CN113698491B - 融合蛋白及靶向cd19的car及其应用 - Google Patents

融合蛋白及靶向cd19的car及其应用 Download PDF

Info

Publication number
CN113698491B
CN113698491B CN202110556149.8A CN202110556149A CN113698491B CN 113698491 B CN113698491 B CN 113698491B CN 202110556149 A CN202110556149 A CN 202110556149A CN 113698491 B CN113698491 B CN 113698491B
Authority
CN
China
Prior art keywords
car
leu
val
thr
gly
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.)
Active
Application number
CN202110556149.8A
Other languages
English (en)
Other versions
CN113698491A (zh
Inventor
陈军
沈俊杰
徐艳敏
杨智
洪娟
梅恩典
赵永春
赵文旭
黄霞
齐亚男
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.)
Chongqing Jingzhun Biological Industrial Technology Institute Co ltd
Chongqing Precision Biotech Co ltd
Original Assignee
Chongqing Jingzhun Biological Industrial Technology Institute Co ltd
Chongqing Precision Biotech Co ltd
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 Chongqing Jingzhun Biological Industrial Technology Institute Co ltd, Chongqing Precision Biotech Co ltd filed Critical Chongqing Jingzhun Biological Industrial Technology Institute Co ltd
Publication of CN113698491A publication Critical patent/CN113698491A/zh
Application granted granted Critical
Publication of CN113698491B publication Critical patent/CN113698491B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—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
    • 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
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4636—Immune checkpoint inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46448—Cancer antigens from embryonic or fetal origin
    • A61K39/464482—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464493—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
    • 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • 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
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • C12N15/79—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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 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 A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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 A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Oncology (AREA)
  • General Engineering & Computer Science (AREA)
  • Gynecology & Obstetrics (AREA)
  • Pregnancy & Childbirth (AREA)
  • Reproductive Health (AREA)
  • Plant Pathology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Developmental Biology & Embryology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)

Abstract

本发明属于肿瘤细胞免疫治疗技术领域，具体涉及一种融合蛋白及其靶向CD19的CAR及其应用。所述融合蛋白为SIRPγ融合蛋白，包含胞外段、跨膜结构和胞内信号区。该融合蛋白和靶向CD19的CAR结构连接共同或单独转染免疫细胞,该免疫细胞在缺氧环境下活性增强，而且以逆转肿瘤微环境，破除肿瘤组织中抑制性信号对CAR‑T功能的影响，实现了CAR‑T治疗有效性，同时能够保证一定的安全性。

