains a JAK binding motif and a STAT5-related motif) is disclosed.
  • these domains are optionally fused directly or indirectly, starting at the N-terminus and in the above order.
  • these domains within the intracellular segment are fused in reverse order.
  • extracellular domain that can bind via an antigen-binding molecule
  • transmembrane domain iii) the transmembrane domain
  • cytoplasmic domain of the interleukin (IL) receptor chain optionally at least one complementary cytoplasm.
  • a chimeric receptor comprising an intracellular segment comprising one or more intracellular signaling domains comprising a domain.
  • these domains are optionally fused directly or indirectly, starting at the N-terminus and in the above order.
  • these domains within the intracellular segment are fused in reverse order.
  • the IL receptor chain is proximal to the transmembrane domain and / or near or forms the N-terminus of the chimeric receptor intracellular segment. In other embodiments, the IL receptor chain is close to or forms the C-terminus of the intracellular segment of the chimeric receptor. In some embodiments, the IL receptor chain is upstream or N-terminal to the CD3 zeta intracellular signaling domain comprising the exogenous STAT3-related motif YXXQ.
  • the chimeric receptor expressing cell is via endogenous TCR by a given antigen present in the MHC complex and / or endogenous. It can be activated by CD80 / 86 molecules via CD28, eg, by B cells.
  • cells expressing the chimeric receptors of the present disclosure are higher relative to cells having a predetermined / preselected antigen on their surface to which the chimeric receptor binds via an antigen binding molecule, for example, compared to parent cells that do not express the chimeric receptor.
  • cells expressing the chimeric receptor receptor of the present disclosure exert an antigen-binding molecule-dependent and cancer antigen-tumor antigen-dependent antitumor effect by administering an antigen-binding molecule. offer. Therefore, antitumor effect in humans is also expected.
  • the chimeric receptor of the present disclosure In the cells expressing the chimeric receptor of the present disclosure, it is sufficient that the chimeric receptor of the present disclosure is expressed, and other transductions may be carried out.
  • the intracellular segment of the chimeric receptor according to the present disclosure may contain one or more intracellular signaling domains, and when the extracellular domain existing in the same molecule binds (interacts) with its cognate antigen / ligand. It is a proteinaceous molecule that can transmit signals to cells.
  • the chimeric receptor intracellular segment comprises a CD3 zeta intracellular signaling domain comprising an exogenous STAT3-related motif.
  • the intracellular segment of the chimeric receptor comprises one or more intracellular signaling domains selected from the cytoplasmic domain and / or the cytoplasmic costimulatory domain of the IL receptor chain, said intracellular segment being endogenous.
  • it comprises an extrinsic JAK binding motif and a STAT5-related motif.
  • the primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex.
  • the CD3 zeta intracellular signaling domain provides the primary cytoplasmic signal.
  • the primary cytoplasmic signaling sequence may include a signaling motif known as the immunoreceptor tyrosine activation motif (ITAM) [Nature, vol. 338, pp. 383-384 (1989)].
  • ITAM immunoreceptor tyrosine activation motif
  • ITIM immune receptor tyrosine inhibitory motif
  • intracellular signal transduction domains having ITAM and / or ITIM can be used.
  • the intracellular domain of the CD3 zeta comprises an immunoreceptor tyrosine activation motif (ITAM).
  • ITAM immunoreceptor tyrosine activation motif
  • Examples of intracellular signaling domains with available ITAMs are, for example, in place of or in place of the CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a. , CD79b, and intracellular signaling domain with ITAM derived from CD66d.
  • examples of an intracellular domain containing one or more ITAMs include amino acid numbers 52 to 164 of the CD3 zeta (NCBI RefSeq: NP_932170.1), Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1). Amino acid numbers 45-86, Fc epsilon RI beta (NCBI RefSeq: NP_000130.1) amino acid numbers 201-244, CD3 gamma (NCBI RefSeq: NP_00000461.1) amino acid numbers 139-182, CD3 delta (NCBI RefSeq: NP_000723).
  • the amino acid residue represented by "X" in the STAT3-related motif YXXQ can be any natural amino acid, including any modified natural amino acid that retains a STAT3 bond.
  • the amino acid X is independently selected from leucine, arginine, histidine, phenylalanine, lysine, proline, methionine, valine, glutamine, threonine, aspartic acid.
  • the amino acid X is arginine.
  • the amino acid X is histidine.
  • the two amino acid residues flanking the tyrosine residue of the STAT3-related motif YXXQ are arginine-histidine.
  • the exogenous STAT3-related motif is YRHQ.
  • the extrinsic STAT3-related motif YXXQ may be introduced into any part of the intracellular domain of the CD3 zeta, but in certain embodiments, the YXXQ-related motif is inserted near the C-terminal region.
  • many endogenous YXXQ motifs are thought to be located near or within 100aa from the C-terminus.
  • GP130 and LIFR studies have shown that the YXXQ motif near the C-terminal region is more functional when it is more proximal (Schmitz J et al. J Immunol. 2000; 164: 848-54). ; Tomida M et al. Blood. 1999; 93: 1934-41).
  • the exogenous STAT3-related motif YXXQ is introduced into any portion of the intracellular domain of the CD3 zeta within the 200 amino acid residue from the C-terminus of the chimeric receptor.
  • STAT3-related motifs are within 200 amino acid residues, within 150 amino acid residues, within 100 amino acid residues, within 90 amino acid residues, within 80 amino acid residues, within 70 amino acid residues, from the C end of the chimeric receptor. It is introduced into 60 amino acid residues, 50 amino acid residues, 40 amino acid residues, 30 amino acid residues, 20 amino acid residues or 10 amino acid residues.
  • the exogenous STAT3-related motif is introduced at a location other than ITAM.
  • the CD3 zeta intracellular domain comprising an extrinsic STAT3-related motif comprises at least one ITAM motif. In one embodiment, the CD3 zeta intracellular domain comprising an exogenous STAT3-related motif comprises two ITAM motifs. In a further embodiment, the CD3 zeta intracellular domain comprising an exogenous STAT3-related motif comprises three ITAM motifs.
  • the extrinsic STAT3-related motif is the amino acid residue Leu-His-Met at amino acid residues 104-106 of the intracellular signaling domain of the CD3 zeta to tyrosine at residue 104 and its tyrosine residue at 105 and It can be introduced by substituting with any other two amino acid residues adjacent at number 106.
  • the amino acid residue 104-105-106 of the intracellular signal transduction domain of the CD3 zeta corresponds to the amino acid residue 156-157-158 of the full-length CD3 zeta (eg, NCBI RefSeq: NP_932170.1).
  • the chimeric receptor in certain embodiments comprises an intracellular segment comprising an IL receptor chain and one or more intracellular signaling domains selected from the cytoplasmic domain of the cytoplasmic co-stimulation domain.
  • the cytoplasmic domain of the IL receptor chain can be selected from any chain of IL receptors, eg, IL-2 receptor beta chain (NCBI REFSEQ: NP_000869.1) (IL-21 receptor). Amino acid numbers 266 to 551 of the alpha chain (NCBI REFSEQ: NP_06857.1) amino acid numbers 256 to 538, amino acid numbers 284 to 369 of the common IL-2 receptor gamma chain (NCBI REFSEQ: NP_000197.1), IL-7R.
  • NCBI REFSEQ: NP_000869.1 IL-2 receptor beta chain
  • IL-21 receptor IL-21 receptor
  • a cytoplasmic domain comprising 257-825 can be used. The entire region of the cytoplasmic domain of the IL receptor chain is available.
  • a terminal cleavage fragment of the cytoplasmic domain of the IL receptor (ILR) chain can also be used.
  • the terminal cleavage fragment comprises up to 250 amino acids of the ILR cytoplasmic domain, or is 50-200 amino acids or 80-150 amino acids.
  • the cytoplasmic domain of the IL receptor chain and optionally the end-cleaving fragment of said cytoplasmic domain of the IL receptor chain, is at least a STAT-related motif, optionally a STAT5-related motif, and a JAK-binding motif (box-1). Also known as a motif).
  • the cytoplasmic domain of the IL receptor chain or a terminal cleavage fragment thereof comprises a STAT5-related motif and a JAK-binding motif.
  • the cytoplasmic domain and / or end-cleavage fragment of the IL receptor chain is a variant that induces a variant having the same function, eg, STAT signaling, and optionally STAT5 signaling and / or JAK signaling.
  • the body is included.
  • the cytoplasmic domain of the IL-2 receptor (IL-2R) beta chain can be used.
  • the IL-2R beta chain cytoplasmic domain that can be used in the present disclosure include amino acid numbers 266-551 of the IL-2R beta chain (NCBI RefSeq: NP_000869.1).
  • a peptide having any of the sequences of amino acids 266-337 and 530-551 is included.
  • a terminal cleavage fragment of the cytoplasmic domain of the IL-2R beta chain can be used.
  • This terminal cleavage fragment is i) a JAK binding motif (eg, NCBI RefSeq: NP_000869.1 amino acid numbers 278-286), also called a BOX-1 motif, which allows association with the tyrosine kinase JAK1, and ii) STAT. It may include related motifs, and optionally STAT5 or STAT3 related motifs. Other parts of the IL receptor chain can be modified, for example, by conservative amino acid variations.
  • the intracellular segment may contain an exogenous JAK binding motif, or a signaling molecule comprising a JAK binding motif.
  • the JAK binding motif is on IL2R gamma (IL2RG), erythropoietin receptor (EpoR), thrombopoietin receptor (TpoR), granulocyte macrophage colony stimulator receptor (GM-CSFR), and growth hormone receptor (GHR).
  • the IL-2R beta chain comprises three functional STAT5 binding motifs used for STAT5 association, YFFF, YCTF and YLSL. Mutations in these tyrosine residues can negate the IL-2 reactivity of the IL-2R beta chain (Friedmann et al., 1996).
  • the erythropoietin receptor (EpoR) comprises two tyrosine residues that mediate STAT5 activation, namely Y343 and Y401, both of which have been described to have a YXXL motif (Klingmuller et al., 1996). ). Therefore, YXXL can be a preferred motif for mobilization of STAT5.
  • the STAT5-related motif is an IL-2R beta chain STAT5-related motif and comprises tyrosine residue-510 (tyrosine residue 510 is amino acid number 536 of NCBI RefSeq: NP_000869.1). Become.
  • the STAT5-related motifs can be derived from IL2R gamma, EpoR, TpoR, GM-CSFR and GHR.
  • the STAT5-related motif of the IL-2R beta chain comprises the amino acid residue YXXL.
  • the amino acid residue represented by "X" in the STAT5-related motif can be any natural amino acid, including any modified natural amino acid that retains a STAT5 bond.
  • the intracellular segment comprises one or more JAK binding motifs that can be placed or introduced into any of the intracellular signaling domains.
  • the cytoplasmic domain of the IL-21 receptor (IL-21R) alpha chain can be used.
  • the cytoplasmic domain of the IL-21R alpha chain used in the present disclosure include an intracellular signaling domain comprising amino acid numbers 256-538 (NCBI RefSeq: NP_0685710.) Of the IL-21R alpha chain. ..
  • a terminal cleavage fragment of the cytoplasmic domain of the IL-21R alpha chain can be used.
  • the terminally cleaved fragment contains a box-1 motif (NCBI RefSeq: amino acid numbers 266-274 of NP_068570.1) required for association with the tyrosine kinase JAK1 and also contains STAT-related motifs.
  • the STAT-related motif is on the tyrosine residue-500 (NCBI RefSeq: amino acid number 519 of NP_000869.1) and the tyrosine residue 500 required for STAT1 / 3 association, i.e., on the C-terminal side of YLRQ. It contains 3 adjacent residues.
  • intracellular signaling domains include cytoplasmic regions derived from TCR complexes and / or costimulatory molecules, and any variants having the same function as their sequences.
  • Other examples include the cytoplasmic signaling domains listed in Table 2 of Sadelain et al 2009, which is incorporated herein by reference.
  • Activation of native T cells induces antigen-dependent primary activation (eg, provided by the CD3 zeta, eg, primary cytoplasmic signal) via two different types of intracellular signaling domains, namely the TCR complex. And transmitted by a domain that acts antigen-independently to provide a secondary or co-stimulating signal (secondary cytoplasmic signal).
  • antigen-dependent primary activation eg, provided by the CD3 zeta, eg, primary cytoplasmic signal
  • secondary cytoplasmic signal secondary cytoplasmic signal
  • amino acid numbers 207-235 of CD8 alpha (NCBI RefSeq: NP_0017599.3), amino acid numbers 196-210 of CD8 beta (GenBank: AAA3564.1), amino acid numbers 180 of CD28 (NCBI RefSeq: NP_006130.1).
  • ⁇ 220 Amino acid numbers 214 to 255 of CD137 (4-1BB, NCBI RefSeq: NP_001552.2), Amino acid numbers 241 to 277 of CD134 (OX40, NCBI RefSeq: NP_003318.1), and ICOS (NCBI RefSeq: NP_036224. ) Includes peptides having any of the sequences of amino acids 166-199, and variants thereof having the same function as those of these peptides.
  • an intracellular segment having one or more, eg, 2 or 3 intracellular signaling domains, in addition to the intracellular signaling domain of the CD3 zeta comprising an extrinsic STAT3-related motif.
  • a chimeric receptor comprising.
  • the present disclosure also includes a chimeric receptor comprising two or more intracellular segments having the same intracellular signaling domain in series.
  • the present disclosure is a chimeric receptor in which the cytoplasmic domain of the IL receptor is on the N-terminal side of the intracellular signaling domain of the CD3 zeta, ie, the cytoplasmic domain of the IL receptor and the intracellular signaling of the CD3 zeta.
  • the domain provides a chimeric receptor comprising those linked in this order from the N-terminal side.
  • the present disclosure also presents the chimeric receptor obtained by further adding the intracellular domain of CD28 (eg, the cytoplasmic costimulatory domain of CD28) to the chimeric receptor described above, i.e., the intracellular signaling domain of CD28 and IL. It also includes a chimeric receptor comprising the cytoplasmic domain of the receptor and the intracellular signaling domain of the CD3 zeta comprising the extrinsic STAT3 motif linked in this order from the N-terminal side.
  • CD28 eg, the cytoplasmic costimulatory domain of CD28
  • the chimeric receptor comprises a CD3 zeta intracellular signal comprising an extrinsic STAT3-related motif and an intracellular signaling domain selected from the cytoplasmic and cytoco-stimulatory domains of the interleukin receptor chain. It comprises an intracellular segment comprising a transduction domain, wherein at least one of the intracellular signaling domains comprises an endogenous or extrinsic JAK binding motif and a STAT5-related motif.
  • the chimeric receptor is the cytoplasm of an IL receptor chain fragment comprising a CD3 zeta intracellular signaling domain with an extrinsic STAT3-related motif and an endogenous or extrinsic JAK binding motif and a STAT5-related motif. It comprises a domain and a cytoplasmic co-stimulatory domain of CD28.
  • an oligopeptide linker or polypeptide linker can be inserted between domains of an intracellular segment such that the domains are linked there and / or they are linked to other domains.
  • a linker having a length of 2 to 10 amino acids can be used.
  • a linker having a glycine-serine continuous sequence can be used.
  • the linker IDGGGGGSGGGGSGGGGS can be inserted between the CD28 cytoplasmic domain and the partial cytoplasmic IL-2 receptor beta domain.
  • the linker KLGGSGP can be inserted between the partially cytoplasmic IL-2 accepting beta domain and the intracellular domain of the CD3 zeta chain.
  • it comprises i) an extracellular domain capable of binding to the immunoreceptor recognition site of an antigen-binding molecule, ii) a transmembrane domain, and iii) a cytoplasmic domain of an interleukin receptor chain and optionally a complementary cytoplasmic domain.
  • a chimeric receptor comprising an intracellular segment, comprising one or more intracellular signaling domains, is provided.
  • the cytoplasmic domain of the IL receptor chain may be selected from any chain of IL receptors described herein. The entire region of the cytoplasmic domain of the IL receptor chain is available. Alternatively, terminal cleavage fragments of said cytoplasmic domain of the IL receptor chain can also be used. Examples of the full length and its terminally truncated fragments are shown herein.
  • the terminal cleavage fragment comprises at least one tyrosine kinase-related motif (also known as a box-1 motif) and a STAT (signal transduction transcriptional activator) -related motif described herein. May be good.
  • the terminal cleavage fragment comprises up to 250 amino acids of the ILR cytoplasmic domain, or is 50-200 amino acids or 80-150 amino acids.
  • the STAT-related motif of the IL-2R beta chain comprises tyrosine residue-510 (tyrosine residue 510 is amino acid number 536 of NCBI RefSeq: NP_000869.1).
  • the STAT-related motif comprises a tyrosine residue 510 and four residues flanking the C-terminal side of the tyrosine residue 510, i.e., YLSLQ.
  • STAT-related motifs are also known, such as IL-6 YXXQ, and in some cases YXPQ, IL-10 YXXQ, IL-12 YLPSNID, IL-2 YLSLQ, YCTFP, YFFFH, IL-7.
  • Any STAT signaling domain can be used and / or introduced into the ILR chain.
  • the chimeric receptor intracellular segment comprises at least one complementary signaling domain other than that present within the IL receptor.
  • intracellular signaling domains include cytoplasmic regions and / or costimulatory molecules from the TCR complex, and any variant having the same function as such a sequence.
  • Other examples include the cytoplasmic signaling domains listed in Table 2 of Sadelain et al 2009, which is incorporated herein by reference.
  • the present disclosure includes a chimeric receptor comprising an intracellular segment comprising one or more, eg, 2 or 3 intracellular signaling domains, in addition to the cytoplasmic domain of the IL receptor.
  • the chimeric receptor comprises the cytoplasmic domain of the IL receptor and the intracellular signaling domain of the CD3 zeta.
  • the chimeric receptor comprises the cytoplasmic domain of the IL receptor, the intracellular signaling domain of the CD3 zeta and the cytoplasmic co-stimulation domain of CD28.
  • the chimeric receptor comprises an intracellular segment comprising the CD3 zeta intracellular signaling domain, one or more cytoplasmic costimulatory domains, wherein said intracellular segment is a JAK binding motif, the intracellular segment.
  • the intracellular segment is a JAK binding motif, the intracellular segment.
  • the chimeric receptor of the present disclosure comprises a transmembrane domain.
  • the transmembrane domain may be derived from a natural polypeptide or may be artificially designed.
  • Transmembrane domains from native polypeptides can be obtained from membrane binding or transmembrane proteins.
  • a transmembrane domain of CD154, or GITR can be used.
  • transmembrane domains are predominantly polypeptides comprising hydrophobic residues such as leucine and valine.
  • phenylalanine, tryptophan and valine triplets can be found at each end of the synthetic transmembrane domain.
  • a short chain oligopeptide or polypeptide linker eg, a linker with a length of 2-10 amino acids, can be placed between the transmembrane domain and the intracellular segment described herein.
  • a linker sequence having a glycine-serine continuous sequence can be used.
  • transmembrane domain having any of the sequences of amino acid numbers 153 to 179 of CD28 (NCBI RefSeq: NP_006130.1) can be used as the transmembrane domain.
  • the spacer domain can be placed between the extracellular domain and the transmembrane domain, or between the intracellular segment and the transmembrane domain.
  • the spacer domain means any oligopeptide or polypeptide that serves to link the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular segment.
  • the spacer domain comprises up to 300 amino acids, eg, about 10-100 amino acids, or about 25-50 amino acids.
  • the spacer domain preferably has a sequence that promotes the binding of the chimeric receptor to the antigen via an antigen-binding molecule and enhances signal transduction to cells.
  • amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine within potential glycosylation sites, which should be used as the amino acids that make up the spacer domain. Can be done.
  • the spacer domain is amino acid number 118-178 of CD8 alpha (NCBI RefSeq: NP_0017599.3), i.e., the hinge region of CD8 alpha, amino acid numbers 135-195 of CD8 beta (GenBank: AAA3564.1).
  • a polypeptide comprising or consisting of amino acid numbers 315 to 396 of CD4 (NCBI RefSeq: NP_0000607.1), amino acids 114 to 152 of CD28 (NCBI RefSeq: NP_006130.1), or a portion thereof. ..
  • the spacer domain may be an artificially synthesized sequence.
  • the chimeric receptors of the present disclosure can be designed to form polymers, especially dimers.
  • cysteine is inserted into the spacer domain and / or transmembrane domain to polymerize (dimerize) the chimeric receptor, eg, via a disulfide bond.
  • the signal peptide sequence can be linked to the N-terminal.
  • the signal peptide sequence is present at the N-terminus of many secretory and membrane proteins and has a length of 15-30 amino acids.
  • Many of the protein molecules having the intracellular domain described herein are membrane proteins and have a signal peptide sequence.
  • Signal peptides derived from such secretory proteins and membrane proteins can be used as signal peptides for the chimeric receptors of the present disclosure. Any signal peptide can be used.
  • the signal peptide can be an oncostatin M signal peptide.
  • the signal peptide may be of human origin or may be of non-human origin, such as insect cells or viruses. In certain embodiments, the signal peptide is a human signal peptide.
  • the present disclosure provides nucleic acids encoding the chimeric receptors described herein.
  • the nucleic acid encoding the chimeric receptor can be easily prepared by a conventional method from the amino acid sequence of the indicated chimeric receptor.
  • the nucleotide sequence encoding the amino acid sequence can be obtained from the NCBI RefSeq ID or GenBank accession number described above for the amino acid sequence of each domain and is disclosed using standard molecular biological and / or chemical procedures.
  • Nucleic acids can be made. For example, nucleic acids can be synthesized based on nucleotide sequences, and the nucleic acids of the present disclosure can be prepared by combining DNA fragments obtained from a cDNA library using a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the nucleic acid of the present disclosure can be linked to another nucleic acid so that it is expressed under the control of a suitable promoter.
  • suitable promoters include the expression of genes or operably linked constructs by the action of promoters, drugs, etc. (eg, tetracycline or doxorubicin) that constitutively promote the expression of genes or operably linked constructs. Includes a promoter to induce.
  • the nucleic acids of the present disclosure can also be linked to nucleic acids comprising promoters or other regulatory elements that cooperate with transcription initiation sites, such as enhancer or terminator sequences, for efficient transcription of the nucleic acid. ..
  • a gene that can be a marker for confirming the expression of the nucleic acid may be incorporated.
  • the nucleic acid is a codon-optimized nucleic acid for expression in a particular host.
  • the method for producing a cell expressing the chimeric receptor of the present disclosure comprises the step of introducing a nucleic acid encoding the chimeric receptor described herein into the cell. This step is performed ex vivo.
  • cells can be transformed with a viral or non-viral vector carrying the nucleic acids of the present disclosure ex vivo to produce cells expressing the chimeric receptors of the present disclosure.
  • cells derived from mammals for example, human cells or cells derived from non-human mammals such as monkeys, mice, rats, pigs, horses, or dogs can be used.
  • the mammal is a human.
  • a chimeric receptor a nucleic acid encoding the chimeric receptor, a cell expressing the chimeric receptor, and a composition containing the cell are provided.
  • one or more of the above may be used in the field of adoptive immunity gene therapy targeting antigens such as tumor antigens and / or in screening or other in vitro assays.
  • the chimeric receptor of the present disclosure can be introduced into a cell, for example, to increase or increase the expression level of the chimeric receptor in the cell. Such cells can exert cytotoxic activity against cells expressing the target antigen.
  • Isolating immune cells from mammals in some cases, the immune cells are T cells
  • Isolated immune cells possibly T cells
  • transfected or transfected with nucleic acids encoding the chimeric receptors described herein and c) optionally transfected or transfected.
  • a method for producing cells expressing the chimeric receptor of the present disclosure which comprises later isolating and / or expanding the chimeric receptor expressing cells (optionally, chimeric receptor expressing T cells).
  • autologous T lymphocytes or autologous NK cells autologous macrophages are activated and / or proliferated in Exvivo prior to reintroduction into the subject.
  • the T lymphocytes or NK cells are allogeneic T lymphocytes or allogeneic NK cells.
  • the allogeneic T lymphocyte is a T lymphocyte in which the expression of the endogenous T cell receptor is blocked or eliminated.
  • allogeneic T lymphocytes or allogeneic NK cells are activated and / or proliferated in Exvivo prior to introduction into the subject.
  • the chimeric receptor is selected by a method selected from the group consisting of retrovirus transduction, lentivirus transduction, DNA electroporation and RNA electroporation, DNA or RNA transfection or gene modification by Gene editing. , T lymphocytes or NK cells or macrophages.
  • NK cells used in the methods of the present disclosure are genetically modified to express membrane-bound IL-15 and 4-1BB ligands (CDI37L), lacking or little expression of major histocompatibility antigens I and / or II molecules. It can be preferentially proliferated by exposure to the cells.
  • Such cell lines are K562 [ATCC, CCL 243; Lozzio et al., Blood 45 (3): 321-334 (1975); Klein et al., Int. J. Cancer 18: 421-431 (1976). ] And Wilms tumor cell line HFWT [Fehniger TA, Caligiuri M A.
  • the cell line used lacks or barely expresses both MHC I and II molecules, such as K562 and HFWT cell lines.
  • Solid supports can be used in place of cell lines.
  • Such a support preferably binds to NK cells and is capable of inducing an early activation event and / or a proliferative response or binding of a molecule having such an action, thereby acting as a scaffold.
  • At least one molecule is attached to its surface.
  • the support may have a CD137 ligand protein, a CD137 antibody, an IL-15 protein or an IL-15 receptor antibody bound to its surface.
  • the support has an IL-15 receptor antibody and a CD137 antibody bound to its surface.
  • T lymphocytes are one or more agents selected from the group consisting of anti-CD3 / CD28, IL-2 and phytohaemagglutinin. Can be activated in the presence.
  • the NK cells are CD137 ligand protein, CD137 antibody, IL-15 protein, IL-15 receptor antibody, IL-2 protein, IL-12 protein, IL-. It can be activated in the presence of one or more agents selected from the group consisting of 21 proteins and K562 cell lines.
  • the cells used in the method of the present disclosure are not particularly limited, and any cell can be used.
  • cells collected, isolated or purified from body fluids, tissues or organs such as blood (peripheral blood, umbilical cord blood, etc.) or bone marrow, or the above-mentioned cells that differentiate or pluripotent stem cells (iPSC).
  • the cells obtained by reprogramming for production can be used (see, for example, Themeli et al 2013).
  • PBMC Peripheral blood mononuclear cells
  • immune cells including, for example, T cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monospheres, NK cells or hematopoietic cells (neutrophils, basal spheres)
  • PBMC Peripheral blood mononuclear cells
  • Umbilical mononuclear cells, fibroblasts, adipose cell precursors, hepatocytes, cutaneous keratinocytes, mesenchymal stem cells, adipose stem cells, various cancer cell lines, or neural stem cells can be used.
  • NK cells or T cells T cell progenitor cells (hematopoietic stem cells, lymphocyte progenitor cells, etc.) or cell populations containing them can be used.
  • T cells include CD8-positive T cells, CD4-positive T cells, regulatory T cells, cytotoxic T cells, and tumor-infiltrating lymphocytes.
  • Cell populations containing T cells and T cell progenitor cells include PBMCs.
  • the above-mentioned cells may be collected from a living body, obtained by expanding culture of cells collected from a living body, or established as a cell line. If transplantation of the produced chimeric receptor-expressing cells or cells differentiated from the produced chimeric receptor-expressing cells into a living body is desired, the nucleic acid is introduced into the living body itself or cells collected from the same kind of living body. be able to.
  • the present disclosure also presents a vector comprising such a polynucleotide, a vector to which such a polynucleotide is operably linked to at least one regulatory element for expression of a chimeric receptor.
  • Including is also provided.
  • useful vectors of the present disclosure include, for example, viral vectors such as retroviral and lentiviral vectors.
  • such a vector also comprises a suicide gene.
  • suicide gene used here refers to a gene that causes the cells expressing the suicide gene to die.
  • a suicide gene can be a gene that sensitizes a cell in which the gene is expressed to a drug, eg, a drug, and causes the cell to die when it comes into contact with or is exposed to the drug. ..
  • Suicide genes are known in the art (eg, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J.
  • HSV simple herpesvirus
  • TK thymidine kinase gene
  • cytosine deaminase purine nucleoside phosphorylase
  • nitroreductase nitroreductase 8
  • the nucleic acid encoding the chimeric receptor of the present disclosure can be introduced into a vector, and the vector can be introduced into cells.
  • retrovirus vectors including oncoretrovirus vectors, lentivirus vectors, and pseudoviral vectors
  • adenovirus vectors including oncoretrovirus vectors, lentivirus vectors, and pseudoviral vectors
  • AAV adeno-associated virus
  • Simian virus vectors Wachsina virus vectors or Sendai virus vectors
  • Epstein-Bar Epstein-Bar
  • Viral vectors such as virus (EBV) vectors and HSV vectors can be used.
  • a viral vector lacking replication ability can be used so as not to self-replicate in infected cells.
  • non-viral vectors are also described in this disclosure in WO96 / 10038, WO97 / 18185, WO97 / 25329, WO97 / 30170 and WO97 / 31934 (which are incorporated herein by reference). As such, it can be used in combination with condensing agents such as liposomes or cationic lipids.
  • the nucleic acids of the present disclosure can also be introduced into cells by calcium phosphate transduction, DEAE-dextran, electroporation, or particle impact.
  • the methods of the present disclosure select suitable packaging cells based on the LTR sequence and the packaging signal sequence processed by the vector, and retrovirus using the packaging cells. It can be done by making particles.
  • packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP + E-86 and GP + envelopeAm-12 (US Pat. No. 5,278,056), and Psi-Crip [Proceedings of]. The National Academy of Sciences of the United States of America, vol. 85, pp. 6460-6464 (1988)] is included.
  • Retrovirus particles can also be made using 293 cells or 293T cells with high transfection efficiency. Many types of viral vectors produced on the basis of packaging cells that can be used to package retroviruses and retroviral vectors are widely marketed by many companies.
  • the present disclosure also provides a host cell containing the above chimeric receptor.
  • useful host cells include T lymphocytes and NK cells, which can be autologous or allogeneic (endogenous T cell receptors have been removed or retained).
  • the host cell is an autologous T lymphocyte isolated from a patient with cancer.
  • such autologous T lymphocytes are activated and proliferated in Exvivo.
  • the chimeric receptor of the present disclosure can be introduced into a host cell by any method known in the art.
  • particularly useful methods include retroviral transduction, lentiviral transduction and DNA and mRNA electroporation.
  • mRNA electroporation results in the effective expression of the chimeric receptors of the present disclosure in T lymphocytes.
  • references describing retroviral transduction are Anderson et al., US Pat. No. 5,399,346; Mann et al., Cell 33: 153 (1983); Temin et al., US Pat. No. 4,650,764; Temin et al., US Pat. No. 5,124,263; Dougherty et al.
  • Host cell activation and proliferation are used to allow the integration of viral vectors into the genome and the expression of genes encoding the chimeric receptors of the present disclosure.
  • mRNA electroporation is used, activation and proliferation are not required (although electroporation is more effective when performed on activated cells).
  • host cells T lymphocytes or NKT cells
  • mRNA electroporation when the receptor is transiently expressed (typically 3-5 days). It has the potential to develop the chimeric receptors of the present disclosure for extended periods of time.
  • viral transduction is complex, expensive and difficult to carry out, while mRNA electroporation is much simpler and much easier to carry out.
  • transient expression is useful if there is potential for toxicity and should be useful in the early stages of clinical trials due to the potential for side effects.
  • One aspect is the method of making cells disclosed herein comprising transfecting or transducing cells with the nucleic acids or vectors described herein.
  • the isolated immune cell is an isolated T cell.
  • the isolated cells are CD3 + , optionally prior to transduction or transfection, and optionally soluble or membrane-bound anti-CD3 antibodies such as OKT3 or mOKT3, and / or. Stimulated by APC.
  • the APC is an artificial APC (aAPC).
  • the APC expresses a membrane-type anti-CD3 monoclonal antibody.
  • the transfection or transduction step is repeated.
  • the transfection or transduction step can be performed twice, three times, or four times, or, for example, until sufficient expression levels are achieved.
  • the transfection or transduction step can be performed 5 times.
  • cells are transfected or transduced for at least 2 consecutive days.
  • cells are transfected or transduced for 2 consecutive days, 3 consecutive days, or 4 consecutive days.
  • chimeric receptor transduced cells are stimulated with irradiated cells expressing a given antigen.
  • chimeric receptor transduced T cells are irradiated with effector: target ratio 100: 1, 75: 1, 50: 1, 25: 1, 20: 1, 15: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: Stimulate at 6, 1: 7, 1: 8, 1: 9, 1:10, 1:15, 1:20, 1:25, 1:50 or 1: 100. It is preferably 1: 1.
  • the cells expressing the chimeric receptor of the present disclosure are cells in which the nucleic acid encoding the chimeric receptor described in the present specification is introduced and expressed by the production method described in the present specification.
  • the cells of the present disclosure bind to a specific antigen via a chimeric receptor and an antigen-binding molecule, whereby a signal is transmitted to the cell, and as a result, the cell is activated.
  • Activation of cells expressing the chimeric receptor depends on the type of host cell and the intracellular domain of the chimeric receptor, and as indicators, for example, cytokine release, cell proliferation rate improvement, or cell surface molecule changes. Can be confirmed based on. For example, the release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) from activated cells causes the destruction of antigen-expressing target cells.
  • cytokine release or changes in cell surface molecules stimulate other immune cells such as B cells, dendritic cells, NK cells, and macrophages.
  • the T lymphocytes used in the methods of the present disclosure are most preferably isolated first from blood samples and preferably exvivo using standard methods such as anti-CD3 / CD28 beads, IL-2 or phytohaemagglutinin.
  • the patient's own cells ie, autologous cells
  • allogeneic T lymphocytes can be used (preferably allogeneic T lymphocytes in which the expression of endogenous T cell receptors is blocked or eliminated). See Torikai et al., Blood, 2012 119: 5697-5705.
  • T lymphocytes and NK cells After isolation (and optionally activation and / or proliferation), T lymphocytes and NK cells from the patient are characterized with a polynucleotide (or a vector containing such a polynucleotide) encoding the chimeric receptor of the present disclosure. Introduce (or electroperforate) so that the chimeric receptor is expressed on the cell surface of T or NK cells. Modified cells can then be administered to the patient (eg, 1 day after therapeutic antibody infusion).
  • chimeric receptor-expressing cells nucleic acids, vectors, cells or compositions described herein for treating a disease is provided.
  • Another aspect comprises a method of treating or preventing a disease in a mammal comprising administering to the mammal in need thereof an effective amount of the cells or compositions disclosed herein. The method.
  • compositions comprising cells expressing the chimeric receptor as an active ingredient are parenteral in intradermal, intramuscular, subcutaneous, intraperitoneal, intranasal, intraarterial, intravenous, intratumoral, or efferent lymph vessels. Administration can be, for example, by injection or infusion, but the route of administration is not limited.
  • a method of incorporating the nucleic acid encoding the chimeric receptor of the present disclosure into a living body using a viral vector or the like and directly expressing the chimeric receptor examples include, but are not limited to, adenovirus. It is also possible to directly incorporate the nucleic acid encoding the chimeric receptor into the living body by an electroporation method or a method of directly administering the nucleic acid without using a viral vector, and to secrete and express the chimeric receptor. It is also possible to continuously secrete the chimeric receptor in the living body by administering the genetically modified cell to the living body.
  • a patient is a T cell or T cell containing a therapeutically effective dose of a therapeutically effective dose ranging from about 105 (1E + 05 ) to 10 10 (1E + 10) or more cells / body weight kilograms (cells / Kg). It can be treated by infusion of NK cells. The infusion may be repeated as often and as many times as possible until the desired response is achieved, as long as the patient is tolerable. Appropriate infusion doses and schedules will vary from patient to patient, but the treating physician can determine for a particular patient. Typically, an initial dose of about 106 (1E + 06) cells / Kg is infused and increased to 10 8 (1E + 08) or more cells / Kg.
  • IL-2 can be used in combination to proliferate the injected cells after injection. The amount of IL-2 can be about 1-5 ⁇ 10 6 (1-5E + 06) international units / square meter of body surface.
  • the pharmaceutical composition comprises cells expressing the chimeric receptor as an active ingredient, and may further contain a suitable excipient.
  • excipients include the above-mentioned pharmaceutically acceptable excipients for compositions comprising the nucleic acids of the present disclosure as active ingredients, various cell culture media, and isotonic sodium chloride.
  • a further aspect of this disclosure provides pharmaceutical compositions.
  • the present disclosure comprises (i) a polynucleotide encoding a chimeric receptor of the present disclosure or a vector comprising such a polynucleotide and (ii) a pharmaceutical composition comprising a pharmaceutically acceptable carrier or additive. Provide things.
  • Suitable additives for use in the pharmaceutical compositions of the present disclosure are well known to those of skill in the art, eg, tissue culture medium (eg, to allow cells to survive in Exvivo) or aqueous saline solution (eg, cells are injected into a patient). When you do).
  • tissue culture medium eg, to allow cells to survive in Exvivo
  • aqueous saline solution eg, cells are injected into a patient. When you do).
  • a detailed description of pharmaceutically acceptable additives is available from Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • the disease to which the pharmaceutical composition of the present invention is administered is not particularly limited as long as the disease is sensitive to the therapy used.
  • the disease is cancer.
  • the subject is suspected of having or has cancer.
  • the cancer is a blood cancer or a solid tumor.
  • hematological malignancies are leukemia, lymphoma or myeloma.
  • solid tumors include adenocarcinoma, squamous cell carcinoma, glandular epithelial cancer, undifferentiated cancer, large cell cancer, small cell cancer, skin cancer, breast cancer, prostate cancer, bladder cancer, etc.
  • the disease is an inflammatory / autoimmune disease (asthma, eczema), hepatitis, and an infectious disease such as tuberculosis, MRSA, the cause of which is a virus, bacterium, or fungus such as influenza and HIV. VRE, and deep-seated fungal disease.
  • the subject is suspected of having these inflammatory diseases or has inflammatory diseases.
  • compositions of the present disclosure that bind to antigens processed by cells that are desired to be reduced or eliminated for the treatment of the above-mentioned diseases, such as tumor antigens, viral antigens, or bacterial antigens, are these diseases. Is administered for the treatment of.
  • one aspect is a method of reducing the number of cells expressing a given antigen in a subject, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition described herein.
  • the chimeric receptor-expressing cells specifically bind to a given antigen via an antigen-binding molecule that binds to the extracellular domain.
  • the cells of the present disclosure can also be used for the prevention of infectious diseases after bone marrow transplantation or radiation exposure, donor lymphocyte transfusion, etc. for the purpose of relieving recurrent leukemia.
  • the chimeric receptors of the present disclosure facilitate T cell therapy that allows the use of a single receptor for a variety of cancer cell types. Considering the antigenic escape mechanism utilized by tumors, it is a strategy that may ultimately be advantageous, and at the same time enables targeting of various antigens (Grupp et al., N. Engl. J. Med. 2013; 368 ( 16): 15091-1518). Antibody-mediated cytotoxicity can be stopped at any time by a simple discontinuation of antibody administration. Since the T cells expressing the chimeric receptor of the present disclosure are activated only by the antibody bound to the target cell, the unbound immunoglobulin exerts no stimulation in the injected T cell. In order to limit any possible autoimmune reactivity, the use of mRNA electroporation for transient expression of chimeric receptors may further enhance clinical safety.
  • the T lymphocyte or NK cell is an autologous T cell or NK cell isolated from the subject.
  • autologous T cells or NK cells are activated and / or proliferated in Exvivo prior to reintroduction into the subject.
  • the T cell or NK cell is an allogeneic T lymphocyte or NK cell.
  • T cells are allogeneic T cells in which the expression of endogenous T cell receptors is blocked or eliminated.
  • allogeneic T cells are activated and / or proliferated in Exvivo prior to introduction into the subject.
  • T lymphocytes can be activated by any method known in the art, for example, in the presence of anti-CD3 / CD28, IL-2 and / or phytohaemagglutinin.