Description

融合蛋白及靶向CD19的CAR及其应用
技术领域
本发明属于肿瘤细胞免疫治疗技术领域，具体涉及一种融合蛋白及靶向CD19的CAR及其应用。
背景技术
CAR-T全称是嵌合抗原受体T细胞免疫疗法，嵌合抗原受体(chimeric antigenreceptor，CAR)是模拟TCR功能的人工受体，由抗原识别域、铰链区和跨膜区及胞内信号域依次连接组成，胞内信号域通常为CD3ζ链或FcRγ，或与一种或多种共刺激分子相连，如4-1BB，CD28，ICOS(CD278)。CAR分子包含的ScFv对肿瘤抗原具有特异性识别的特点，通过铰链区和跨膜区进行T细胞激活细胞传递。目前临床上CAR-T治疗主要运用于血液瘤***的肿瘤治疗。
CAR-T疗法虽然在血液***瘤中取得较好的成绩，但CAR-T在实体瘤中的效果不如血液***瘤，其原因一方面因为CAR-T较难进入实体瘤内部，一方面即便CAR-T细胞进入实体瘤内部也因为肿瘤微环境而不能正常发挥功能，这些都影响CAR-T细胞在实体瘤治疗的疗效；并且由于实体瘤异质程度高，实体瘤靶点往往在正常组织也有所表达，因此还存在脱靶风险等安全问题。虽然目前有构建***CAR、有构建双CAR(构建带有双抗原的CAR-T)或带有激活抑制功能的iCAR的结构以期望提高肿瘤CAR-T治疗的有效性和安全性，但这些CAR结构仍存在安全性或较难被激活的问题。总之CAR-T疗法目前较多研究为血液瘤治疗，实体瘤微环境较为复杂，如肿瘤微环境、肿瘤异质性等特点，使CAR-T疗法的应用受到局限，因此，构建一种在肿瘤微环境中特异启动活化的CAR结构很有必要。酸性和缺氧是肿瘤微环境中的两大物理因素，肿瘤中的大部分的细胞都处在缺氧的环境中，而缺氧改变了肿瘤细胞的糖代谢途径，产生大量的乳酸，造成肿瘤微环境中酸性的特征，而这些酸性可以降低肿瘤细胞凋亡，增强细胞的增殖和生长，并且帮助肿瘤细胞的迁移。缺氧在实体瘤微环境研究比较清楚，如果可以设计在缺氧条件下被激活的可调控启动子，就可以达到在肿瘤微环境中特异性激活下游蛋白的表达从而提高CAR-T细胞的有效性和安全性的目的。另外信号调节蛋白α(SIRPα)是CD47的配体之一，可与CD47结合，抑制巨噬细胞的吞噬作用，目前绝大多数关于CD47的研究均集中于SIRPα。SIRPγ也可与CD47结合，其存在于T细胞表面，胞外区由一个V结构域和两个C1结构域组成，无胞内信号，仅通过CD47传递单向信号，目前很少有研究者针对SIRPγ进行改造，并且CD47作为继PD-1/PD-L1,CTLA-4之后新的免疫检查点，通过向巨噬细胞传递“不要吃我”信号，抑制固有免疫的进行。目前有文献报道采用靶向CD47 ScFv制备CAR-T进行肿瘤治疗。CD47可以促成肿瘤逃逸的免疫微环境，不过由于肿瘤的异质性，单独靶向CD47疗效有限，并且外源的ScFv由于其强的亲和力可能会导致CAR-T细胞过度活化存续性差和非特异性引起安全隐患。因此，采用更安全有效的方案去识别CD47破除肿瘤微环境的逃逸信号十分重要。
CD19是B细胞表面的跨膜蛋白(Cluster of differentiation 19protein，CD19)，它与B细胞活化、信号传导及生长调节密切相关，是B淋巴细胞表面特异性表达的一种功能受体分子，参与B细胞抗原受体(BCR)识别抗原和B细胞内Ca2+的转运，调节B细胞的活化与增殖。CD19作为重要的分子标记物，可用于白血病、淋巴瘤及免疫***疾病的诊断和预后判断。CD19作为靶点的治疗有效性好，安全性高。已有多项临床研究，探索利用CAR-T治疗CD19阳性B细胞恶性肿瘤。但是由于肿瘤微环境的影响，CAR-T在实体瘤的治疗效果有限。虽然目前靶向CD19的CAR-T制品在治疗淋巴细胞白血病上效果显著，但在治疗淋巴瘤上效果有限，构建一种针对肿瘤微环境的CAR-T细胞制品用于淋巴瘤的治疗很有必要。
发明内容
有鉴于此，本发明在于提供一种逆转肿瘤微环境的融合蛋白，其与靶向CD19的CAR结构a构成一种含有该融合蛋白的免疫细胞，该免疫细胞可以靶向杀伤CD47阳性的的肿瘤细胞；破除肿瘤组织中抑制性信号对免疫细胞功能的影响，实现了CAR-T治疗有效性，同时能够保证一定的安全性，其中含有融合蛋白的免疫细胞与不含有融合蛋白的免疫细胞相比较，含有融合蛋白的免疫细胞的体内存续时间会更长并且安全性更高。
进一步，所述融合蛋白为SIRPγ融合蛋白，所述SIRPγ融合蛋白结构包含胞外段、跨膜结构和胞内信号区，所述SIRPγ胞外段氨基酸序列如SEQ ID NO:5或其功能性变体所示；优选地，所述跨膜结构来源于人CD28跨膜区或人CD8来源跨膜区；优选地，所述跨膜结构氨基酸序列如SEQ ID NO:3所示或其功能性变体；或如SEQ ID NO:4所示或其功能性变体。
进一步，所述SIRPγ融合蛋白的结构为SIRPγ-CD28TM-CD28或SIRPγ-CD8TM-4-1BB；优选地，所述SIRPγ融合蛋白SIRPγ-CD28TM-CD28的氨基酸序列如SEQ ID NO:6所示或其功能性变体；或所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的氨基酸序列如SEQ ID NO:7所示或其功能性变体。
进一步，编码所述SIRPγ融合蛋白SIRPγ-CD28TM-CD28的核苷酸序列如SEQ IDNO:8所示；或编码所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的核苷酸序列如SEQ ID NO:9所示。
还进一步提供一种包含前述融合蛋白的表达载体。
进一步，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体中的任一种。
进一步还提供一种包含前述表达载体的免疫细胞，所述免疫细胞是T细胞、T细胞前体或者NK细胞。
进一步还提供一种制备前述免疫细胞的方法，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
进一步，所述免疫细胞包含具有识别肿瘤抗原的嵌合抗原受体结构，所述嵌合抗原受体包含识别肿瘤抗原的胞外段、hinge区、跨膜区和胞内信号区，所述肿瘤抗原为CD19。
本发明目的在于还提供一种包含前述融合蛋白的肿瘤免疫抑制抵抗型CAR，所述肿瘤免疫抑制抵抗型CAR是融合蛋白与CAR结构a结合而成。发明人认为SIRPγ蛋白更适合进行针对CD47进行肿瘤微环境破除的配体。因此，选择SIRPγ蛋白的胞外段进行改造设计，并进一步设计肿瘤免疫抑制抵抗型的CAR；因此本发明创造性的将SIRPγ蛋白联合CAR-T细胞技术应用到肿瘤的免疫治疗当中，即进一步对传统的CAR结构进行改造，提高CAR-T在实体瘤上的治疗药效及CAR-T的安全性。
进一步，所述免疫细胞还包括CAR结构a。所述CAR结构a可以是常规的第一代、第二代、第三代CAR结构，也可以是改进的双CAR、可调控CAR结构(如FRB/FKBP12调控)等新型CAR结构。
进一步，CAR结构a中的铰链区序列可以来源于：IgG、CD8、CD7、CD4；CAR结构中跨膜区可以来源于：CD8、CD28、CD3ε、CD4、CD16、CD137、CD80以及CD86；CAR结构中胞内信号区可来源于：CD3、CD137、CD28、CD27、OX40、ICOS、GITR、CD2、CD40、PD-1、PD1L、B7-H3、淋巴细胞功能相关抗原-1(LFA-1)、ICAM-1、CD7、NKG2C、CD83、CD86以及CD127。
优选地，所述CAR结构a包括CD19单链抗体、CD8铰链区、CD8跨膜区、CD137和CD3ξ双刺激信号；优选地，所述CAR结构a氨基酸序列如SEQ ID NO：1所示或其功能性变体。
具体地，所述融合蛋白通过多顺反子结构与CAR结构a连接，所述多顺反子结构为自剪切多肽或内部核糖体进入位点IRES，所述自剪切多肽为T2A、P2A、E2A或F2A。所述CAR结构a和融合蛋白组成的结构为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ融合蛋白或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ融合蛋白，即为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28。
在某些实施例中，所述CAR结构a带有截短的EGFRt调控标签；在某些实施例中，所述CAR结构a为通用型CAR结构；在某些实施例中，所述CAR结构a带有***基因如iCasp9。
在某些实施例中，所述CAR结构a包含天然杀伤细胞受体(NKR)的一种或多种组分，因而形成NKR-CAR。NKR组分可以是来自以下任何天然杀伤细胞受体的跨膜结构域、铰链结构域或胞质结构域：杀伤细胞免疫球蛋白样受体(KIR)，例如KIR2DL1、KIR2DL2/L3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR2DS1、KIR2DS2、KIR2DS3、KIR2DS4、DIR2DS5、KIR3DL1/S1、KIR3DL2、KIR3DL3、KIR2DP1和KIR3DP1；天然细胞毒性受体(NCR)，例如，NKp30、NKp44、NKp46；免疫细胞受体的信号传导淋巴细胞活化分子(SLAM)家族，例如，CD48、CD229、2B4、CD84、NTB-A、CRA、BLAME和CD2F-10；Fc受体(FcR)，例如，CD16、和CD64；和Ly49受体，例如，LY49A、LY49C。所述的NKR-CAR分子可以与衔接分子或胞内信号结构域(例如，DAP12)相互作用。
更优选的技术方案，所述免疫细胞中还包括缺氧启动子，所述缺氧启动子与CAR结合，所述缺氧可调控启动子的核酸序列包含如SEQ ID NO：2所示的序列。包含缺氧启动子的靶向CD19的CAR能够在缺氧微环境中有效的清除体内肿瘤作用,并且利用缺氧微环境诱导的启动子能够在缺氧环境强化目的基因、蛋白等因子的表达，提高肿瘤的药物疗效和CAR-T治疗的有效性和安全性，而且不仅可以有效的表达于T淋巴细胞，而且在缺氧环境中能够加强CAR分子表达，并且使CAR-T细胞有高的IFN-γ分泌能力，提高CAR-T细胞针对肿瘤靶细胞的杀伤，能够用于肿瘤的靶向治疗.
进一步，所述缺氧可调控启动子，由Hifla调节元件和迷你启动子连接构成；所述迷你启动子选自细胞病毒启动子、HSV胸苷激酶的启动子、猿猴病毒40的启动子、腺病毒晚期启动子和合成启动子中的任一项。
本发明发明目的在于还提供一种核酸序列，所述核酸序列编码前述的肿瘤免疫抑制抵抗型CAR，所述免疫细胞中不包含缺氧启动子，其包含如SEQ ID NO：10所示的序列；或所述免疫细胞中包含缺氧启动子，其包含如SEQ ID NO：11所示的序列。
本发明目的在于还提供一种包含上述核酸序列的重组质粒,所述重组质粒还包括表达载体，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体、质粒中的任一种。
在某些实施例中，所述慢病毒载体选自基本上由以下组成的群组：人免疫缺陷病毒1(HIV-1)、人免疫缺陷病毒2(HIV-2)、维斯纳-梅迪病毒(visna-maedi virus，VMV)病毒、山羊关节炎-脑炎病毒(CAEV)、马传染性贫血病毒(EIAV)、猫免疫缺陷病毒(FIV)、牛免疫缺陷病毒(BIV)和猿猴免疫缺陷病毒(SIV)。
在某些实施例中，载体包含左(5')逆转录病毒LTR、Psi(Ψ)包装信号、中心多嘌呤段/DNA瓣(cPPT/FLAP)、逆转录病毒导出元件、可操作地连接到编码本文所涵盖的CAR的多核苷酸的启动子和右(3')逆转录病毒LTR。
在某些实施例中，CAR包含乙型肝炎病毒转录后调节元件(HPRE)或土拔鼠转录后调节元件(WPRE)以及优化的土拔鼠转录后调节元件(oPRE)。
在某些实施例中，所述5'LTR的启动子经异源启动子置换。
在某些实施例中，所述异源启动子是巨细胞病毒(CMV)启动子、劳斯肉瘤病毒(Rous Sarcoma Virus，RSV)启动子或猿猴病毒40(SV40)启动子。
在某些实施例中，所述5'LTR或3'LTR是慢病毒LTR。
在某些实施例中，所述3'LTR是自我失活(SIN)LTR。
在某些实施例中，所述CAR结构的核酸序列包含优化的Kozark序列。
在某些实施例中，可操作地连接到编码本文所涵盖的CAR的多核苷酸的所述启动子以及以下组成的群组：巨细胞病毒立即早期基因启动子(CMV)、延伸因子1α启动子(EF1-α)、磷酸甘油酸激酶-1启动子(PGK)、泛素-C启动子(UBQ-C)、巨细胞病毒增强子/鸡β-肌动蛋白启动子(CAG)、多瘤病毒增强子/单纯疱疹胸苷激酶启动子(MC1)、β肌动蛋白启动子(β-ACT)、猿猴病毒40启动子(SV40)和骨髓增生肉瘤病毒增强子，阴性对照区缺失的、dl587rev引物结合位点取代的(MND)启动子。
在某些实施例中，包含CAR的载体可以包含分泌型抗PD-1ScFv；在某些实施例中，包含CAR的载体包含PD-1共轭转导肽(如PD-1-CD28-CD137-CD3信号结构)；在某些实施例中，包含CAR的载体多个CAR组合，如2个靶向不同抗原或同一抗原的不同识别位点的CAR组合。
本发明目的在于还提供一种提高缺氧环境下CAR-T细胞杀伤能力的方法，其特征在于，构建一种前任一所述的重组质粒并感染T淋巴细胞，然后作用于靶细胞。
进一步，还提供一种包含上述重组质粒的免疫细胞，所述免疫细胞是T细胞、T细胞前体或者NK细胞。
在某些实施例中，所述免疫细胞可以表达其它活性剂，例如，增强CAR表达细胞活性的活性剂。活性剂可以是阻断抑制性分子的活性剂。抑制性分子如PD1可以在一些实施方案中降低CAR表达细胞发动免疫效应子反应的能力。抑制性分子包括PD1、PD-L1、CTLA4、TIM3、LAG3、VISTA、BTLA、TIGIT,LAIR1、CD160、2B4、CEACAM(CEACAM-1、CEACAM-3、CEACAM-5)、LAG3、VISTA、BTLA、TIG、LAIR1、CD160、2B4、CD80、CD86、B7-H3(CD276)、B7-H4(VTCN1)、HVEM(TNFRSF14或CD270)、KIR、A2aR、MHC I类、MHC II类、GAL9、腺苷、TGFR(TGFRβ)和TGFRβ。所述抑制性分子的胞外结构域可以融合到跨膜结构域和胞内信号传导结构域，比如PD1CAR。