  • NK cells are selected from the group consisting, for example, CD137 ligand protein, CD137 antibody, IL-15 protein, IL-15 receptor antibody, IL-2 protein, IL-12 protein, IL-21 protein and K562 cell line. It can be activated by any method known in the art in the presence of one or more agents. See, for example, US Pat. Nos. 7,435,596 and 8,026,097 for a description of useful methods for growing NK cells.
  • a therapeutically effective amount of a PD-1 / PD-L1 signal inhibitor and / or a VEGF signal inhibitor is targeted following the introduction (or reintroduction) of T cells or NK cells into the subject.
  • a PD-1 / PD-L1 signal inhibitor and / or a VEGF signal inhibitor is targeted following the introduction (or reintroduction) of T cells or NK cells into the subject.
  • compositions and methods described herein can be used in combination with other types of treatment for cancer such as chemotherapy, surgery, radiation, gene therapy and the like. Such treatment may be applied simultaneously or sequentially (in any order) with immunotherapy according to the present disclosure.
  • the appropriate therapeutically effective dose of each pharmaceutical composition can be reduced by additive or synergistic action.
  • the treatments of the present disclosure include, for example, therapeutic vaccines (including, but not limited to, GVAX, DC vaccines, etc.), checkpoint inhibitors (including, but not limited to, pharmaceutical compositions that block CTLA4, PD1, LAG3, TIM3, etc. Can be combined with other immunomodulatory treatments such as (but not limited to) or activators (including, but not limited to, pharmaceutical compositions that enhance 41BB, OX40, etc.).
  • therapeutic vaccines including, but not limited to, GVAX, DC vaccines, etc.
  • checkpoint inhibitors including, but not limited to, pharmaceutical compositions that block CTLA4, PD1, LAG3, TIM3, etc.
  • activators including, but not limited to, pharmaceutical compositions that enhance 41BB, OX40, etc.
  • compositions of the present disclosure may also include one or more additional active compounds, preferably those having complementary activities that do not adversely affect each other, if required for the particular indication to be treated.
  • additional active compounds include, for example, PD-1 / PD-L1 signal inhibitors and VEGF signal inhibitors.
  • the pharmaceutical composition of the present disclosure further comprises a monoclonal antibody capable of exerting cytotoxicity against cancer cells (eg, rituximab, trastuzumab, hu14.18K322A, etc.) or other antitumor molecule (eg, eg, Fc moiety). Further comprises a mixed component consisting of a ligand (eg, a cytokine, an immune cell receptor) bound to a tumor surface receptor in combination with the Fc portion of an immunoglobulin or Fc-containing DNA or RNA.
  • the appropriate dose of antibody to be used depends on the type of cancer to be treated, the severity and course of the disease, previous treatment, the patient's clinical history and response to the antibody and the discretion of the treating physician.
  • the antibody can be administered to the patient at one time or in a series of multiple treatments.
  • the progress of treatment according to the present disclosure can be easily monitored by conventional techniques and assays.
  • the administration of the antibody can be carried out by any suitable route, including systemic administration and direct administration to the site of the disease (eg, to the primary tumor).
  • Non-limiting examples of other therapeutic agents useful in combination with the immunotherapies of the present disclosure include: (i) Antiangiogenic agents (eg, TNP-470, thrombospondin-1, thrombospondin-1, tissue metalloproteinase inhibitors (TIMP1 and TIMP2), prolactin (16Kd fragment), angiostatin (38Kd fragment of plasminogen). ), Endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placenta proliferin-related proteins and those described in Carmeliet and Jain (2000);
  • Antiangiogenic agents eg, TNP-470, thrombospondin-1, thrombospondin-1, tissue metalloproteinase inhibitors (TIMP1 and TIMP2), prolactin (16Kd fragment), angiostatin (38Kd fragment of plasminogen).
  • Endostatin eg, bFGF soluble receptor,
  • VEGF antagonists or VEGF receptors such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralized anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinase and any combination thereof.
  • Body antagonist VEGF antagonists or VEGF receptors such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralized anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinase and any combination thereof.
  • Body antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralized anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinase and any combination thereof.
  • pyrimidine analogs (5-fluorouracil, floxuridine, capesitabin, gemcitabine and citarabin), purine analogs, folic acid antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenocin (cladribin)).
  • Chemotherapeutic compounds such as; natural products such as binca alkaloids (binbrastin, bincristin and binorerbin), taxan (pacritaxel, docetaxel), bincristin, binbrastin, nocodazole, etoposide and microtube disruptors such as navelvin, epipods.
  • Phyllotoxin etoposide, teniposide
  • DNA damage agents actinomycin, amsacline, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambusyl, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin , Hexamethylmelamine, Oxaliplatin, Iphosphamide, Melfaran, Mechloretamine, Mitomycin, Mitoxanthron, Nitrosouria, Plicamycin, Procarbazine, Taxol, Taxotere, Teniposide, Triethylenethiophosphoroamide and Etoposide (VP16) Proliferative / threaded mitotic inhibitors; antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin),
  • Substances enzymes (L-asparaginase that systemically metabolizes L-asparagin and removes cells that do not have the ability to synthesize their own asparagin); antiplatelet agents; nitrogen mustards (mechloretamine, cyclophosphamide and analogs, mel) Faran, chlorambusyl), ethyleneimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfane, nitrosoureas (carmustin (BCNU) and analogs, streptosocin), trazens-dacarbazine (DTIC)
  • Antiproliferative / anti-thread splitting alkylating agents anti-proliferative / anti-thread splitting antimetabolites such as folic acid analogs (methotrexate); platinum-coordinating complexes (cisplatin, carboplatin), procal Vazine, hydroxyurea, mitotan, amino
  • Example 1 Chimera receptor having an extracellular domain of an immunoreceptor in the extracellular domain A binding domain on the surface of an immune cell and exhibiting agonistic activity against an immunoreceptor involved in the activation of the immune cell.
  • an antigen-binding molecule having a binding domain to the target antigen on the target cell the immune cell expressing the immunoreceptor and the target cell expressing the target antigen are cross-linked by the antigen-binding molecule, and the immune cell is used. Is known to be activated.
  • a chimeric receptor having an antigen-binding molecule that exerts an agonistic activity on an immunoreceptor and an immunoreceptor recognized by the antigen-binding molecule in an extracellular domain (Chimeric Receptor: hereinafter also referred to as "CR").
  • CR Chimeric Receptor
  • the treatment method comprises 1) a binding domain that exerts an agonistic activity on an immunoreceptor on the surface of an immune cell and involved in the activation of the immune cell, and a target in a subject in need of treatment.
  • the antigen-binding molecule cross-links the CR-expressing immune cell with the target cell, including administering a CR-transfected immune cell having an extracellular domain of an immunoreceptor to which the binding molecule can bind. And activate CR-expressing cells.
  • the type of the target antigen can be changed by changing the antigen-binding molecule of the first composition, and it is necessary to recreate the immune cells expressing CR. No. Therefore, the production of such CR cells is more versatile and useful than the production of immune cells that directly recognize an antigen specifically or selectively expressed in a target cell.
  • the immune cells transformed with CR not only the immune cells transformed with CR but also the endogenous immune cells that endogenously express the immune receptor are simultaneously mobilized by the antigen-binding molecule in a target antigen-dependent manner.
  • the endogenous immune cells are also activated by the agonist activity of the antigen-binding molecule, and it is expected that the drug efficacy will be enhanced.
  • Example 2 Preparation of bispecific antibody (2-1) Preparation of homoantibody
  • the antibody shown in Table 1 was prepared.
  • the heavy chain constant region has been modified to reduce binding to the Fc ⁇ receptor and to hetero-associate the two heavy chains.
  • the full-length gene encoding the base sequence of the heavy chain and the light chain of each antibody was prepared by a method known to those skilled in the art using PCR or the like.
  • the obtained plasmid fragment was inserted into an animal cell expression vector to prepare a heavy chain expression vector and a light chain expression vector.
  • the base sequence of the obtained expression vector was determined by a method known to those skilled in the art.
  • the prepared plasmid was transiently introduced into FreeStyle 293 cells (Invitrogen) derived from human fetal renal cell carcinoma cells or Expi293 cells (Invitrogen) to express the antibody. After the obtained culture supernatant was recovered, the culture supernatant was obtained through a 0.22 ⁇ m filter (Millipore). From the obtained culture supernatant, rProtein A Sepharose (R) Fast Flow (GE Healthcare Japan) or Protein G Sepharose (R) 4 Fast Flow (GE Healthcare Japan) was used by a method known to those skilled in the art. The antibody was purified.
  • the absorbance at 280 nm was measured using a spectrophotometer, and the antibody concentration was calculated from the obtained value using the extinction coefficient calculated by a method such as PACE (Protein Science (1995) 4). , 2411-2423).
  • the antibodies in the present specification are named according to the following rules; (Heavy chain variable region)-(Heavy chain constant region) / (Light chain variable region)-(Light chain constant region)
  • H0000-F760nN17 / GL4-k0a means that the heavy chain variable region is H0000, the heavy chain constant region is F760nN17, the light chain variable region is GL4, and the light chain constant region is k0a.
  • variable region of an antibody may be indicated according to the following rules; (Heavy chain variable region) / (Light chain variable region)
  • Bispecific antibodies herein are named according to the following rules; AA (first antigen heavy chain) / XX (first antigen light chain) // BB (second antigen heavy chain) / YY (second antigen light chain)
  • Example 3 Evaluation of activation ability of chimeric receptor-expressing cells having an extracellular domain of CD137 in the extracellular domain (3-1) Construction of lentiviral vector Human CD137 extracellular domain, human CD8 hinge and transmembrane domain , And the chimeric receptor CD137-CD8-CD28-CD137-CD3 zeta (referred to as FIG. 2, CD137-CR1) consisting of the intracellular signal domain of the third generation chimeric antigen receptor (CD28-CD137-CD3 zeta). A wrench virus vector was prepared. As the lentiviral vector skeleton, pCDH-CMV-MCS-T2A-copGFP (Sytem Bioscience) was used. FIG.
  • FIG. 3 is a schematic representation of the sequence order of the vector construct and the frame-by-frame components from the 5'end to the 3'end.
  • Codon-optimized human CD137 extracellular domain Genbank NM001561.6, Leu24 to Grn186
  • human CD8 alpha chain hinge and transmembrane regions Genbank NM001768.7, Ph128 to Asn210
  • human CD28 molecules Genbank NM001768.7, Ph128 to Asn210.
  • Genbank NM006139.4, Arg180-Ser220 human CD137 molecules (Genbank NM001561.6, Arg209-Leu255), and human CD3 zeta molecules (Genbank NM000734.4, Arg52-Arg164) were ligated.
  • the gene encoding CD137-CR1 (SEQ ID NO: 24) was synthesized by a method known to those skilled in the art. This sequence was ligated to the MCS of the pCDH-CMV-MCS-T2A-copGFP vector to construct a lentiviral vector co-expressing CD137-CR1 and copGFP.
  • NFAT-RE-luc2 Jurkat cells (Promega). ) was transduced with the CD137-CR1-copGFP vector.
  • the luciferase gene is inserted downstream of the transcription factor natural factor of activated T cells (NFAT) response element, and the activation of the NFAT pathway can be quantified by detecting the luminescence of luciferase.
  • Transduction was performed by the lentiviral method using the lentiviral vector constructed in Example 3-1. Specifically, first, the above-mentioned CD137-CR1-copGFP vector and the packaging plasmid mix were transfected into the 293Ta cell line (Genecopoeia) using the Lenti-Pac TM HIV Transfection Packing Kit (Genecopoeia), and the CD137-CR1 was used. A lentivirus into which the -copGFP vector was introduced was produced. The supernatant containing the lentivirus was recovered 48 to 72 hours after transfection and concentrated using Lenti- XTM concentrator (Takara Bio).
  • NFAT-RE-luc2 Jurkat cells were obtained.
  • NFAT-RE-luc2 Jurkat cells transduced with the pCDH-CMV-MCS-T2A-copGFP vector expressing only copyGFP without ligating CD137-CR1 were simultaneously produced.
  • SK-pca60 cells in which human GPC3 was stably expressed in SK-HEP1 (ATCC), which is a human liver cancer cell line, were used.
  • SK-pca60 cells were seeded in 384-well flat bottom plates (Corning) in 10 ⁇ L / well increments (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • 5 ⁇ L / well of CD137-CR1-copGFP Jurkat cells or NFAT-RE-luc2 Jurkat cells expressing only copGFP without expressing the chimeric receptor as a negative target were mixed (1E + 04 (1 ⁇ 10 4 )). Cell / well).
  • NCI-H929 As the tumor cell line expressing human GPRC5D, NCI-H929 (ATCC) derived from human plasmacytoma was used. First, NCI-H929 cells were seeded in 384-well flat bottom plates (Corning) in 10 ⁇ L / well increments (1E + 04 (1 ⁇ 10 4 ) cells / well). Next, 5 ⁇ L / well of CD137-CR1-copGFP Jurkat cells or NFAT-RE-luc2 Jurkat cells expressing only copGFP without expressing the chimeric receptor as a negative target were mixed (1E + 04 (1 ⁇ 10 4 )). Cell / well).
  • Bispecific antibodies were then added at a final concentration of 0 or 10 ⁇ g / mL in 5 ⁇ L / well increments. After 24 hours, luciferase activity was measured using Bio-Glo Luciferase (Promega). Luciferase activity was measured by EnVision TM Xcite (PerkinElmer) and the results are shown in FIG.
  • CD137-CR1-copGFP Jurkat cells As a result, activation was detected only in the presence of bispecific antibody in CD137-CR1-copGFP Jurkat cells.
  • NFAT-RE-luc2 Jurkat cells that did not express the chimeric receptor and expressed only copyGFP were not detected to be activated even in the presence of bispecific antibody. From this, CD137-CR1-copGFP-T cells are activated in a bispecific antibody-dependent manner, exhibit cytotoxic activity against cells expressing the tumor antigen GPRC5D, and induce an antitumor effect. was suggested.
  • Example 3-2 (3-6) Evaluation of Activation Ability of CD137-CR1-copGFP Jurkat Cell When Target Antigen is IL-6R
  • the CD137-CR1-copGFP Jurkat cell prepared in Example 3-2 expresses the target antigen.
  • the ability of Jurkat cells to be activated was analyzed under the condition that a bispecific antibody consisting of an antibody against the target antigen and an anti-CD137 antibody was present after being co-cultured with the target cells.
  • human IL-6R was selected as the target antigen of the model, and a bispecific antibody (MRAH.v1-F760mnN17 / MRAL) consisting of an anti-IL-6R antibody and an anti-CD137 antibody prepared in Example 2-2 was selected.
  • MRAH.v1-F760mnN17 / MRAL a bispecific antibody consisting of an anti-IL-6R antibody and an anti-CD137 antibody prepared in Example 2-2 was selected.
  • .V1-k0.v1 // hCD137VH-F760
  • IL-6R-CHO cells Z8AGBA-01-C26-CN-006 cells (IL-6R-CHO cells) in which the full length of human IL-6R was stably expressed in CHO cells were used.
  • IL-6R-CHO cells were seeded in 384-well flat bottom plates (Corning) in 10 ⁇ L / well increments (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • 5 ⁇ L / well of CD137-CR1-copGFP Jurkat cells or NFAT-RE-luc2 Jurkat cells expressing only copGFP without expressing the chimeric receptor as a negative target were mixed (1E + 04 (1 ⁇ 10 4 )). Cell / well).
  • Bispecific antibodies were then added at a final concentration of 0 or 10 ⁇ g / mL in 5 ⁇ L / well increments. After 24 hours, luciferase activity was measured using Bio-Glo TM Luciferase (Promega). Luciferase activity was measured by EnVision TM Xcite (PerkinElmer) and the results are shown in FIG. As a result, activation was detected only in the presence of bispecific antibodies in CD137-CR1-copGFP Jurkat cells. On the other hand, NFAT-RE-luc2 Jurkat cells that did not express the chimeric receptor and expressed only copyGFP were not detected to be activated even in the presence of bispecific antibody. This suggests that CD137-CR1-copGFP-T cells are activated in a bispecific antibody-dependent manner and exhibit cytotoxic activity against cells expressing the target antigen IL-6R.
  • FIG. 7 is a schematic diagram of the arrangement order of the vector construct and the components in frame units from the 5'end to the 3'end.
  • CD137-CR1 was ligated with an F2A peptide (derived from foot and mouth disease virus) and an eGFP molecule (nucleotide sequence 5521-6237bp, Genebank KF957646.1). The gene was synthesized by a method known to those of skill in the art and ligated to pMSGV1 to generate the CD137-CR1-eGFP retroviral vector.
  • CD137-CR1-eGFP is expressed using the retroviral transduction method of human T cells and the retroviral vector constructed in Example 4-1. Genetically modified T cells were generated. Specifically, first, by Lipofectamine (R) 2000 or 3000 (Thermo Fisher Scientific), the above-mentioned CD137-CR1-eGFP vector and pAmpho plasmid (Takara Bio) are used in the GP2-293 packaging cell line (Takara Bio). Transfected into a retrovirus into which the CD137-CR1-eGFP vector was introduced. Forty-eight hours after transfection, the supernatant containing the retrovirus was recovered to produce transduction plates adsorbed on two 24-well plates.
  • Example 4-4 Evaluation of Cytotoxicity of CD137-CR1-eGFP Expressed T Cells Using the Number of Residual Tumor Cells as an Index
  • the cytotoxic activity of CD137-CR1-eGFP-expressing T cells prepared in Example 4-2 is CytoFLEX. It was evaluated by (Beckman Coulter).
  • SK-Hep1 was prepared as a negative target
  • SK-pca60 which stably expressed human GPC3 in SK-Hep1
  • Negative subjects and target cells were seeded on a 6-well plate with 1E + 05 (1 ⁇ 105) cells.
  • CD137-CR1-eGFP-expressing cells were used as effector cells and mixed with 1E + 05 cells so that the ratio of effector cells to target cells (E: T) was 1: 1.
  • the bispecific antibody H0000-F760mnN17 / GL4-k0a // hCD137VH-F760mnP17 / hCD137VL-k0
  • Example 2-2 the bispecific antibody (H0000-F760mnN17 / GL4-k0a // hCD137VH-F760mnP17 / hCD137VL-k0) prepared in Example 2-2 consisting of the anti-GPC3 antibody and the anti-CD137 antibody was 10 ⁇ g per well. Added in.
  • bispecific antibodies (IC17HdK-F760mnN17 / IC17L-k0 // hCD137VH-F760mnP17 / hCD137VL-k0 / hCD137VL-k0 / IC17H IC17L-k0 // H0000-F760mnP17 / GL4-k0a) was added in an amount of 10 ⁇ g per well. 48 hours after the addition, CD137-CR1-eGFP expressing T cells and target cells were recovered. The recovered cells were stained with dead cells using Zombie Aqua TM Fixable Viability Kit (BioLegend, 423104) and T cells with anti-human CD45 antibody (BioLegend, 304039).
  • Cytotoxicity was assessed by the number of residual cancer cells.
  • the number of residual cancer cells was calculated from the number of cells after co-culture and the proportion of cells in the CD45-negative fraction in living cells. The results are shown in FIG. 8A. Only under the condition that the target cells were SK-pca60 and a bispecific antibody consisting of an anti-GPC3 antibody and an anti-CD137 antibody was added, the number of residual tumor cells was significantly reduced.
  • CD137-CR1-eGFP-expressing cells as effector cells, 1E + 04 (1 ⁇ 10 4 ) and 3E + 04 (3 ⁇ ) so that the ratio of effector cells to target cells (E: T) is 1: 1 and 1: 3. 10 4 )
  • the cells were mixed.
  • the bispecific antibody H0000-F760mnN17 / GL4-k0a // hCD137VH-F760mnP17 / hCD137VL-k0
  • Example 2-2 consisting of the anti-GPC3 antibody and the anti-CD137 antibody had a final concentration per well. It was added to 10 ⁇ g / mL.
  • the cytotoxic activity of CD137-CR1-eGFP-expressing cells was monitored in real time by measuring the electrical resistance value over time.
  • Cell proliferation inhibitory activity was calculated by the following formula.
  • Cell proliferation inhibitory activity (%) 100x (A-B) / (A-1) (A: Mean value of Delta Cell Index of wells without antibody, B: Delta Cell Index of sample)
  • B Delta Cell Index of sample
  • Example 5 Chimera receptor having an immunoreceptor whose ability to bind to a ligand is attenuated in the extracellular domain
  • An immune cell transformed with a chimeric receptor having an extracellular full length of the immune receptor is the immune receptor. It can be activated independently of the target antigen by binding to the ligand to the body (Fig. 9). This is problematic as it causes damage to non-target cells. In order to solve this problem, it was considered that the immune receptors in the extracellular domain were artificially modified to reduce the ability to bind to the ligand.
  • Example 6 Evaluation of activation ability of chimeric receptor-expressing cells having a CD137 fragment in the extracellular domain (6-1) Construction of a lentiviral vector expressing trCD137-CR
  • the extracellular domain of human CD137 is cysteine-rich. It is known that CRD2 and CRD3 play important roles in binding to CD137L, which is composed of domains (CRDs) 1 to 4 and is a ligand. Therefore, as a modified CR that attenuated the binding to CD137L, trCD137-CR (SEQ ID NO: 25) in which CRD3 and CRD4 of the human CD137 extracellular domain were deleted was prepared.
  • the gene encoding trCD137-CR was synthesized by a method known to those skilled in the art. This sequence was ligated to the MCS of the pCDH-CMV-MCS-T2A-copGFP vector to construct a lentiviral vector co-expressing trCD137-CR and copGFP.
  • Transduction was performed by the lentiviral method using the lentiviral vector constructed in Example 6-1. Specifically, first, the above-mentioned trCD137-CR vector and the packaging plasmid mix were transfected into the 293Ta cell line (Genecopoeia) using the Lenti-Pac TM HIV Expression Packing Kit (Genecopoeia), and trCD137-CR-copP. A vector-introduced lentivirus was produced. The supernatant containing the lentivirus was recovered 48 to 72 hours after transfection and concentrated using Lenti- XTM concentrator (Takara Bio).
  • NFAT-RE-luc2 Jurkat cells were obtained.
  • trCD137-CR-copGFP Jurkat cells were obtained.
  • SK-pca60 cells in which human GPC3 was stably expressed in SK-HEP1 (ATCC), which is a human liver cancer cell line, were used.
  • SK-pca60 cells were seeded in 384-well flat bottom plates (Corning) in 10 ⁇ L / well increments (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • trCD137-CR-copGFP Jurkat cells or NFAT-RE-luc2 Jurkat cells expressing only copGFP without expressing the chimeric receptor as a negative target were mixed at 5 ⁇ L / well each (1E + 04 (1 ⁇ 10 4 )). Cell / well).
  • trCD137-CR-copGFP Jurkat cells As a result, antibody concentration-dependent activation of bispecific antibodies was detected in trCD137-CR-copGFP Jurkat cells.
  • NFAT-RE-luc2 Jurkat cells that did not express the chimeric receptor and expressed only copyGFP were not detected to be activated even in the presence of bispecific antibody. This suggests that trCD137-CR-copGFP-T cells are activated in a bispecific antibody-dependent manner, exhibit cytotoxic activity against cells expressing tumor antigens, and induce antitumor effects. rice field.
  • Example 7 Evaluation of activation ability of chimeric receptor-expressing cells having a CD137 variant in the extracellular domain (7-1) Construction of lentiviral vector I64 or V71 on CRD2 of human CD137 is mutated to Arg. All of these mutants are known to have diminished binding to CD137L.
  • CD137-CR2 The gene encoding CD137-CR2 was synthesized by a method known to those skilled in the art. This sequence was ligated to the MCS of the pCDH-CMV-MCS-T2A-copGFP vector to construct a lentiviral vector co-expressing CD137-CR2 and copGFP.
  • Transduction was performed by the lentiviral method using the lentiviral vector constructed in Example 7-1. Specifically, first, the above-mentioned CD137-CR2 vector and the packaging plasmid mix were transfected into the 293Ta cell line (Genecopoeia) using the Lenti-Pac TM HIV Transfection Packing Kit (Genecopoeia), and the CD137-CR2-copGFP was used. A vector-introduced lentivirus was produced. The supernatant containing the lentivirus was recovered 48 to 72 hours after transfection and concentrated using Lenti-X TM concentrator (Takara Bio).
  • NFAT-RE-luc2 Jurkat cells were obtained.
  • NFAT-RE-luc2 Jurkat cells transduced with the pCDH-CMV-MCS-T2A-copGFP vector expressing only copyGFP without ligating CD137-CR2 were simultaneously produced.
  • Example 7-2 (7-4) Evaluation of Activation Ability of CD137-CR2-copGFP Jurkat Cell by Reporter Assay
  • the CD137-CR2-copGFP Jurkat cell prepared in Example 7-2 was co-cultured with a tumor cell expressing a tumor antigen.
  • the ability of Jurkat cells to activate was analyzed under the condition that a bispecific antibody consisting of an antibody against a tumor antigen and an anti-CD137 antibody was present.
  • human GPC3 was selected as the model tumor antigen
  • a bispecific antibody consisting of an anti-GPC3 antibody and an anti-CD137 antibody (H0000-F760nN17 / GL4-k0a // hCD137VH-) prepared in Example 2-2 was selected.
  • F760mnP17 / hCD137VL-k0 was used in the experiment.
  • SK-pca60 cells were seeded in 384-well flat bottom plates (Corning) in 10 ⁇ L / well increments (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • CD137-CR2-copGFP Jurkat cells As a result, antibody concentration-dependent activation of bispecific antibodies was detected in CD137-CR2-copGFP Jurkat cells.
  • NFAT-RE-luc2 Jurkat cells that did not express the chimeric receptor and expressed only copyGFP were not detected to be activated even in the presence of bispecific antibody. This suggests that CD137-CR2-copGFP-T cells are activated in a bispecific antibody-dependent manner, exhibit cytotoxic activity against cells expressing tumor antigens, and induce antitumor effects. rice field.
  • Example 8 Evaluation of activation ability of chimeric receptor-expressing cells in the presence of ligand-expressing cells It was investigated whether a chimeric receptor having an immune receptor in the extracellular domain is activated by binding to a ligand for the immune receptor regardless of the presence of a target antigen. Specifically, CD137-CR1-copGFP Jurkat cells prepared in Example 3-2, trCD137-CR-copGFP Jurkat cells prepared in Example 6-2, or CD137 prepared in Example 7-2. -CR2-copGFP Jurkat cells were co-cultured with cells expressing human CD137L and analyzed whether Jurkat cells were activated.
  • Raji cells As the cell line expressing human CD137L, Raji cells (ATCC) derived from human B-cell lymphoma were used. First, a suspension of Raji cells or a medium containing no Raji cells as a negative subject was added to a 384-well flat bottom plate (Corning) at a rate of 10 ⁇ L / well (1E + 04 (1 ⁇ 10 4 ) cells / well). Then, CD137-CR1-copGFP Jurkat cells, trCD137-CR-copGFP Jurkat cells, or CD137-CR2-copGFP Jurkat cells were mixed at 5 ⁇ L / well each (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • luciferase activity was measured using Bio-Glo Luciferase (Promega). Luciferase activity was measured by EnVision TM Xcite (PerkinElmer) and the results are shown in FIGS. 12A-12C.
  • Example 9 Optimization of CD137 variant with diminished ligand binding ability
  • immune cells transformed with a chimeric receptor having the full length of the extracellular domain (CD137) of the immune receptor are targeted by binding to an in vivo ligand (CD137L) for the immune receptor.
  • CD137L in vivo ligand
  • Being able to activate antigen-independently can cause non-target antigen-specific cell damage.
  • the immune receptors in the extracellular domain were artificially modified to weaken the binding to the in vivo ligand.
  • One method is to delete a part of the extracellular domain as described in Example 6.
  • a plasmid having a gene encoding the sequence of the prepared variant was introduced into a human embryonic kidney cell-derived Expi 293 strain (Invitrogen) by a lipofection method.
  • the desired CD137 variant was obtained from the supernatant cultured for 4 days by purification using rProtein A Sepharose (R) Fast Flow (Amersham Biosciences) by a method known to those skilled in the art.
  • the absorbance of the purified variant solution at 280 nm was measured using a spectrophotometer. From the obtained measured values, the concentration of the purified antibody was calculated using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4,2411-2423).
  • CD137L as an antigen CD137L is expressed and purified by a method known to those skilled in the art using a combination of heavy chain and light chain plasmids shown in Table 3, and then 37 by Lys-C (Roche). It was cut by being treated at ° C for 1 hour. Each fragment was passed through a MabSelect SuRe (Cytiva) affinity column and the unbound components were subjected to gel permeation chromatography. As a result, CD137L was separated.
  • the chip was regenerated with 3M MgCl 2 and the antibody was repeatedly captured and measured.
  • the dissociation constant KD (mol / L) for each variant of the anti-CD137 antibody fragment was calculated using the Biacore TM T200 Evolution Software Version 2.0 and the 1: 1 Binding model.
  • N42, Q43, I44 of CRD1 are C48, P50, F53, D63, I64, D87, and even CRD3 L95 and M101 were found.
  • Example 9-2-3 Among the positions found in Example 9-2-2, a modification capable of reducing binding to CD137L was searched for by comprehensively introducing a one-amino acid modification at the position of M101.
  • the prepared variant was evaluated for its binding activity to CD137L by the same method as the interaction analysis described in Example 9-2-2.
  • a modification called M101D (BB0139: SEQ ID NO: 120) was newly found as a modification capable of reducing CD137L binding.
  • ECM binding evaluation of BB0000 and BB0077 When the prepared variant has non-specific binding activity to mammalian cells, cytotoxic activity is exhibited non-specifically to cells. There is a possibility that it will be done. Therefore, the binding property to the Extracellular Matrix (ECM), which is known as a system for evaluating the amount of non-specific binding of each variant in vitro, was evaluated. Non-specific binding of BB0000 and BB0077 was evaluated in this assay system (US Patent Application Publication No. 2014/0080153). As a result, BB0077 was confirmed to bind to ECM very strongly, suggesting that it may cause a non-specific reaction in vivo (Fig. 14).
  • ECM Extracellular Matrix
  • the height of the isoelectric point of arginine introduced by modification was noted as the cause of non-specific binding. It is considered that non-specific binding occurs by attracting the positive charge of arginine to the negative charge charged on the cell membrane surface or ECM, and it has no positive charge and is bulky enough to inhibit the binding with the ligand. It was confirmed whether non-specific binding can be reduced by modifying the amino acids with aspartic acid, glutamine, and glutamic acid. Specifically, variants in which aspartic acid, glutamine, and glutamic acid were introduced into one or both of I64 and V71 mutated in BB0077 were prepared. A list of modifications and SEQ ID NOs introduced for each name and BB0000 produced is shown in Table 4 below.
  • Example 10 Evaluation of activation ability of chimeric receptor-expressing cells having a CD137 variant having a attenuated ligand-binding ability in the extracellular domain (10-1) Construction of lentiviral vector The construction of Example 9 was carried out. Among the CD137 variants with reduced binding to CD137L, 6 species of BB0127, BB0128, BB0131, BB0133, BB0134, and BB0139 are used as extracellular domains, and the hinge region and transmembrane region of the human CD8 alpha chain (Genbank NM001768.7,).
  • Transduction was performed by a lentiviral method using the lentiviral vector constructed in Example 10-1. Specifically, first, a lentiviral vector and a packaging plasmid mix were transfected into a 293Ta cell line (Genecopoeia) using a Lenti-Pac TM HIV Expression Packing Kit (Genecopoeia) to generate a lentivirus. The supernatant containing the lentivirus was recovered 48 to 72 hours after transfection.
  • the supernatant containing the recovered lentivirus and the polybrene solution (Nacalai Tesque) were added to NFAT-RE-luc2 Jurkat cells and centrifuged at 3000 rpm for 60 minutes to transduce NFAT-RE. -Luc2 Jurkat cells were obtained.
  • Example 10-4 Evaluation of activation ability by reporter assay
  • the Jurkat cells expressing the chimeric receptor prepared in Example 10-2 were co-cultured with the tumor cells expressing the tumor antigen, and antibody and anti-antibody against the tumor antigen were obtained.
  • the ability to activate Jurkat cells in the presence of a bispecific antibody consisting of a CD137 antibody was analyzed.
  • human GPC3 was selected as the model tumor antigen
  • a bispecific antibody consisting of an anti-GPC3 antibody and an anti-CD137 antibody (H0000-F760nN17 / GL4-k0a // hCD137VH-) prepared in Example 2-2 was selected.
  • F760mnP17 / hCD137VL-k0 was used in the experiment.
  • SK-pca60 cells in which human GPC3 was stably expressed in SK-HEP1 (ATCC), which is a human liver cancer cell line, were used.
  • SK-pca60 cells were seeded in 384-well flat bottom plates (Corning) in 5 ⁇ L / well increments (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • the CD137-CR2-copGFP Jurkat cell (extracellular domain is BB0077) prepared in Example 7-2 and the parent strain (Mock) of the NFAT-RE-luc2 Jurkat cell into which the gene has not been introduced as a negative target, respectively.
  • 10 ⁇ L / well were mixed (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • Bispecific antibodies were then added at a final concentration of 0 or 10 ⁇ g / mL in 5 ⁇ L / well increments.
  • luciferase activity was measured using Bio-Glo Luciferase (Promega). Luciferase activity was measured by EnVision TM Xcite (PerkinElmer) and the results are shown in FIG.
  • T cells expressing a chimeric receptor having a CD137 variant in the extracellular domain are activated in a bispecific antibody-dependent manner, exhibit cytotoxic activity against cells expressing a tumor antigen, and are anti-virus. It was suggested to induce a tumor effect.
  • a suspension of Raji cells or a medium containing no Raji cells as a negative subject was added to a 384-well flat bottom plate (Corning) at a rate of 5 ⁇ L / well (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • Jurkat cells expressing the chimeric receptor prepared in Example 10-2 and CD137-CR1-copGFP Jurkat cells (extracellular domain is BB0000) prepared in Example 3-2 as positive subjects.
  • Example 10-2 the Jurkat cells expressing the chimeric receptor having BB0127, BB0128, BB0131, BB0133, BB0134, or BB0139 in the extracellular domain prepared in Example 10-2 were referred to as CD137-CR2-copGFP Jurkat cells. Similarly, activation by co-culture with Raji cells was not detected. This suggests that the CD137 variant found in Example 9 can suppress target antigen-independent activation by binding to a ligand.
  • Example 11 Evaluation of agonistic action by bispecific antibody (11-1) Construction of lentiviral vector expressing CD137-CR3 having no intracellular signal domain of CD137 In this system, antigen binding having an agonistic action It is considered that administration of the molecule can induce agonist activity to host immune cells and CR-expressing cells to be administered at the same time via endogenous immune receptors. Therefore, it was examined whether an antigen-binding molecule having a CD137 agonist action induces CD137 agonist activity on cells expressing an endogenous immune receptor.
  • cells expressing CR that do not have the CD137 intracellular signal domain were prepared. Specifically, a lentivirus expressing a chimeric receptor CD137-CD8-CD3 zeta (FIG. 19, referred to as CD137-CR3) consisting of a human CD137 extracellular domain, a human CD8 hinge and transmembrane domain, and a CD3 zeta. A vector was created. As the wrench virus vector skeleton, pCDH-CMV-MCS-T2A-copGFP (Sytem Bioscience) was used.
  • CD137 extracellular domain (Genbank NM001561.6, Leu24-Gln186), human CD8 alpha chain hinge and transmembrane regions (Genbank NM001768.7, Ph128-Asn210), and human CD3 zeta molecule.
  • the cytoplasmic region of (Genbank NM000734.4, Arg52-Arg164) was ligated.
  • the gene encoding CD137-CR3 (SEQ ID NO: 33) was synthesized by a method known to those skilled in the art. This sequence was ligated to the MCS of the pCDH-CMV-MCS-T2A-copGFP vector to construct a lentiviral vector co-expressing CD137-CR3 and copGFP.
  • NF-kB-Luc2 NF-kB-Luc2 / 4-1BB Jurkat cells
  • NF-kB-copGFP vector was transfected into 4-1BB Jurkat cells (Promega).
  • the cell has a luciferase gene inserted downstream of the transcription factor nuclear factor-kappa B (NF-kB) response element, and by detecting the luminescence of luciferase, The activation of the NF-kB pathway can be quantified.
  • NF-kB transcription factor nuclear factor-kappa B
  • Transduction was performed by a lentiviral method using the lentiviral vector constructed in Example 11-1. Specifically, first, the above-mentioned CD137-CR3-copGFP vector and the packaging plasmid mix were transfected into the 293Ta cell line (Genecopoeia) using the Lenti-Pac TM HIV Transfection Packing Kit (Genecopoeia), and the CD137-CR3 A lentivirus into which the -copGFP vector was introduced was produced. The supernatant containing the lentivirus was recovered 48 to 72 hours after transfection and concentrated using Lenti-X TM concentrator (Takarabio).
  • the supernatant containing the recovered lentivirus and the polybrene solution (Nakalaitesk) were added to NF-kB-Luc2 / 4-1BB Jurkat cells and centrifuged at 3000 rpm for 60 minutes to produce CD137-CR3.
  • -NF-kB-Luc2 / 4-1BB Jurkat cells (CD137-CR3-copGFP Jurkat cells) transduced with the copGFP vector were obtained.
  • NF-kB-Luc2 / 4-1BB Jurkat cells (CD137-CR1 Jurkat cells) transduced with the CD137-CR1 vector, and NF that does not express the chimeric receptor and expresses only copyGFP.
  • -KB-Luc2 / 4-1BB Jurkat cells were simultaneously produced.
  • SK-pca60 cells in which human GPC3 was stably expressed in SK-HEP1 (ATCC), which is a human liver cancer cell line, were used.
  • SK-pca60 cells were seeded in 384-well flat bottom plates (Corning) in 10 ⁇ L / well increments (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • CD137-CR3-copGFP Jurkat cells, CD137-CR1 Jurkat cells, and NF-kB-Luc2 / 4-1BB Jurkat cells expressing only copGFP were mixed at 5 ⁇ L / well each (1E + 04 (1 ⁇ 10 4 )). Cells / wells).
  • Bispecific antibodies were then added at a final concentration of 0 or 16 ⁇ g / mL in 5 ⁇ L / well increments. After 24 hours, luciferase activity was measured using Bio-Glo TM Luciferase (Promega). Luciferase activity was measured by EnVision TM Xcite (PerkinElmer) and the results are shown in FIG.
  • CD137-CR1 is fused with CD137 as an intracellular domain, and it is considered that the agonist activity is induced not only via the CD137-mediated agonist activity on the cell membrane but also via the gene-introduced CR.
  • the NF-kB-Luc2 / 4-1BB Jurkat cell expressing CD137-CR3 to which CD137 is not fused as the intracellular domain and the NF-kB-Luc2 / 4-1BB Jurkat cell expressing only copGFP the cell membrane It was considered that the agonist activity was induced via the above CD137. From the above, it was shown that in the system, an antigen-binding molecule having an agonistic action can induce agonist activity on cells expressing an endogenous immune receptor regardless of the presence or absence of CR expression.
  • Example 12 Evaluation of activation ability of chimeric receptor-expressing cells having CD28 in the extracellular region (12-1) Construction of lentiviral vector A lentiviral vector expressing a chimeric receptor fused with the extracellular domain of human CD28 (Genbank NM006139.4, Asn19 to Pro152) was prepared. Two types were produced: a construct using human CD8 for the transmembrane domain (CD28-CR1) and a construct using human CD28 (CD28-CR2) (FIG. 21). As the lentiviral vector skeleton, pCDH-CMV-MCS-T2A-copGFP (Sytem Bioscience) was used.
  • CD28-CR1 SEQ ID NO: 34
  • CD28-CR2 SEQ ID NO: 35
  • a lentiviral vector was constructed by ligating this sequence to the MCS of the pCDH-CMV-MCS-T2A-copGFP vector.
  • the above-mentioned CD28-CR1-copGFP or CD28-CR2-copGFP vector and the packaging plasmid mix are transferred to the 293Ta cell line (Genecopoeia) using the Lenti-Pac TM HIV Transfection Packing Kit (Genecopoeia). Ejection produced a lentivirus into which the CD28-CR1-copGFP or CD28-CR2-copGFP vector was introduced. The supernatant containing the lentivirus was recovered 48 to 72 hours after transfection and concentrated using Lenti-X TM concentrator (Takara Bio).
  • NFAT-RE-luc2 Jurkat cells are added to NFAT-RE-luc2 Jurkat cells and centrifuged at 3000 rpm for 60 minutes to produce CD28-CR1-copGFP or CD28.
  • -NFAT-RE-luc2 Jurkat cells transduced with the CR2-copGFP vector were obtained.
  • NFAT-RE-luc2 Jurkat cells into which the pCDH-CMV-MCS-T2A-copGFP vector expressing only copyGFP was transduced were simultaneously produced.
  • Example 12-4 Evaluation of activation ability by reporter assay
  • the CD28-CR1-copGFP-expressing Jurkat cells prepared in Example 12-2 and the CD28-CR2-copGFP-expressing Jurkat cells express tumor antigens, respectively.
  • the ability to activate Jurkat cells was analyzed under the condition that a bispecific antibody consisting of an antibody against a tumor antigen and an anti-CD28 antibody was present.
  • human GPC3 was selected as the model tumor antigen
  • a bispecific antibody consisting of an anti-GPC3 antibody and an anti-CD28 antibody H0000-F760mnN17 / GL4-k0a // TGN1412VH-
  • F760mnP17.v1 / TGN1412VL-KT0.v1 F760mnP17.v1 / TGN1412VL-KT0.v1
  • a bispecific antibody IC17HdK-F760mnN17 / IC17L-k0 / VN14 / IC17L-k0 / TG14
  • KLH anti-Keyhole limpet hemocyanin
  • F760mnP17.v1 / TGN1412VL-KT0.v1 or IC17HdK-F760mnN17 / IC17L-k0 // H0000-F760mnP17 / GL4-k0a was used in the experiment.
  • SK-pca60 cells in which human GPC3 was stably expressed in SK-HEP1 (ATCC), which is a human liver cancer cell line, were used.
  • SK-pca60 cells were seeded in 384-well flat bottom plates (Corning) in 10 ⁇ L / well increments (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • 5 ⁇ L / well of CD28-CR1-copGFP Jurkat cells, CD28-CR2-copGFP Jurkat cells, or NFAT-RE-luc2 Jurkat cells expressing only copGFP without expressing the chimeric receptor as a negative target were mixed. (1E + 04 (1 ⁇ 10 4 ) cells / well).
  • luciferase activity was measured using Bio-Glo TM Luciferase (Promega). Luciferase activity was measured by EnVision TM Xcite (PerkinElmer) and the results are shown in FIG.
  • both CD28-CR1-copGFP Jurkat cells and CD28-CR2-copGFP Jurkat cells were weakly activated under the condition that no antibody was added or the condition that a negative target bispecific antibody was added.
  • it was strongly activated for the first time in the presence of a bispecific antibody consisting of an anti-GPC3 antibody and an anti-CD28 antibody.
  • NFAT-RE-luc2 Jurkat cells expressing only copyGFP without expressing the chimeric receptor were not detected to be activated even in the presence of a bispecific antibody consisting of an anti-GPC3 antibody and an anti-CD28 antibody.
  • T cells expressing a chimeric receptor having CD28 in the extracellular region are activated in a bispecific antibody-dependent manner, exhibit cytotoxic activity against cells expressing a tumor antigen, and have an antitumor effect. It was suggested to induce.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Hematology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/JP2021/028230 2020-07-31 2021-07-30 キメラ受容体を発現する細胞を含む医薬組成物 Ceased WO2022025220A1 (ja)