本发明还提供一种前任一所述的免疫细胞或肿瘤免疫抑制抵抗型CAR或前任一所述的核酸序列或前任一所述的重组质粒制备肿瘤药物中的应用。所述肿瘤免疫抑制抵抗型CAR、核酸序列、重组质粒包含或不包含启动子。
进一步，所述肿瘤为恶性肿瘤，包括急性淋巴样白血病、慢性淋巴细胞白血病、慢性髓性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、***癌、结直肠癌、乳腺癌、卵巢癌、***、胰腺癌、肺癌、肾癌、肝癌、脑癌和皮肤癌；所述肿瘤高表达CD19、CD20、CD123、CD22、BCMA、ROR1、CEA、间皮素、PSCA、PSMA、c-Met、GPC-3、Her2、EGFRvIII、GD-2、NY-ESO-1TCR、MAGE A3TCR中的一种或多种。
本发明目的在于还提供一种药物组合物，所述药物组合物包括：前任一所述的免疫细胞或肿瘤免疫抑制抵抗型CAR和可增强CAR表达活性的活性剂和/或治疗剂。
进一步，所述活性剂和/或治疗剂包括环孢素(cyclosporin)、硫唑嘌呤(azathioprine)、甲氨蝶呤(methotrexate)、霉酚酸酯(mycophenolate)和FK506、抗体或其它免疫清除剂(immunoablativeagents)例如CAMPATH、抗CD3抗体或其它抗体治疗、环磷酰胺(cytoxan)、氟达拉滨(fludarabine)、环孢素(cyclosporin)、FK506、雷帕霉素(rapamycin)、霉酚酸(mycophenolicacid)、类固醇(steroids)、FR901228、细胞因子。
本发明中，所述“功能性变体”通常是指包括与其具有基本上相同的功能(例如，可以具备所述嵌合抗原受体的性质)，且与其具有至少85％(例如，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的氨基酸序列。在某些实施方式中，所述氨基酸序列的变体为与其具有基本上相同的功能。
本发明有益效果在于
本发明提供的包含逆转肿瘤微环境的融合蛋白的CAR-T细胞或免疫细胞可以逆转肿瘤微环境，靶向杀伤CD47阳性的的肿瘤细胞；破除肿瘤组织中抑制性信号对CAR-T功能的影响，实现了CAR-T治疗有效性，同时能够保证一定的安全性，其中含有融合蛋白的CAR与不含有融合蛋白的CAR相比较，含有融合蛋白的CAR的结构的体内存续时间会更长并且安全性更高。
本发明提供的包含缺氧启动子与融合蛋白的靶向CD19的免疫细胞，能够在缺氧微环境中有效的清除体内肿瘤作用,并且利用缺氧微环境诱导的启动子能够在缺氧环境强化目的基因、蛋白等因子的表达，提高肿瘤的药物疗效和CAR-T治疗的有效性和安全性，而且不仅可以有效的表达于T淋巴细胞，而且在缺氧环境中能够加强CAR分子表达，并且使CAR-T细胞有高的IFN-γ分泌能力，提高CAR-T细胞针对肿瘤靶细胞的杀伤，能够用于肿瘤的靶向治疗。
本发明提供的CAR-T细胞或免疫细胞在缺氧环境下活性增强，可以用于制备***药物中的过继细胞治疗，对CAR-T的临床应用以及肿瘤联合治疗新策略的开发具有重要的指导意义。
附图说明
图1为流式检测各实验组CAR-T的阳性率情况。
图2为各实验组细胞杀伤情况。
图3为各实验组IFN-γ因子分泌情况。
图4为各实验组小鼠体内生物发光情况。
图5为各实验组小鼠体内肿瘤生长情况。
图中，5HCD19-BBZ是指胞内具有缺氧启动子、来源4-1BB(简写为BB)胞内域的共刺激信号和CD3ζ的靶向CD19的CAR结构；SIRPγ-28TM-28(SIRPγ-28)是指胞内仅有来源CD28胞内域信号的融合肽；5HCD19-BBZ-SIRPγ-28指的是指的是如下结构的CAR：5HCD19-8H-8TM-CD137-CD3ζ；SIRPγ-28+5HCD19-BBZ是指是包含缺氧启动子的CAR融合蛋白分别表达两个载体转染免疫细胞获得的CAR-T。
具体实施方式
所举实施例是为了更好地对本发明进行说明，但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
本发明实施例中，体内验证使用小鼠为NOD.Cg-PrkdcscidII2rgtm1Sug/JicCrl，简称NOG小鼠，由日本实验动物研究所(CIEA)的Mamoru Ito培育而成，为国际上CAR-T体内相关成瘤实验最常见品系。
本发明实施例中，各实验组结构中：5HCD19-BBZ是指胞内具有缺氧启动子、来源4-1BB(简写为BB)胞内域的共刺激信号和CD3ζ的靶向CD19的CAR结构；SIRPγ-28TM-28(简写SIRPγ-28)是指胞内仅有来源CD28胞内域信号的融合肽；SIRPγ-28+5HCD19-BBZ是指是包含缺氧启动子的CAR融合蛋白分别表达两个载体转染免疫细胞获得的CAR-T。5HCD19-BBZ-P2A-SIRPγ-28是指是包含缺氧启动子的CAR融合蛋白共同表达一个载体转染免疫细胞获得的CAR-T。
本发明实施例中，采用磷酸钙法包装慢病毒的方法为：用含10％FBS(w/v)的DMEM培养基培养293T细胞至较佳状态，包装质粒(RRE:REV:2G)和表达质粒按一定比列加入到1.5的离心管中，加入CaCl2和2×HBS，混匀后室温静置后加入到处理好的293T细胞培养液中，3-5h后再次换液至10mL含10％FBS的DMEM培养基，48h或72h后收集细胞上清，纯化病毒。
本发明实施例中，抗体为：Protein-L-PE，Protein-L可识别抗体轻链，CAR抗原识别区的ScFv序列的轻链可被Protein-L识别，因此利用Protein-L可检测CAR阳性率和CAR表达强度。SIRPγ-28后有GFP标签，以GFP阳性率确定其表达情况。
本发明实施例中，IFN-γ检测采用BD IFN-γ试剂盒检测，实验步骤依据产品说明书进行。
本发明实施例中，检测不同CAR-T对靶细胞杀伤能力的方法为：利用ACEAxCELLigence RTCA MP仪器进行，实验步骤依据仪器说明书进行。ACEA xCELLigence RTCAMP原理为对附着于孔底的肿瘤细胞以电阻指数为数据每15分钟记录一次，通过电阻指数判断贴壁的靶细胞的增殖或者死亡情况。利用电阻指数分析结果公式为：CAR-T细胞杀伤率＝基线电阻指数-实时电阻指数。
实施例1质粒构建
以SIRPγ及CD47全长的质粒、pL-CAG-2AGFP、pL-CAG-PD1-CD28-2ACherry、pL-CAG-PD1-BB-2Acherry为模板，CD19靶点CAR结构。构建获得载体：SIRPγ-28、5HCD19-BBZ、5HCD19-BBZ-SIRPγ-28。通过测序比对验证后，结构建成功。
实施例2制备慢病毒及感染T淋巴细胞
采用磷酸钙法包装慢病毒，获得实施例1中3个病毒颗粒(SIRPγ-28、5HCD19-BBZ、5HCD19-BBZ-P2A-SIRPγ-28)。
利用梯度离心法进行淋巴细胞分离，离心后，取第二层白色淋巴细胞层，生理盐水洗涤，加入含有10％FBS的RPMI 1640完全培养基培养，获得人PBMC细胞。获得的PBMC细胞经抗CD3、CD28单克隆抗体活化24h后，按一定的感染复数(MOI)感染已活化的PBMC，在病毒感染的第8天利用流式检测CAR-T的阳性率，结果如图1以及下表1所示。
表1流式检测各实验组CAR-T的阳性率
结构 ％of CD3+T Cells
Control T 0.26
SIRPγ-28 51.25
5HCD19-BBZ 53.15
5HCD19-BBZ-P2A-SIRPγ-28 54.89
SIRPγ-28+5HCD19-BBZ 18.04
实施例3体外药效学评价
以Control T为对照组，实验组设置SIRPγ-28组、5HCD19-BBZ组、5HCD19-BBZ-P2A-SIRPγ-28组，以Nam6-Luc-GFP(CD19阳性)、K562-Luc-GFP(CD19阴性)为靶细胞，通过体外杀伤和体外因子分泌验证体外有效性。结果如图2及下表2所示，采用一个共表达的载体转染免疫细胞获得的产品(5HCD19-BBZ-P2A-SIRPγ-28)与采用两个分别表达的载体共转染免疫细胞获得的产品(SIRPγ-28+5HCD19-BBZ)的体外杀伤显著高于SIRPγ-28组和5HCD19-BBZ组，对阴性细胞无杀伤。
和图3及表3所示，采用一个共表达的载体转染免疫细胞获得的产品(5HCD19-BBZ-P2A-SIRPγ-28)与采用两个分别表达的载体共转染免疫细胞获得的产品(SIRPγ-28+5HCD19-BBZ)的因子分泌显著高于高于SIRPγ-28组和5HCD19-BBZ组。
表2各实验组细胞杀伤情况
结构 Specific Lysis(％)
Control T 37.2867
SIRPγ-28 28.6995
5HCD19-BBZ 84.3876
5HCD19-BBZ-P2A-SIRPγ-28 97.5187
SIRPγ-28+5HCD19-BBZ 97.4608
表3各实验组IFN-γ因子分泌情况
结构 IFN-γ(pg/ml)
Control T 135
SIRPγ-28 453.97
5HCD19-BBZ 17296.67
5HCD19-BBZ-P2A-SIRPγ-28 21820
SIRPγ-28+5HCD19-BBZ 14363.33
实施例4体内药效学评价
选用NCG小鼠(雌性，6周龄)，以1×106Cells/只剂量皮下注射(s.c.)Nalm6-Luc-GFP细胞建立体内荷瘤模型，荷瘤后8d以1×107CAR-T Cells/只剂量尾静脉注射(i.v.)给予不同组别(Control T、5HCD19-BBZ、5HCD19-BBZ-P2A-SIRPγ-28)CAR-T。通过活体成像观察肿瘤体内生长情况，体内评价不同CAR-T对淋巴瘤治疗效果。结果如图4和图5所示，相比Control T组和5HCD19-BBZ组，5HCD19-BBZ-P2A-SIRPγ-28体内抗肿瘤效果明显，能明显清除肿瘤。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 重庆精准生物技术有限公司、重庆精准生物产业技术研究院有限公司
<120> 融合蛋白及靶向CD19的CAR及其应用
<130> 2021-3-4
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 470
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser
130 135 140
Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Thr Arg Leu
180 185 190
Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Val Leu Thr Met Thr
195 200 205
Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Ser Ser
225 230 235 240
Val Thr Val Ser Ser Leu Glu Thr Thr Thr Pro Ala Pro Arg Pro Pro
245 250 255
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
260 265 270
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
275 280 285
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
290 295 300
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
305 310 315 320
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
325 330 335
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
340 345 350
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
355 360 365
Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
370 375 380
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
385 390 395 400
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
405 410 415
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
420 425 430
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
435 440 445
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
450 455 460
Gln Ala Leu Pro Pro Arg
465 470
<210> 2
<211> 277
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atcccacagt gcatacgtgg gctccaacag gtcctcttgt cgagccacag tgcatacgtg 60
ggctccaaca ggtcctcttg tcgagccaca gtgcatacgt gggctccaac aggtcctctt 120
gtcgagccac agtgcatacg tgggctccaa caggtcctct tgtcgagcca cagtgcatac 180
gtgggctcca acaggtcctc ttgtcgagat ctggtaggcg tgtacggtgg gaggtctata 240