Priority Applications (10)

Application Number Priority Date Filing Date Title
CN202180058518.9A CN116194124A (zh) 2020-07-31 2021-07-30 包含表达嵌合受体的细胞的药物组合物
KR1020237006010A KR20230048059A (ko) 2020-07-31 2021-07-30 키메라 수용체를 발현하는 세포를 포함하는 의약 조성물
EP21850145.0A EP4197545A4 (en) 2020-07-31 2021-07-30 Pharmaceutical composition comprising a cell for expressing a chimeric receptor
MX2023001120A MX2023001120A (es) 2020-07-31 2021-07-30 Composicion farmaceutica que comprende una celula que expresa un receptor quimerico.
CA3190649A CA3190649A1 (en) 2020-07-31 2021-07-30 Pharmaceutical composition comprising cell expressing chimeric receptor
IL300225A IL300225A (en) 2020-07-31 2021-07-30 A pharmaceutical composition including chimeric receptor expressing cells
BR112023000537A BR112023000537A2 (pt) 2020-07-31 2021-07-30 Composições farmacêuticas incluindo célula que expressa o receptor quimérico
AU2021317974A AU2021317974A1 (en) 2020-07-31 2021-07-30 Pharmaceutical Composition comprising Cell Expressing Chimeric Receptor
JP2022539585A JPWO2022025220A1 (https=) 2020-07-31 2021-07-30
US18/018,807 US20230322898A1 (en) 2020-07-31 2021-07-30 Pharmaceutical composition comprising cell expressing chimeric receptor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2020-131116 2020-07-31
JP2020131116 2020-07-31

Publications (1)

Publication Number Publication Date
WO2022025220A1 true WO2022025220A1 (ja) 2022-02-03

Family

ID=80036292

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2021/028230 Ceased WO2022025220A1 (ja) 2020-07-31 2021-07-30 キメラ受容体を発現する細胞を含む医薬組成物

Country Status (12)

Country Link
US (1) US20230322898A1 (https=)
EP (1) EP4197545A4 (https=)
JP (1) JPWO2022025220A1 (https=)
KR (1) KR20230048059A (https=)
CN (1) CN116194124A (https=)
AU (1) AU2021317974A1 (https=)
BR (1) BR112023000537A2 (https=)
CA (1) CA3190649A1 (https=)
IL (1) IL300225A (https=)
MX (1) MX2023001120A (https=)
TW (1) TW202220677A (https=)
WO (1) WO2022025220A1 (https=)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023174925A1 (en) * 2022-03-14 2023-09-21 Novimmune Sa Bispecific gpc3xcd28 and gpc3xcd3 antibodies and their combination for targeted killing of gpc3 positive malignant cells

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2020257748A1 (en) * 2019-04-19 2021-11-18 Chugai Seiyaku Kabushiki Kaisha Chimeric receptor recognizing modification site of antibody

Citations (85)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773719A (en) 1966-12-06 1973-11-20 Hoffmann La Roche 2-aminoxy-2'-acyl-acetanilide
EP0058481A1 (en) 1981-02-16 1982-08-25 Zeneca Limited Continuous release pharmaceutical compositions
EP0133988A2 (de) 1983-08-02 1985-03-13 Hoechst Aktiengesellschaft Regulatorische Peptide enthaltende pharmazeutische Präparate mit protrahierter Freisetzung und Verfahren zu deren Herstellung
US4650764A (en) 1983-04-12 1987-03-17 Wisconsin Alumni Research Foundation Helper cell
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
EP0239400A2 (en) 1986-03-27 1987-09-30 Medical Research Council Recombinant antibodies and methods for their production
WO1988001649A1 (en) 1986-09-02 1988-03-10 Genex Corporation Single polypeptide chain binding molecules
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1992001047A1 (en) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
WO1992003918A1 (en) 1990-08-29 1992-03-19 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5124263A (en) 1989-01-12 1992-06-23 Wisconsin Alumni Research Foundation Recombination resistant retroviral helper cell and products produced thereby
WO1992020791A1 (en) 1990-07-10 1992-11-26 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
WO1993006213A1 (en) 1991-09-23 1993-04-01 Medical Research Council Production of chimeric antibodies - a combinatorial approach
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
WO1993011236A1 (en) 1991-12-02 1993-06-10 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
WO1993012227A1 (en) 1991-12-17 1993-06-24 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1993017706A1 (en) 1992-03-11 1993-09-16 Agracetus, Inc. Genetic vaccine for immunodeficiency viruses
WO1993019172A1 (en) 1992-03-24 1993-09-30 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US5278056A (en) 1988-02-05 1994-01-11 The Trustees Of Columbia University In The City Of New York Retroviral packaging cell lines and process of using same
WO1994002602A1 (en) 1992-07-24 1994-02-03 Cell Genesys, Inc. Generation of xenogeneic antibodies
WO1994011523A2 (en) 1992-11-13 1994-05-26 Idec Pharmaceuticals Corporation Fully impaired consensus kozac sequences for mammalian expression
WO1994025585A1 (en) 1993-04-26 1994-11-10 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1995001438A1 (en) 1993-06-30 1995-01-12 Medical Research Council Sbp members with a chemical moiety covalently bound within the binding site; production and selection thereof
WO1995001937A1 (fr) 1993-07-09 1995-01-19 Association Gradient Procede de traitement de residus de combustion et installation de mise en ×uvre dudit procede
WO1995007358A1 (en) 1993-07-30 1995-03-16 University Of Medicine & Dentistry Of New Jersey Efficient gene transfer into primary lymphocytes
US5399346A (en) 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
WO1995015388A1 (en) 1993-12-03 1995-06-08 Medical Research Council Recombinant binding proteins and peptides
WO1995033844A1 (de) 1994-06-03 1995-12-14 GSF - Forschungszentrum für Umwelt und Gesundheit GmbH Verfahren zur herstellung von heterologen bispezifischen antikörpern
WO1996002576A1 (en) 1994-07-13 1996-02-01 Chugai Seiyaku Kabushiki Kaisha Reconstituted human antibody against human interleukin-8
WO1996010038A1 (en) 1994-09-28 1996-04-04 Apollon, Inc. Multifunctional molecular complexes for gene transfer to cells
WO1996027011A1 (en) 1995-03-01 1996-09-06 Genentech, Inc. A method for making heteromultimeric polypeptides
WO1996033735A1 (en) 1995-04-27 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1997018185A1 (fr) 1995-11-14 1997-05-22 Rhone-Poulenc Rorer S.A. Lipopolymamines comme agents de transfection et leurs applications pharmaceutiques
WO1997025329A1 (fr) 1996-01-10 1997-07-17 Rhone-Poulenc Rorer S.A. DERIVES DE 5H,10H-IMIDAZO[1,2-a]INDOLO[3,2-e]PYRAZINE-4-ONE, LEUR PREPARATION ET LES MEDICAMENTS LES CONTENANT
WO1997030170A1 (de) 1996-02-15 1997-08-21 Boehringer Ingelheim International Gmbh Zusammensetzung für die transfektion höherer eukaryotischer zellen
WO1997031934A2 (de) 1996-02-29 1997-09-04 Chemicon Laboratories Gmbh Neue metabolisierbare lipopolyamine, deren darstellung und anwendung
WO1998050431A2 (en) 1997-05-02 1998-11-12 Genentech, Inc. A method for making multispecific antibodies having heteromultimeric and common components
WO2000042072A2 (en) 1999-01-15 2000-07-20 Genentech, Inc. Polypeptide variants with altered effector function
WO2002020565A2 (en) 2000-09-08 2002-03-14 Universität Zürich Collections of repeat proteins comprising repeat modules
WO2002032925A2 (en) 2000-10-16 2002-04-25 Phylos, Inc. Protein scaffolds for antibody mimics and other binding proteins
WO2003000883A1 (fr) 2001-06-22 2003-01-03 Chugai Seiyaku Kabushiki Kaisha Inhibiteurs de proliferation cellulaire renfermant un anticorps anti-glypicane 3
WO2003029462A1 (en) 2001-09-27 2003-04-10 Pieris Proteolab Ag Muteins of human neutrophil gelatinase-associated lipocalin and related proteins
WO2003104453A1 (ja) 2002-06-05 2003-12-18 中外製薬株式会社 抗体作製方法
WO2004022754A1 (ja) 2002-09-04 2004-03-18 Chugai Seiyaku Kabushiki Kaisha MRL/lprマウスを用いた抗体の作製
WO2004044011A2 (en) 2002-11-06 2004-05-27 Avidia Research Institute Combinatorial libraries of monomer domains
WO2004065611A1 (ja) 2003-01-21 2004-08-05 Chugai Seiyaku Kabushiki Kaisha 抗体の軽鎖スクリーニング方法
WO2005040229A2 (en) 2003-10-24 2005-05-06 Avidia, Inc. Ldl receptor class a and egf domain monomers and multimers
WO2006006693A1 (ja) 2004-07-09 2006-01-19 Chugai Seiyaku Kabushiki Kaisha 抗グリピカン３抗体
US20060025576A1 (en) 2000-04-11 2006-02-02 Genentech, Inc. Multivalent antibodies and uses therefor
WO2006019447A1 (en) 2004-07-15 2006-02-23 Xencor, Inc. Optimized fc variants
WO2006106905A1 (ja) 2005-03-31 2006-10-12 Chugai Seiyaku Kabushiki Kaisha 会合制御によるポリペプチド製造方法
WO2006132352A1 (ja) 2005-06-10 2006-12-14 Chugai Seiyaku Kabushiki Kaisha sc(Fv)2を含有する医薬組成物
WO2007114325A1 (ja) 2006-03-31 2007-10-11 Chugai Seiyaku Kabushiki Kaisha 二重特異性抗体を精製するための抗体改変方法
WO2008016854A2 (en) 2006-08-02 2008-02-07 The Uab Research Foundation Methods and compositions related to soluble monoclonal variable lymphocyte receptors of defined antigen specificity
WO2008119353A1 (en) 2007-03-29 2008-10-09 Genmab A/S Bispecific antibodies and methods for production thereof
US7435596B2 (en) 2004-11-04 2008-10-14 St. Jude Children's Research Hospital, Inc. Modified cell line and method for expansion of NK cell
WO2009011941A2 (en) 2007-04-04 2009-01-22 The Government Of U.S.A., As Represented By The Secretary, Departmetnt Of Health & Human Services Monoclonal antibodies against dengue and other viruses with deletion in fc region
WO2009089004A1 (en) 2008-01-07 2009-07-16 Amgen Inc. Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
WO2009125825A1 (ja) 2008-04-11 2009-10-15 中外製薬株式会社 複数分子の抗原に繰り返し結合する抗原結合分子
WO2011028952A1 (en) 2009-09-02 2011-03-10 Xencor, Inc. Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens
WO2011131746A2 (en) 2010-04-20 2011-10-27 Genmab A/S Heterodimeric antibody fc-containing proteins and methods for production thereof
WO2012023053A2 (en) 2010-08-16 2012-02-23 Novimmune S.A. Methods for the generation of multispecific and multivalent antibodies
WO2012058768A1 (en) 2010-11-05 2012-05-10 Zymeworks Inc. Stable heterodimeric antibody design with mutations in the fc domain
WO2012082841A2 (en) 2010-12-14 2012-06-21 University Of Maryland, Baltimore Universal anti-tag chimeric antigen receptor-expressing t cells and methods of treating cancer
WO2013047752A1 (ja) 2011-09-30 2013-04-04 中外製薬株式会社 抗原の消失を促進する抗原結合分子
WO2013063702A1 (en) 2011-11-04 2013-05-10 Zymeworks Inc. Stable heterodimeric antibody design with mutations in the fc domain
WO2013120629A1 (en) 2012-02-15 2013-08-22 Curevac Gmbh Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein
US20130336973A1 (en) 2012-05-10 2013-12-19 Zymeworks Inc. Heteromultimer Constructs of Immunoglobulin Heavy Chains with Mutations in the Fc Domain
WO2014018572A2 (en) 2012-07-23 2014-01-30 Zymeworks Inc. Immunoglobulin constructs comprising selective pairing of the light and heavy chains
US20140080153A1 (en) 2011-01-07 2014-03-20 Chugai Seiyaku Kabushiki Kaisha Method for improving physical properties of antibody
WO2015156268A1 (ja) 2014-04-07 2015-10-15 中外製薬株式会社 免疫活性化抗原結合分子
WO2016040441A1 (en) 2014-09-09 2016-03-17 Unum Therapeutics Chimeric receptors and uses thereof in immune therapy
WO2016047722A1 (ja) 2014-09-26 2016-03-31 中外製薬株式会社 細胞傷害誘導治療剤
EP2101823B1 (en) 2007-01-09 2016-11-23 CureVac AG Rna-coded antibody
WO2017161333A1 (en) 2016-03-18 2017-09-21 Unum Therapeutics Modified chimeric receptors and uses thereof in immune therapy
WO2018002358A1 (en) 2016-06-30 2018-01-04 F. Hoffmann-La Roche Ag Improved adoptive t-cell therapy
WO2018177966A1 (en) 2017-03-27 2018-10-04 F. Hoffmann-La Roche Ag Improved antigen binding receptors
WO2019116046A1 (en) * 2017-12-15 2019-06-20 Autolus Limited Cell
WO2019129220A1 (en) * 2017-12-28 2019-07-04 Nanjing Legend Biotech Co., Ltd. Multispecific chimeric receptors comprising nkg2d domain and methods of use thereof
WO2019140425A1 (en) 2018-01-15 2019-07-18 Pfizer Inc. Methods of administering chimeric antigen receptor immunotherapy in combination with 4-1bb agonist
JP2019537429A (ja) * 2016-09-23 2019-12-26 メルス ナムローゼ フェンノートシャップ 細胞によって発現される生物活性を調節する結合分子
JP2020504132A (ja) * 2017-01-10 2020-02-06 ザ ジェネラル ホスピタル コーポレイション キメラ抗原受容体を発現するｔ細胞