taagcagagc tcgtttagtg aaccgtcaga tcactag 277
<210> 3
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 4
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 5
<211> 362
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu Leu
1 5 10 15
Leu Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu
20 25 30
Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr
35 40 45
Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val
50 55 60
Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln
65 70 75 80
Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys
85 90 95
Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro Ala
100 105 110
Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Glu
115 120 125
Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly Ala
130 135 140
Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr Pro
145 150 155 160
Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg
165 170 175
Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe
180 185 190
Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg
195 200 205
Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val
210 215 220
Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly
225 230 235 240
Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu Val
245 250 255
Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys Gln
260 265 270
Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu Asn
275 280 285
Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys
290 295 300
Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp
305 310 315 320
Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln
325 330 335
Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln Lys
340 345 350
Asp Gln Ser Ser Asp Ala Thr Pro Leu Glu
355 360
<210> 6
<211> 430
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu Leu
1 5 10 15
Leu Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu
20 25 30
Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr
35 40 45
Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val
50 55 60
Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln
65 70 75 80
Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys
85 90 95
Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro Ala
100 105 110
Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Glu
115 120 125
Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly Ala
130 135 140
Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr Pro
145 150 155 160
Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg
165 170 175
Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe
180 185 190
Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg
195 200 205
Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val
210 215 220
Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly
225 230 235 240
Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu Val
245 250 255
Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys Gln
260 265 270
Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu Asn
275 280 285
Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys
290 295 300
Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp
305 310 315 320
Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln
325 330 335
Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln Lys
340 345 350
Asp Gln Ser Ser Asp Ala Thr Pro Leu Glu Phe Trp Val Leu Val Val
355 360 365
Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe
370 375 380
Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp
385 390 395 400
Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr
405 410 415
Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
420 425 430
<210> 7
<211> 428
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu Leu
1 5 10 15
Leu Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu
20 25 30
Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr
35 40 45
Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val
50 55 60
Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln
65 70 75 80
Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys
85 90 95
Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro Ala
100 105 110
Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Glu
115 120 125
Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly Ala
130 135 140
Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr Pro
145 150 155 160
Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg
165 170 175
Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe
180 185 190
Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg
195 200 205
Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val
210 215 220
Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly
225 230 235 240
Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu Val
245 250 255
Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys Gln
260 265 270
Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu Asn
275 280 285
Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys
290 295 300
Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp
305 310 315 320
Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln
325 330 335
Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln Lys
340 345 350
Asp Gln Ser Ser Asp Ala Thr Pro Leu Glu Ile Tyr Ile Trp Ala Pro
355 360 365
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
370 375 380
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
385 390 395 400
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
405 410 415
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
420 425
<210> 8
<211> 1290
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgcctgtcc cagcctcctg gccccatcct cctggtcctt tcctgcttct gactctactg 60
ctgggactta cagaagtggc aggtgaggag gagctacaga tgattcagcc tgagaagctc 120
ctgttggtca cagttggaaa gacagccact ctgcactgca ctgtgacctc cctgcttccc 180
gtgggacccg tcctgtggtt cagaggagtt ggaccaggcc gggaattaat ctacaatcaa 240
aaagaaggcc acttccccag ggtaacaaca gtttcagacc tcacaaagag aaacaacatg 300
gacttttcca tccgcatcag tagcatcacc ccagcagatg tcggcacata ctactgtgtg 360
aagtttcgaa aagggagccc tgagaacgtg gagtttaagt ctggaccagg cactgagatg 420
gctttgggtg ccaaaccctc tgcccccgtg gtattgggcc ctgcggcgag gaccacacct 480
gagcatacag tgagtttcac ctgtgagtcc catggcttct ctcccagaga catcaccctg 540
aaatggttca aaaatgggaa tgagctctca gacttccaga ccaacgtgga ccccacagga 600
cagagtgtgg cctacagcat ccgcagcaca gccagggtgg tactggaccc ctgggacgtt 660
cgctctcagg tcatctgcga ggtggcccat gtcaccttgc agggggaccc tcttcgtggg 720
actgccaact tgtctgaggc catccgagtt ccacccacct tggaggttac tcaacagccc 780
atgagggtgg ggaaccaggt aaacgtcacc tgccaggtga ggaagttcta cccccagagc 840
ctacagctga cctggtcgga gaatggaaac gtgtgccaga gagaaacagc ctcgaccctt 900
acagagaaca aggatggtac ctacaactgg acaagctggt tcctggtgaa catatctgac 960
caaagggatg atgtggtcct cacctgccag gtgaagcatg atgggcagct ggcggtcagc 1020
aaacgccttg ccctagaggt cacagtccac cagaaggacc agagctcaga tgctacccct 1080
ctcgagttct gggtgctggt cgtggtgggt ggcgtgctgg cctgctacag cctgctggtg 1140
acagtggcct tcatcatctt ttgggtgagg agcaagcgga gcagaggcgg ccacagcgac 1200
tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 1260
ccaccacgcg acttcgcagc ctatcgctcc 1290
<210> 9
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgcctgtcc cagcctcctg gccccatcct cctggtcctt tcctgcttct gactctactg 60
ctgggactta cagaagtggc aggtgaggag gagctacaga tgattcagcc tgagaagctc 120
ctgttggtca cagttggaaa gacagccact ctgcactgca ctgtgacctc cctgcttccc 180
gtgggacccg tcctgtggtt cagaggagtt ggaccaggcc gggaattaat ctacaatcaa 240
aaagaaggcc acttccccag ggtaacaaca gtttcagacc tcacaaagag aaacaacatg 300
gacttttcca tccgcatcag tagcatcacc ccagcagatg tcggcacata ctactgtgtg 360
aagtttcgaa aagggagccc tgagaacgtg gagtttaagt ctggaccagg cactgagatg 420
gctttgggtg ccaaaccctc tgcccccgtg gtattgggcc ctgcggcgag gaccacacct 480
gagcatacag tgagtttcac ctgtgagtcc catggcttct ctcccagaga catcaccctg 540
aaatggttca aaaatgggaa tgagctctca gacttccaga ccaacgtgga ccccacagga 600
cagagtgtgg cctacagcat ccgcagcaca gccagggtgg tactggaccc ctgggacgtt 660
cgctctcagg tcatctgcga ggtggcccat gtcaccttgc agggggaccc tcttcgtggg 720
actgccaact tgtctgaggc catccgagtt ccacccacct tggaggttac tcaacagccc 780
atgagggtgg ggaaccaggt aaacgtcacc tgccaggtga ggaagttcta cccccagagc 840
ctacagctga cctggtcgga gaatggaaac gtgtgccaga gagaaacagc ctcgaccctt 900
acagagaaca aggatggtac ctacaactgg acaagctggt tcctggtgaa catatctgac 960
caaagggatg atgtggtcct cacctgccag gtgaagcatg atgggcagct ggcggtcagc 1020
aaacgccttg ccctagaggt cacagtccac cagaaggacc agagctcaga tgctacccct 1080
ctcgagatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1140
gttatcaccc tttactgcaa acggggcaga aagaaactcc tgtatatatt caaacaacca 1200
tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 1260
gaagaagaag gaggatgtga actg 1284
<210> 10
<211> 1473
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggctctgc cagtgacagc tctgctgctg cctctggctc tgctgctgca cgcagctaga 60
cccgacatcc agatgaccca gagcccttct tctctgagcg ccagcgtggg agacagagtg 120
accatcactt gcagggccag ccaggacatc agcaagtacc tgaattggta ccagcagaag 180
ccaggcaagg cccctagact gctgatctac cacacaagca gactgcacag cggagtgcct 240
agcagattca gcggcagcgg aagcggaacc gactacaccc tgaccatcag cagcctgcag 300
ccagaggact tcgccaccta ctactgccag cagggcaaca cactgcctta caccttcggc 360
ggaggcacaa gactggagat caagggcagc acaagcggaa gcggcaaacc aggaagcgga 420
gaaggaagca ccaagggaca ggtgcagctg caggaaagcg gaccaggact ggtgaagcct 480
tctcagaccc tgagcctgac ttgcaccgtg tcaggagtgt ccctgccaga ttacggcgtg 540
tcttggatca gacagccccc aggaaaggcc ctggagtggc tgggagtgat ttggggaagc 600
gagaccacct actacaacag cagcctgaag acccggctga ccatcagcaa ggacaacagc 660
aagaaccagg tggtgctgac catgaccaac atggaccccg tggacaccgc cacctactat 720
tgcgccaagc actactacta cggcggaagc tacgccatgg actattgggg ccagggaagc 780
agcgtgaccg tgtctagcct cgagaccacg acgccagcgc cgcgaccacc aacaccggcg 840
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900
ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960
gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 1020
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1080
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1140
gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1200
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgc 1473
<210> 11
<211> 1756
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atcccacagt gcatacgtgg gctccaacag gtcctcttgt cgagccacag tgcatacgtg 60
ggctccaaca ggtcctcttg tcgagccaca gtgcatacgt gggctccaac aggtcctctt 120
gtcgagccac agtgcatacg tgggctccaa caggtcctct tgtcgagcca cagtgcatac 180
gtgggctcca acaggtcctc ttgtcgagat ctggtaggcg tgtacggtgg gaggtctata 240
taagcagagc tcgtttagtg aaccgtcaga tcactaggct agcatggctc tgccagtgac 300
agctctgctg ctgcctctgg ctctgctgct gcacgcagct agacccgaca tccagatgac 360
ccagagccct tcttctctga gcgccagcgt gggagacaga gtgaccatca cttgcagggc 420
cagccaggac atcagcaagt acctgaattg gtaccagcag aagccaggca aggcccctag 480
actgctgatc taccacacaa gcagactgca cagcggagtg cctagcagat tcagcggcag 540
cggaagcgga accgactaca ccctgaccat cagcagcctg cagccagagg acttcgccac 600
ctactactgc cagcagggca acacactgcc ttacaccttc ggcggaggca caagactgga 660
gatcaagggc agcacaagcg gaagcggcaa accaggaagc ggagaaggaa gcaccaaggg 720
acaggtgcag ctgcaggaaa gcggaccagg actggtgaag ccttctcaga ccctgagcct 780
gacttgcacc gtgtcaggag tgtccctgcc agattacggc gtgtcttgga tcagacagcc 840
cccaggaaag gccctggagt ggctgggagt gatttgggga agcgagacca cctactacaa 900
cagcagcctg aagacccggc tgaccatcag caaggacaac agcaagaacc aggtggtgct 960
gaccatgacc aacatggacc ccgtggacac cgccacctac tattgcgcca agcactacta 1020
ctacggcgga agctacgcca tggactattg gggccaggga agcagcgtga ccgtgtctag 1080
cctcgagacc acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca 1140
gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag 1200
ggggctggac ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt 1260
ccttctcctg tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta 1320
tatattcaaa caaccattta tgagaccagt acaaactact caagaggaag atggctgtag 1380
ctgccgattt ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag 1440
cgcagacgcc cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg 1500
acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg 1560
aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat 1620
ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga 1680
tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca 1740
ggccctgccc cctcgc 1756