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3144388B1 (en) * 2014-05-13 2020-07-01 Chugai Seiyaku Kabushiki Kaisha T cell-redirecting antigen-binding molecule for cells having immunosuppression function
SI3194434T1 (sl) * 2014-09-15 2019-11-29 Molmed Spa Himerni antigenski receptorji
CN113862292B (zh) * 2015-01-29 2024-12-27 明尼苏达大学董事会 嵌合抗原受体、组合物以及方法
TW201632559A (zh) * 2015-02-22 2016-09-16 索倫多醫療公司 結合ｃｄ１３７之抗體治療劑
AU2016297014B2 (en) * 2015-07-21 2021-06-17 Novartis Ag Methods for improving the efficacy and expansion of immune cells
CN111511762B (zh) * 2017-08-21 2025-05-06 天演药业公司 抗cd137分子及其用途
AU2018329741B2 (en) * 2017-09-08 2025-02-20 Poseida Therapeutics, Inc. Compositions and methods for chimeric ligand receptor (CLR)-mediated conditional gene expression
US11851497B2 (en) * 2017-11-20 2023-12-26 Compass Therapeutics Llc CD137 antibodies and tumor antigen-targeting antibodies and uses thereof
KR102752985B1 (ko) * 2017-12-20 2025-01-10 포세이다 테라퓨틱스, 인크. Vcar 조성물 및 사용 방법
JP2024508938A (ja) * 2021-03-12 2024-02-28 ザ・ジョンズ・ホプキンス・ユニバーシティ 操作を受けた免疫細胞及びその使用
KR20240095012A (ko) * 2022-11-29 2024-06-25 (주) 테라베스트 Cd137 세포외 도메인을 포함하는 융합단백질, 이를 발현하는 면역세포 및 이의 용도

Patent Citations (87)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773719A (en) 1966-12-06 1973-11-20 Hoffmann La Roche 2-aminoxy-2'-acyl-acetanilide
EP0058481A1 (en) 1981-02-16 1982-08-25 Zeneca Limited Continuous release pharmaceutical compositions
US4650764A (en) 1983-04-12 1987-03-17 Wisconsin Alumni Research Foundation Helper cell
EP0133988A2 (de) 1983-08-02 1985-03-13 Hoechst Aktiengesellschaft Regulatorische Peptide enthaltende pharmazeutische Präparate mit protrahierter Freisetzung und Verfahren zu deren Herstellung
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
EP0239400A2 (en) 1986-03-27 1987-09-30 Medical Research Council Recombinant antibodies and methods for their production
WO1988001649A1 (en) 1986-09-02 1988-03-10 Genex Corporation Single polypeptide chain binding molecules
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5278056A (en) 1988-02-05 1994-01-11 The Trustees Of Columbia University In The City Of New York Retroviral packaging cell lines and process of using same
US5124263A (en) 1989-01-12 1992-06-23 Wisconsin Alumni Research Foundation Recombination resistant retroviral helper cell and products produced thereby
US5399346A (en) 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
WO1992001047A1 (en) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
WO1992020791A1 (en) 1990-07-10 1992-11-26 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
WO1992003918A1 (en) 1990-08-29 1992-03-19 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1993006213A1 (en) 1991-09-23 1993-04-01 Medical Research Council Production of chimeric antibodies - a combinatorial approach
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
WO1993011236A1 (en) 1991-12-02 1993-06-10 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
WO1993012227A1 (en) 1991-12-17 1993-06-24 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1993017706A1 (en) 1992-03-11 1993-09-16 Agracetus, Inc. Genetic vaccine for immunodeficiency viruses
WO1993019172A1 (en) 1992-03-24 1993-09-30 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
WO1994002602A1 (en) 1992-07-24 1994-02-03 Cell Genesys, Inc. Generation of xenogeneic antibodies
WO1994011523A2 (en) 1992-11-13 1994-05-26 Idec Pharmaceuticals Corporation Fully impaired consensus kozac sequences for mammalian expression
WO1994025585A1 (en) 1993-04-26 1994-11-10 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1995001438A1 (en) 1993-06-30 1995-01-12 Medical Research Council Sbp members with a chemical moiety covalently bound within the binding site; production and selection thereof
WO1995001937A1 (fr) 1993-07-09 1995-01-19 Association Gradient Procede de traitement de residus de combustion et installation de mise en ×uvre dudit procede
WO1995007358A1 (en) 1993-07-30 1995-03-16 University Of Medicine & Dentistry Of New Jersey Efficient gene transfer into primary lymphocytes
WO1995015388A1 (en) 1993-12-03 1995-06-08 Medical Research Council Recombinant binding proteins and peptides
WO1995033844A1 (de) 1994-06-03 1995-12-14 GSF - Forschungszentrum für Umwelt und Gesundheit GmbH Verfahren zur herstellung von heterologen bispezifischen antikörpern
WO1996002576A1 (en) 1994-07-13 1996-02-01 Chugai Seiyaku Kabushiki Kaisha Reconstituted human antibody against human interleukin-8
WO1996010038A1 (en) 1994-09-28 1996-04-04 Apollon, Inc. Multifunctional molecular complexes for gene transfer to cells
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
WO1996027011A1 (en) 1995-03-01 1996-09-06 Genentech, Inc. A method for making heteromultimeric polypeptides
WO1996033735A1 (en) 1995-04-27 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1997018185A1 (fr) 1995-11-14 1997-05-22 Rhone-Poulenc Rorer S.A. Lipopolymamines comme agents de transfection et leurs applications pharmaceutiques
WO1997025329A1 (fr) 1996-01-10 1997-07-17 Rhone-Poulenc Rorer S.A. DERIVES DE 5H,10H-IMIDAZO[1,2-a]INDOLO[3,2-e]PYRAZINE-4-ONE, LEUR PREPARATION ET LES MEDICAMENTS LES CONTENANT
WO1997030170A1 (de) 1996-02-15 1997-08-21 Boehringer Ingelheim International Gmbh Zusammensetzung für die transfektion höherer eukaryotischer zellen
WO1997031934A2 (de) 1996-02-29 1997-09-04 Chemicon Laboratories Gmbh Neue metabolisierbare lipopolyamine, deren darstellung und anwendung
WO1998050431A2 (en) 1997-05-02 1998-11-12 Genentech, Inc. A method for making multispecific antibodies having heteromultimeric and common components
WO2000042072A2 (en) 1999-01-15 2000-07-20 Genentech, Inc. Polypeptide variants with altered effector function
US20060025576A1 (en) 2000-04-11 2006-02-02 Genentech, Inc. Multivalent antibodies and uses therefor
WO2002020565A2 (en) 2000-09-08 2002-03-14 Universität Zürich Collections of repeat proteins comprising repeat modules
WO2002032925A2 (en) 2000-10-16 2002-04-25 Phylos, Inc. Protein scaffolds for antibody mimics and other binding proteins
WO2003000883A1 (fr) 2001-06-22 2003-01-03 Chugai Seiyaku Kabushiki Kaisha Inhibiteurs de proliferation cellulaire renfermant un anticorps anti-glypicane 3
WO2003029462A1 (en) 2001-09-27 2003-04-10 Pieris Proteolab Ag Muteins of human neutrophil gelatinase-associated lipocalin and related proteins
WO2003104453A1 (ja) 2002-06-05 2003-12-18 中外製薬株式会社 抗体作製方法
WO2004022754A1 (ja) 2002-09-04 2004-03-18 Chugai Seiyaku Kabushiki Kaisha MRL/lprマウスを用いた抗体の作製
WO2004044011A2 (en) 2002-11-06 2004-05-27 Avidia Research Institute Combinatorial libraries of monomer domains
WO2004065611A1 (ja) 2003-01-21 2004-08-05 Chugai Seiyaku Kabushiki Kaisha 抗体の軽鎖スクリーニング方法
WO2005040229A2 (en) 2003-10-24 2005-05-06 Avidia, Inc. Ldl receptor class a and egf domain monomers and multimers
WO2006006693A1 (ja) 2004-07-09 2006-01-19 Chugai Seiyaku Kabushiki Kaisha 抗グリピカン３抗体
WO2006019447A1 (en) 2004-07-15 2006-02-23 Xencor, Inc. Optimized fc variants
US7435596B2 (en) 2004-11-04 2008-10-14 St. Jude Children's Research Hospital, Inc. Modified cell line and method for expansion of NK cell
US8026097B2 (en) 2004-11-04 2011-09-27 St. Jude Children's Research Hospital Expansion of NK cells and therapeutic uses thereof
WO2006106905A1 (ja) 2005-03-31 2006-10-12 Chugai Seiyaku Kabushiki Kaisha 会合制御によるポリペプチド製造方法
WO2006132352A1 (ja) 2005-06-10 2006-12-14 Chugai Seiyaku Kabushiki Kaisha sc(Fv)2を含有する医薬組成物
WO2007114325A1 (ja) 2006-03-31 2007-10-11 Chugai Seiyaku Kabushiki Kaisha 二重特異性抗体を精製するための抗体改変方法
WO2008016854A2 (en) 2006-08-02 2008-02-07 The Uab Research Foundation Methods and compositions related to soluble monoclonal variable lymphocyte receptors of defined antigen specificity
EP2101823B1 (en) 2007-01-09 2016-11-23 CureVac AG Rna-coded antibody
WO2008119353A1 (en) 2007-03-29 2008-10-09 Genmab A/S Bispecific antibodies and methods for production thereof
WO2009011941A2 (en) 2007-04-04 2009-01-22 The Government Of U.S.A., As Represented By The Secretary, Departmetnt Of Health & Human Services Monoclonal antibodies against dengue and other viruses with deletion in fc region
WO2009089004A1 (en) 2008-01-07 2009-07-16 Amgen Inc. Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
WO2009125825A1 (ja) 2008-04-11 2009-10-15 中外製薬株式会社 複数分子の抗原に繰り返し結合する抗原結合分子
WO2011028952A1 (en) 2009-09-02 2011-03-10 Xencor, Inc. Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens
WO2011131746A2 (en) 2010-04-20 2011-10-27 Genmab A/S Heterodimeric antibody fc-containing proteins and methods for production thereof
WO2012023053A2 (en) 2010-08-16 2012-02-23 Novimmune S.A. Methods for the generation of multispecific and multivalent antibodies
WO2012058768A1 (en) 2010-11-05 2012-05-10 Zymeworks Inc. Stable heterodimeric antibody design with mutations in the fc domain
WO2012082841A2 (en) 2010-12-14 2012-06-21 University Of Maryland, Baltimore Universal anti-tag chimeric antigen receptor-expressing t cells and methods of treating cancer
US20140080153A1 (en) 2011-01-07 2014-03-20 Chugai Seiyaku Kabushiki Kaisha Method for improving physical properties of antibody
WO2013047752A1 (ja) 2011-09-30 2013-04-04 中外製薬株式会社 抗原の消失を促進する抗原結合分子
WO2013063702A1 (en) 2011-11-04 2013-05-10 Zymeworks Inc. Stable heterodimeric antibody design with mutations in the fc domain
WO2013120629A1 (en) 2012-02-15 2013-08-22 Curevac Gmbh Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein
US20130336973A1 (en) 2012-05-10 2013-12-19 Zymeworks Inc. Heteromultimer Constructs of Immunoglobulin Heavy Chains with Mutations in the Fc Domain
WO2014018572A2 (en) 2012-07-23 2014-01-30 Zymeworks Inc. Immunoglobulin constructs comprising selective pairing of the light and heavy chains
WO2015156268A1 (ja) 2014-04-07 2015-10-15 中外製薬株式会社 免疫活性化抗原結合分子
WO2016040441A1 (en) 2014-09-09 2016-03-17 Unum Therapeutics Chimeric receptors and uses thereof in immune therapy
WO2016047722A1 (ja) 2014-09-26 2016-03-31 中外製薬株式会社 細胞傷害誘導治療剤
WO2017161333A1 (en) 2016-03-18 2017-09-21 Unum Therapeutics Modified chimeric receptors and uses thereof in immune therapy
WO2018002358A1 (en) 2016-06-30 2018-01-04 F. Hoffmann-La Roche Ag Improved adoptive t-cell therapy
JP2019537429A (ja) * 2016-09-23 2019-12-26 メルス ナムローゼ フェンノートシャップ 細胞によって発現される生物活性を調節する結合分子
JP2020504132A (ja) * 2017-01-10 2020-02-06 ザ ジェネラル ホスピタル コーポレイション キメラ抗原受容体を発現するｔ細胞
WO2018177966A1 (en) 2017-03-27 2018-10-04 F. Hoffmann-La Roche Ag Improved antigen binding receptors
WO2019116046A1 (en) * 2017-12-15 2019-06-20 Autolus Limited Cell
WO2019129220A1 (en) * 2017-12-28 2019-07-04 Nanjing Legend Biotech Co., Ltd. Multispecific chimeric receptors comprising nkg2d domain and methods of use thereof
WO2019140425A1 (en) 2018-01-15 2019-07-18 Pfizer Inc. Methods of administering chimeric antigen receptor immunotherapy in combination with 4-1bb agonist

Non-Patent Citations (133)

* Cited by examiner, † Cited by third party
Title
"Cell Technology suppl. Handbook", 1994, GAKKEN MEDICAL SHUJUNSHA CO., article "Adhesion Factor Handbook"
"GenBank", Database accession no. KF957646.1
"Genbank", Database accession no. NM000734.4
"Harrison's Principles of Internal Medicine", 2001, MCGRAW HILL
"Methods in Molecular Biology", vol. 66, 1996, article "Epitope Mapping Protocols"
"NCBI", Database accession no. NP _036224.1
"New Comprehensive Biochemistry", vol. 18, 1988, ELSEVIER SCIENCE PUBLISHERS, article "Hormones and their Actions Part II", pages: 1 - 46
"Remington's Pharmaceutical Science", 1980
"Remington's Pharmaceutical Sciences", 1991, MACK PUB. CO.
"Remington's The Science and Practice of Pharmacy", 2000, LIPPINCOTT WILLIAMS AND WILKINS
"The Merck Manual of Diagnosis and Therapy", 1992, MERCK RESEARCH LABORATORIES
ADOLPH: "Viral Genome Method", 1996, CRC PRESS
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10
ANAL. BIOCHEM., vol. 162, no. 1, 1987, pages 156 - 1111
ANNU. REV. BIOPHYS. BIOMOL. STRUCT., vol. 35, 2006, pages 225 - 249
ANTIBODIES, vol. 4, no. 3, 2015, pages 141 - 156
BATZER ET AL., NUCLEIC ACID RES., vol. 19, 1991, pages 5081
BIO/TECHNOLOGY, vol. 12, no. 7, 1994, pages 699 - 702
BIOCHEMISTRY, vol. 18, no. 24, 1979, pages 5294 - 5299
BIOPOLYMERS, vol. 22, 1983, pages 547 - 556
BLOOD, vol. 109, 2007, pages 1185 - 1192
BLOOD, vol. 76, no. 1, 1990, pages 31 - 35
BRENNAN ET AL., SCIENCE, vol. 229, no. 81, 1985
C. EUR. J. IMMUNOL., vol. 6, no. 7, 1976, pages 511 - 519
CAROLINE J.: "Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research", 2004, HUMANA PRESS
CELL, vol. 57, no. 2, 1989, pages 277 - 285
CELL, vol. 69, no. 6, 1992, pages 1067 - 1070
CELL, vol. 8, no. 3, 1976, pages 405 - 415
CHEMTECH., vol. 12, 1982, pages 98 - 105
CHEN ET AL., J. MOL. BIOL., vol. 293, 1999, pages 865 - 881
CHIN SM ET AL., NAT COMMUN., vol. 9, 2018, pages 4679
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHRISTOPH ET AL., NATURE BIOTECHNOLOGY, vol. 31, 2013, pages 753 - 758
CLARK, M., CHEMICAL IMMUNOLOGY, vol. 65, 1997, pages 88 - 110
DALBADIE-MCFARLAND, G. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 79, 1982, pages 6409 - 13
DAO ET AL., SCI TRANSL MED, vol. 5, no. 176, 2013, pages 176 - 33
DAVIS ET AL., IMMUNOLOGICAL REVIEWS, vol. 190, 2002, pages 123 - 136
EMBO J., vol. 12, no. 7, 1993, pages 2645 - 53
FANTL ET AL., ANNU. REV. BIOCHEM., vol. 62, 1993, pages 453 - 481
FEHNIGER T ACALIGIURI M A., INT REV IMMUNOL, vol. 20, no. 3-4, 2001, pages 503 - 534
FLOWER DR., BIOCHIM. BIOPHYS. ACTA, vol. 1422, no. 3, 1999, pages 207 - 234
GREENWOOD JCLARK MWALDMANN H., EUR. J. IMMUNOL., vol. 23, 1993, pages 1098 - 1104
GRUPP ET AL., N. ENGL. J. MED., vol. 368, no. 16, 2013, pages 1509 - 1518
HARADA H ET AL., EXP HEMATOL, vol. 32, no. 7, 2004, pages 614 - 621
HARADA H. ET AL., JPN. J. CANCER RES, vol. 93, 2002, pages 313 - 319
HASHIMOTO-GOTOH, TMIZUNO, TOGASAHARA, YNAKAGAWA, M.: "An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis.", GENE, vol. 152, 1995, pages 271 - 275, XP004042690, DOI: 10.1016/0378-1119(94)00750-M
HODI FS ET AL., N ENGL J MED., vol. 363, 2010, pages 711
HOLLINGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 6448
HOUOT R ET AL., BLOOD., vol. 114, 2009, pages 3431
HUM. ANTIBOD. HYBRIDOMAS, vol. 1, no. 1, 1990, pages 47 - 54
INT J CANCER., vol. 103, no. 4, 2003, pages 455 - 65
J CLIN INVEST., vol. 32, no. 8, August 1953 (1953-08-01), pages 746 - 768
J IMMUNOL. METHODS, vol. 231, no. 1-2, 1999, pages 177 - 189
J IMMUNOL., vol. 162, no. 2, 1999, pages 897 - 902
J PHARM SCI., vol. 97, no. 10, October 2008 (2008-10-01), pages 4167 - 83
J. BIOMED. MATER. RES., vol. 15, 1981, pages 267 - 277
J. EXP. MED., vol. 148, no. 1, 1978, pages 313 - 323
J. IMMUNOL. METHODS, vol. 35, no. 1-2, 1980, pages 1 - 21
J. IMMUNOL., vol. 123, no. 4, 1979, pages 1548 - 1550
J. RHEUMATOL, vol. 34, 2007, pages 11
JOURNAL OF IMMUNOLOGY, vol. 152, no. 11, 1994, pages 5368 - 5374
JUNE CHSADELAIN M, N ENGL J MED., vol. 379, 2018, pages 64
KARCHES CH ET AL., CLIN CANCER RES., vol. 25, 2019, pages 5890
KLEIN ET AL., INT. J. CANCER, vol. 18, 1976, pages 421 - 431
KOHLER, MILSTEIN, METHODS ENZYMOL., vol. 73, 1981, pages 3 - 46
KOSTELNY ET AL., J. IMMUNOL., vol. 148, no. 5, 1992, pages 1547 - 1553
KRAMER WFRITZ HJ: "Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods", ENZYMOL, vol. 154, 1987, pages 350 - 367
KRAMER, WDRUTSA, VJANSEN, HWKRAMER, BPFLUGFELDER, MFRITZ, HJ: "The gapped duplex DNA approach to oligonucleotide-directed mutation construction.", NUCLEIC ACIDS RES., vol. 12, 1984, pages 9441 - 9456, XP002026371
KUDO K ET AL., CANCER RES., vol. 74, 2014, pages 93
KUNKEL, PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 488 - 492
KUNKEL, TA: "Rapid and efficient site-specific mutagenesis without phenotypic selection.", PROC NATL ACAD SCI USA., vol. 82, 1985, pages 488 - 492, XP002052322, DOI: 10.1073/pnas.82.2.488
KUO ET AL., BLOOD, vol. 82, 1993, pages 845
LABRIJN ET AL., PROC. NATL. ACAD. SCI., vol. 110, 2013, pages 5145
LOZZIO ET AL., BLOOD, vol. 45, no. 3, 1975, pages 321 - 334
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MANN ET AL., CELL, vol. 33, 1983, pages 153
MARDIANA S ET AL., CANCER RES., vol. 77, 2017, pages 1296
MARK, D. F. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 5662 - 6
MERCHANT AM ET AL., NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 677 - 681
METHODS ENZYMOL., vol. 323, 2000, pages 325 - 40
MICROBIOLOGY AND IMMUNOLOGY, vol. 81, 1978, pages 1 - 7
MILONE ET AL., MOL. THER., vol. 17, no. 8, 2009, pages 1453 - 1464
MILSTEIN C ET AL., NATURE, vol. 305, 1983, pages 537 - 540
MIYAJIMA ET AL., ANNU. REV. IMMUNOL., vol. 10, 1992, pages 295 - 331
MOL CELL BIOL., vol. 8, 1988, pages 466 - 472
MORGAN AJONES NDNESBITT AMCHAPLIN LBODMER MWEMTAGE JS., IMMUNOLOGY, vol. 86, 1995, pages 319 - 324
MULLBERG ET AL., J. IMMUNOL., vol. 152, no. 10, 1994, pages 5368 - 4968
NAT BIOTECHNOL., vol. 32, no. 2, February 2014 (2014-02-01), pages 191 - 8
NATURE, vol. 276, no. 5685, 1978, pages 269 - 270
NATURE, vol. 277, no. 5692, 1979, pages 131 - 133
NATURE, vol. 313, no. 6005, 1985, pages 756 - 761
NATURE, vol. 338, 1989, pages 383 - 384
NUCLEIC ACIDS RES., vol. 17, no. 8, 1989, pages 2919 - 2932
OHTSUKA ET AL., J. BIOL. CHEM., vol. 260, 1985, pages 2605 - 2608
PROC NATL ACAD SCI USA., vol. 86, no. 1, 1989, pages 27 - 31
PROC NATL ACAD SCI USA., vol. 89, no. 12, 1992, pages 5640 - 5644
PROC. NATL. ACAD. SCI. U.S.A., vol. 100, no. 11, 2003, pages 6353 - 6357
PROC. NATL. ACAD. SCI. U.S.A., vol. 85, no. 16, 1988, pages 5879 - 5883
PROC. NATL. ACAD. SCI. USA, vol. 103, no. 11, 2006, pages 4005 - 4010
PROC. NATL. ACAD. SCI. USA, vol. 85, no. 23, 1988, pages 8998 - 9002
PROC. NATL. ACAD. SCI. USA., vol. 85, no. 10, 1988, pages 3435 - 3439
PROC. NATL. ACAD. SCI. USA., vol. 87, no. 22, 1990, pages 8702 - 8706
PROC. NATL. ACAD. SCI. USA., vol. 91, no. 2, 1994, pages 459 - 463
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 85, 1988, pages 6460 - 6464
PROTEIN ENGINEERING DESIGN & SELECTION, vol. 23, 2010, pages 195 - 202
PROTEIN SCIENCE, vol. 4, 1995, pages 2411 - 2423
RIDGWAY JB ET AL., PROTEIN ENGINEERING, vol. 9, no. 3, 1996, pages 617 - 621
ROBERT C ET AL., N ENGL J MED., vol. 372, 2015, pages 320
ROSSOLINI ET AL., MOL. CELL. PROBES, vol. 8, 1994, pages 91 - 98
SASTRY ET AL., J VIROL., vol. 85, no. 5, 2011, pages 1935 - 1942
SATO, K. ET AL., CANCER RES., vol. 53, 1993, pages 851 - 856
SCHMITZ J ET AL., J IMMUNOL., vol. 164, 2000, pages 848 - 54
SCIENCE, vol. 240, no. 4855, 1988, pages 1038 - 1041
See also references of EP4197545A4
SERGEEVA ET AL., BOOD, vol. 117, no. 16, 2011, pages 4262 - 4272
SMITH ET AL., CELL, vol. 77, no. 3, 1994, pages 391 - 400
TAGA ET AL., FASEB J., vol. 6, 1992, pages 3387 - 3396
TAMADA ET AL., CLIN CANCER RES., vol. 18, 2012, pages 6436
TAMADA K ET AL., CLIN CANCER RES, vol. 18, 2012, pages 6436 - 6445
TASSEV ET AL., CANCER GENE THER, vol. 19, no. 2, 2012, pages 84 - 100
THOMSON P D RMONTVALE N.J., PHYSICIAN'S DESK REFERENCE, 2005
TOMIDA M ET AL., BLOOD, vol. 93, 1999, pages 1934 - 41
TORIKAI ET AL., BLOOD, vol. 119, 2012, pages 5697 - 5705
TRAUNECKER ET AL., EMBO J., vol. 10, 1991, pages 3655
TUTT ET AL., J. IMMUNOL., vol. 147, no. 60, 1991
ULLRICH ET AL., CELL, vol. 60, no. 2, 1990, pages 225 - 234
URBANSKA K ET AL., J TRANSL MED., vol. 12, 2014, pages 347
VANDAMME ET AL., EUR. J. BIOCHEM., vol. 192, no. 3, 1990, pages 767 - 775
VERMA ET AL., J IMMUNOL, vol. 184, no. 4, 2010, pages 2156 - 2165
WANG, A. ET AL., SCIENCE, vol. 224, 1984, pages 1431 - 3
WILLEMSEN ET AL., GENE THER, vol. 8, no. 21, 2001, pages 1601 - 1608
ZOLLER, M. J.SMITH, M., NUCLEIC ACIDS RES., vol. 10, 1982, pages 6487 - 500
ZOLLER, MJSMITH, M.: "Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.", METHODS ENZYMOL., vol. 100, 1983, pages 468 - 500, XP001088749, DOI: 10.1016/0076-6879(83)00074-9

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023174925A1 (en) * 2022-03-14 2023-09-21 Novimmune Sa Bispecific gpc3xcd28 and gpc3xcd3 antibodies and their combination for targeted killing of gpc3 positive malignant cells
US12122850B2 (en) 2022-03-14 2024-10-22 LamKap Bio gamma AG Bispecific GPC3xCD28 and GPC3xCD3 antibodies and their combination for targeted killing of GPC3 positive malignant cells

Also Published As

Publication number Publication date
EP4197545A1 (en) 2023-06-21
JPWO2022025220A1 (https=) 2022-02-03
KR20230048059A (ko) 2023-04-10
TW202220677A (zh) 2022-06-01
BR112023000537A2 (pt) 2023-01-31
CN116194124A (zh) 2023-05-30
IL300225A (en) 2023-03-01
EP4197545A4 (en) 2025-04-09
MX2023001120A (es) 2023-02-22
US20230322898A1 (en) 2023-10-12
CA3190649A1 (en) 2022-02-03
AU2021317974A1 (en) 2023-03-16

Similar Documents

Publication Publication Date Title
JP7777183B2 (ja) Ｃｄ３およびｃｄ１３７に結合することができるが、同時には結合しない、抗原結合分子
JP7357616B2 (ja) Ｃｄ３およびｃｄ１３７に結合する改変された抗体可変領域を含む抗原結合分子
US11236168B2 (en) Mouse FcγammaRII-specific Fc antibody
JP2024105621A (ja) 改変された抗体可変領域を含む抗原結合分子
CN113905757B (zh) 抗体切割位点结合分子
EP3546574A1 (en) Antigen-binding domain, and polypeptide including conveying section
KR20160056940A (ko) 폴리펩티드 이종 다량체의 제조방법
TW201420604A (zh) ＦｃγＲＩＩｂ特異性Ｆｃ區域變異體
US20200369781A1 (en) Polypeptide including antigen-binding domain and carrying section
KR20220035369A (ko) 고 친화도 항-cd3 항체, 및 이의 생성 및 사용 방법
US20230322898A1 (en) Pharmaceutical composition comprising cell expressing chimeric receptor
HK40090466A (zh) 包含表达嵌合受体的细胞的药物组合物
EP4729126A1 (en) Nk cell-derived extracellular vesicles armed with modified monoclonal antibodies to target cancer cells, and use thereof
RU2850715C2 (ru) Антигенсвязывающая молекула, содержащая измененную вариабельную область антитела
HK40061688B (zh) 抗体切割位点结合分子
WO2025169945A1 (ja) 安定性が改善されたｃｄ３標的抗原結合分子
HK40061688A (en) Antibody cleavage site-binding molecule
HK40102459A (zh) 用於通用细胞疗法的武装nk细胞

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21850145

Country of ref document: EP

Kind code of ref document: A1

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112023000537

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 3190649

Country of ref document: CA

Ref document number: 2022539585

Country of ref document: JP

Kind code of ref document: A

Ref document number: 112023000537

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20230111

ENP Entry into the national phase

Ref document number: 20237006010

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2021850145

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2021850145

Country of ref document: EP

Effective date: 20230228

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021317974

Country of ref document: AU

Date of ref document: 20210730

Kind code of ref document: A