Claims (9)

1.一种肿瘤免疫抑制抵抗型 CAR，其特征在于，所述肿瘤免疫抑制抵抗型 CAR包括SIRPγ融合蛋白和CAR结构a；所述CAR结构a包括CD19单链抗体、CD8铰链区、CD8跨膜区、CD137和CD3ξ双刺激信号；所述SIRPγ融合蛋白结构包含胞外段、跨膜结构和胞内信号区，所述SIRPγ的胞外段氨基酸序列如SEQ ID NO:5所示；所述SIRPγ融合蛋白的结构为SIRPγ- CD28TM-CD28或SIRPγ- CD8TM-4-1BB； SIRPγ融合蛋白SIRPγ- CD28TM-CD28的氨基酸序列如SEQ ID NO:6所示；或SIRPγ融合蛋白SIRPγ- CD8TM-4-1BB的氨基酸序列如SEQID NO:7所示；所述SIRPγ融合蛋白通过自剪切肽与所述CAR结构a连接。
2.根据权利要求1所述的肿瘤免疫抑制抵抗型 CAR，其特征在于，编码所述SIRPγ融合蛋白SIRPγ- CD28TM-CD28的核苷酸序列如SEQ ID NO:8所示；或编码所述SIRPγ融合蛋白SIRPγ- CD8TM-4-1BB的核苷酸序列如SEQ ID NO:9所示。
3.根据权利要求1所述的肿瘤免疫抑制抵抗型 CAR，其特征在于，所述CAR结构a还包括缺氧启动子，所述缺氧启动子的核酸序列如SEQ ID NO：2所示。
4.根据权利要求1所述的肿瘤免疫抑制抵抗型 CAR，其特征在于，所述CAR结构a氨基酸序列如SEQ ID NO：1所示。
5.一种制备免疫细胞的方法，其特征在于，将重组质粒转染细胞；重组质粒包含分离的核酸分子，分离的核酸分子编码权利要求1所述的肿瘤免疫抑制抵抗型 CAR，所述CAR结构a不包含缺氧启动子，其序列如SEQ ID NO：10 所示；或所述CAR结构a包含缺氧启动子，其序列如SEQ ID NO：11所示。
6.一种非治疗目的提高缺氧环境下CAR-T细胞杀伤能力的方法，其特征在于，构建一种包含重组质粒并感染T淋巴细胞，然后作用于靶细胞；其中，重组质粒包含分离的核酸分子，分离的核酸分子编码权利要求1所述的肿瘤免疫抑制抵抗型 CAR；所述CAR结构a包含缺氧启动子，其序列如SEQ ID NO：11所示。
7.权利要求1至4中任一项所述的肿瘤免疫抑制抵抗型 CAR在制备治疗血液肿瘤的药物中的应用。
8.根据权利要求7所述的应用，其特征在于，所述血液肿瘤为急性淋巴样白血病、慢性淋巴细胞白血病或慢性髓性白血病。
9.一种药物组合物，其特征在于，所述药物组合物包括：权利要求1至4中任一项权利要求所述的肿瘤免疫抑制抵抗型 CAR和可增强CAR表达活性的活性剂和/或治疗剂。
CN202110556149.8A 2020-05-22 2021-05-21 融合蛋白及靶向cd19的car及其应用 Active CN113698491B (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010441127 2020-05-22
CN2020104411272 2020-05-22

Publications (2)

Publication Number Publication Date
CN113698491A CN113698491A (zh) 2021-11-26
CN113698491B true CN113698491B (zh) 2024-04-30

Family

ID=78647990

Family Applications (6)

Application Number Title Priority Date Filing Date
CN202110556149.8A Active CN113698491B (zh) 2020-05-22 2021-05-21 融合蛋白及靶向cd19的car及其应用
CN202110556154.9A Active CN113717288B (zh) 2020-05-22 2021-05-21 逆转肿瘤微环境的融合蛋白及其应用
CN202410037361.7A Pending CN117844761A (zh) 2020-05-22 2021-05-21 逆转肿瘤微环境的融合蛋白及其应用
CN202410037360.2A Pending CN117843812A (zh) 2020-05-22 2021-05-21 逆转肿瘤微环境的融合蛋白及其应用
CN202110556140.7A Active CN113698489B (zh) 2020-05-22 2021-05-21 融合蛋白及其靶向psca的car及其应用
CN202110556147.9A Active CN113698490B (zh) 2020-05-22 2021-05-21 靶向cea的缺氧诱导启动的car结构及免疫细胞和应用

Family Applications After (5)

Application Number Title Priority Date Filing Date
CN202110556154.9A Active CN113717288B (zh) 2020-05-22 2021-05-21 逆转肿瘤微环境的融合蛋白及其应用
CN202410037361.7A Pending CN117844761A (zh) 2020-05-22 2021-05-21 逆转肿瘤微环境的融合蛋白及其应用
CN202410037360.2A Pending CN117843812A (zh) 2020-05-22 2021-05-21 逆转肿瘤微环境的融合蛋白及其应用
CN202110556140.7A Active CN113698489B (zh) 2020-05-22 2021-05-21 融合蛋白及其靶向psca的car及其应用
CN202110556147.9A Active CN113698490B (zh) 2020-05-22 2021-05-21 靶向cea的缺氧诱导启动的car结构及免疫细胞和应用

Country Status (8)

Country Link
US (1) US20230203125A1 (zh)
EP (1) EP4151660A4 (zh)
JP (1) JP2023527194A (zh)
KR (1) KR20230013257A (zh)
CN (6) CN113698491B (zh)
BR (1) BR112022023371A2 (zh)
IL (1) IL298346A (zh)
WO (1) WO2021233411A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023114055A2 (en) * 2021-12-13 2023-06-22 The Regents Of The University Of California SIRPalpha SWITCH RECEPTORS
CN117343906A (zh) * 2022-07-04 2024-01-05 上海优卡迪生物医药科技有限公司 表达重组抗原蛋白的饲养细胞及其制备方法和应用
CN117625545A (zh) * 2022-08-29 2024-03-01 新加坡星汉德生物医药有限公司 经修饰的靶向hbv免疫细胞及其医药用途
CN117467022A (zh) * 2023-09-28 2024-01-30 上海恩凯细胞技术有限公司 嵌合抗原受体及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399318A (zh) * 2016-09-08 2017-02-15 徐家科 一种氧调控性碱性成纤维细胞生长因子修饰细胞的方法
CN107312091A (zh) * 2017-05-02 2017-11-03 重庆精准生物技术有限公司 靶向人cd19抗原的人源化单克隆抗体
CN107531805A (zh) * 2015-03-05 2018-01-02 弗雷德哈钦森癌症研究中心 免疫调节融合蛋白及其用途
CN109152824A (zh) * 2015-11-27 2019-01-04 卡瑟里克斯私人有限公司 经遗传修饰的细胞及其用途
WO2020068752A1 (en) * 2018-09-27 2020-04-02 Celgene Corporation SIRPα BINDING PROTEINS AND METHODS OF USE THEREOF

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201506423D0 (en) 2015-04-15 2015-05-27 Tc Biopharm Ltd Gamma delta T cells and uses thereof
GB201507368D0 (en) 2015-04-30 2015-06-17 Ucl Business Plc Cell
US11725210B2 (en) * 2017-03-17 2023-08-15 Fred Hutchinson Cancer Center Immunomodulatory fusion proteins and uses thereof
WO2019061012A1 (zh) * 2017-09-26 2019-04-04 南京凯地生物科技有限公司 靶向cd47的特异性嵌合抗原受体t细胞的制备及其应用
CN112125976B (zh) * 2018-01-26 2022-06-03 重庆精准生物技术有限公司 改造的铰链及其在构建car骨架中的应用
MX2021000263A (es) * 2018-07-11 2021-05-12 Kahr Medical Ltd Proteina de fusion variante sirpalfa-4-1bbl y procedimientos de uso de la misma.
CN113481168A (zh) * 2018-12-26 2021-10-08 重庆精准生物技术有限公司 增强car-t细胞归巢到实体肿瘤组织能力的试剂及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107531805A (zh) * 2015-03-05 2018-01-02 弗雷德哈钦森癌症研究中心 免疫调节融合蛋白及其用途
CN109152824A (zh) * 2015-11-27 2019-01-04 卡瑟里克斯私人有限公司 经遗传修饰的细胞及其用途
CN106399318A (zh) * 2016-09-08 2017-02-15 徐家科 一种氧调控性碱性成纤维细胞生长因子修饰细胞的方法
CN107312091A (zh) * 2017-05-02 2017-11-03 重庆精准生物技术有限公司 靶向人cd19抗原的人源化单克隆抗体
WO2020068752A1 (en) * 2018-09-27 2020-04-02 Celgene Corporation SIRPα BINDING PROTEINS AND METHODS OF USE THEREOF

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Christopher Ede等.Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.ACS Synthetic Biology.2016,(第5期),第400页左栏和右栏第1段、图2. *
SHINICHIRO TAKAHASHI.Molecular functions of SIRPα and its role in cancer (Review).BIOMEDICAL REPORTS.第9卷第3页第1节，第4页第3节. *

Also Published As

Publication number Publication date
JP2023527194A (ja) 2023-06-27
CN117844761A (zh) 2024-04-09
CN113698489A (zh) 2021-11-26
EP4151660A4 (en) 2024-01-17
WO2021233411A1 (zh) 2021-11-25
CN113698490B (zh) 2024-04-30
KR20230013257A (ko) 2023-01-26
EP4151660A1 (en) 2023-03-22
BR112022023371A2 (pt) 2022-12-20
CN117843812A (zh) 2024-04-09
CN113717288A (zh) 2021-11-30
CN113717288B (zh) 2023-12-12
IL298346A (en) 2023-01-01
CN113698491A (zh) 2021-11-26
CN113698490A (zh) 2021-11-26
US20230203125A1 (en) 2023-06-29
CN113698489B (zh) 2024-04-30

Similar Documents

Publication Publication Date Title
US11851491B2 (en) Compositions and methods for TCR reprogramming using fusion proteins
CN113698491B (zh) 融合蛋白及靶向cd19的car及其应用
WO2023046110A1 (zh) 联合表达CCR2b的工程化免疫细胞及其制备和应用
CN116057181A (zh) 用于修饰和递送淋巴细胞的方法和组合物
US20210317408A1 (en) Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs
WO2020047527A2 (en) Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs
CN111686128A (zh) 一种缺氧可调控的启动子在car-t中的应用
JP2017533729A (ja) Ｔ細胞ベースの免疫療法薬
US11680099B2 (en) Anti-PD-1/CD47 bispecific antibody and application thereof
WO2023072307A1 (zh) 一种靶向cd70的抗原结合片段、单链抗体和嵌合抗原受体及其应用
CN112921035B (zh) 一种调控car-t特异性活化的启动子及其应用
CN113493520A (zh) 靶向双特异性位点的双car及其应用
CN112921034B (zh) 一种调控car-t特异性活化的启动子及其应用
CN113493516A (zh) 靶向双特异性位点的嵌合抗原受体及其应用
EP3922721A9 (en) Hypoxia-regulated promoter and application thereof
CN113493522B (zh) 双特异性嵌合抗原受体及其应用
EP4372088A1 (en) Anti-egfrviii antibody, polypeptide, cell capable of expressing said polypeptide, pharmaceutical composition containing said cell, method for producing said cell, and polynucleotide or vector comprising nucleotide sequence encoding said polypeptide
CA3232203A1 (en) Il5ra cell surface markers
KR20240035506A (ko) 키메라 항원 수용체, 상기 수용체를 발현하는 세포, 상기 세포를 포함하는 의약 조성물, 상기 세포의 제조 방법, 및 상기 키메라 항원 수용체를 코딩하는 염기서열을 포함하는 폴리뉴클레오티드 또는 벡터
JP2023527621A (ja) 哺乳類におけるキメラ抗原受容体媒介性免疫応答を刺激するための方法における使用のための、ｐｄ－ｌ１および／またはｐｄ－ｌ２を含む標的化モジュール
GB2569692A (en) T cell antigen receptor chimera

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant