[go: up one dir, main page]

CN112080527A - 重组表达载体、耗竭减少的嵌合抗原受体t细胞及其应用 - Google Patents

重组表达载体、耗竭减少的嵌合抗原受体t细胞及其应用 Download PDF

Info

Publication number
CN112080527A
CN112080527A CN202010973707.6A CN202010973707A CN112080527A CN 112080527 A CN112080527 A CN 112080527A CN 202010973707 A CN202010973707 A CN 202010973707A CN 112080527 A CN112080527 A CN 112080527A
Authority
CN
China
Prior art keywords
ptpn2
chimeric antigen
antigen receptor
cell
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.)
Pending
Application number
CN202010973707.6A
Other languages
English (en)
Inventor
赵薇
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.)
Nanjing First Hospital
Original Assignee
Nanjing First Hospital
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 Nanjing First Hospital filed Critical Nanjing First Hospital
Priority to CN202010973707.6A priority Critical patent/CN112080527A/zh
Publication of CN112080527A publication Critical patent/CN112080527A/zh
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • 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
    • 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
    • C07K16/2827—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 against B7 molecules, e.g. CD80, CD86
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/60—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
    • 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
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Landscapes

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

Abstract

本发明属于生物技术领域，具体涉及重组表达载体、耗竭减少的嵌合抗原受体T细胞及其应用。本发明涉及一种利用基因编辑技术敲除健康人来源的T细胞PTPN2基因，以减少CD8+阳性T细胞耗竭，与CAR共表达于T细胞上，改善了对肿瘤的检查点阻断应答，促进有效的抗肿瘤免疫。

Description

重组表达载体、耗竭减少的嵌合抗原受体T细胞及其应用
技术领域
本发明属于生物技术领域，具体涉及重组表达载体、耗竭减少的嵌合抗原受T细胞体及其应用。
背景技术
肿瘤可以通过设置免疫检查点，直接或间接抑制细胞毒性CD8+T细胞的激活和功能，从而避开免疫系统。肿瘤微环境可以上调T细胞抑制受体的配体，如肿瘤细胞上的程序性细胞死亡蛋白-1(PD-1)可以抑制T细胞信号传递，促进T细胞的耐受性或衰竭。免疫检查点受体，包括PD-1和细胞毒性T淋巴细胞抗原-4(CTLA-4)，可以通过招募磷酸酶来抵消T细胞受体(TCR)和共刺激受体诱导的激酶信号，从而抑制T细胞反应的持续期。
CAR-T疗法，是通过基因工程对患者自身的免疫T细胞进行改造，插入识别癌细胞抗原的嵌合抗原受体(CAR)，使其具备攻击癌细胞的能力，然后输注回患者体内产生免疫应答，消灭癌症。然而在肿瘤微环境中抗原的持续刺激下，输注回患者体内的CAR-T细胞会变得反应迟钝，抑制受体越来越多，CAR-T细胞失去效应功能，产生T细胞耗竭现象。现有技术没有公开过导致T细胞衰竭的关键机制。
PTPN2是一种非受体型的酪氨酸磷酸酶，其属于第一类基于半胱氨酸的磷酸酶中的经典磷酸酶。PTPN2磷酸酶最初是从T细胞的cDNA文库当中克隆得到，因此也被称为T细胞磷酸酶(T-cell Protein Tyrosine Phosphatase,TCPTP)。PTPN2通过去磷酸化使TCR信号级联近端的酪氨酸激酶失活。PTPN2也拮抗T细胞功能、稳态和分化所需的细胞因子信号。
发明内容
本发明的目的在于提供一种重组表达载体。
本发明的再一目的在于提供上述重组表达载体的应用。
本发明的再一目的在于提供含有上述重组表达载体的、耗竭减少的嵌合抗原受体T细胞。
本发明的再一目的在于提供上述嵌合抗原受体T细胞的制备方法。
本发明的再一目的在于提供上述嵌合抗原抗体T细胞的应用。
根据本发明具体实施方式的重组表达载体，所述重组表达载体共表达sh-PTPN2-1序列与Trop2/PD-L1双特异性嵌合抗原受体的核苷酸序列，其中，sh-PTPN2-1序列能够敲除人源PTPN2基因，其核苷酸序列如SEQ ID No.1所示，其反义链的核苷酸序列如SEQ ID No.2所示。
SEQ ID No.1：
5'-CCGGC GCTATTACTACCTTTAATTCA TTTTTTG-3'；
SEQ ID No.2：
5'-AATTCAAAAAA AATTAAAGGTAGTAATAGCCC-3'。
根据本发明具体实施方式的重组表达载体，所述Trop2/PD-L1双特异性嵌合抗原受体由CD8、人源化抗Trop2、抗PD-L1单链抗体、跨膜区CD8TM、胞内区CD28、CD137和CD3ζ串联构成。
CD8的氨基酸序列如SEQ ID No.3所示：
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGMALPVTALLLPLALLLHAARP
人抗Trop2单链抗体的重链氨基酸序列如SEQ ID No.4所示：
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPGWGFDYWGQGTLVTVSS
人抗Trop2单链抗体的轻链氨基酸序列如SEQ ID No.5所示：
ELQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK
所示抗PD-L1单链抗体的轻链氨基酸序列如SEQ ID No.6所示：
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS
所示抗PD-L1单链抗体的重链氨基酸序列如SEQ ID No.7所示：
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL
跨膜区CD8TM的氨基酸序列如SEQ ID No.8所示：
FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
胞内区CD28的氨基酸序列如SEQ ID No.9所示：
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC
CD137的氨基酸序列如SEQ ID No.10所示：
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
D3ζ的氨基酸序列如SEQ ID No.11所示：
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
根据本发明具体实施方式的重组表达载体，所述sh-PTPN2-1序列的上游还含有hU6p启动子，以驱动sh-PTPN2-1序列的表达。
根据本发明具体实施方式的重组表达载体在制备嵌合抗原受体T细胞以及制备抗肿瘤药物中的应用。
根据本发明具体实施方式的耗竭减少的嵌合抗原受体T细胞，所述嵌合抗原受体T细胞含有能够共表达sh-PTPN2-1序列与Trop2/PD-L1双特异性嵌合抗原受体的核苷酸序列的重组表达载体，其中，sh-PTPN2-1序列的核苷酸序列如SEQ ID No.1所示，其反义链的核苷酸序列如SEQ ID No.2所示。
根据本发明具体实施方式的耗竭减少的嵌合抗原受体T细胞，所述Trop2/PD-L1双特异性嵌合抗原受体由CD8、人源化抗Trop2、抗PD-L1单链抗体、跨膜区CD8TM、胞内区CD28、CD137和CD3ζ串联构成。
根据本发明具体实施方式的耗竭减少的嵌合抗原受体T细胞的制备方法，包括以下步骤：
提取含有重组表达载体的慢病毒，并感染T细胞，使T细胞表达sh-PTPN2-1和Trop2/PD-L1双特异性嵌合抗原受体。
根据本发明具体实施方式的耗竭减少的嵌合抗原受体T细胞在制备抗肿瘤药物以及抗肿瘤药物增效剂方面的应用。
本发明的有益效果：
本发明利用基因编辑技术敲除健康人来源的T细胞PTPN2基因，以减少CD8阳性T细胞耗竭，与CAR共表达于T细胞上，通过促进IFN-γ，TNF-a，IL-2等细胞因子的释放，增强双特异性CAR-T细胞的抗肿瘤活性，改善了对肿瘤的检查点阻断应答，促进有效的抗肿瘤免疫，将CAR-T细胞的抗肿瘤作用从血液细胞肿瘤拓展到实体瘤的范围。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是293T细胞中PTPN2的基因和蛋白的表达情况，其中，A为qRT-PCR检测PTPN2的基因的表达情况，B为WB检测293T细胞中PTPN2蛋白的表达情况；泳道1:NC，泳道2:shRNA-NC，泳道3:shRNA-PTPN2-1，泳道4:shRNA-PTPN2-2，泳道5:shRNA-PTPN2-3；
图2显示敲减PTPN2序列条带的电泳图，泳道1为核酸分子量标准，泳道2为shRNA-PTPN2；泳道3为敲减的对照组；
图3显示本发明的重组表达载体上的CAR结构；
图4是敲减PTPN2 CAR克隆到逆转录病毒载体中，用Eco I和BspEI双酶切鉴定，释放片段为CD8-Trop2/PD-L1 scFv-CD8TM-CD28-CD137-CD3ζ，大小约为1.8kb，泳道1为2000bp的核酸分子量标准，泳道2(Eco I)和3(BspEI)为单酶切的CAR质粒，泳道4和5分别为经BamH I和Xho I双酶切的质粒。
图5是western blot检测本发明构建的sh-PTPN2-CD8-Trop2/PD-L1 scFv-CD8TM-CD28-CD137-CD3ζ转染293T细胞后的表达情况；泳道1为T细胞，泳道2为转染Trop2/PD-L1-CAR载体的293T细胞；泳道3为转染sh-NC-Trop2/PD-L1-CAR载体的293T细胞；泳道4为转染sh-PTPN2-Trop2/PD-L1-CAR载体的293T细胞。
图6显示流式检测本发明构建的sh-PTPN2-Trop2/PD-L1-CAR修饰的T细胞中CAR分子的表达情况；
图7显示检测sh-PTPN2-Trop2/PD-L1-CAR修饰的T细胞中细胞因子的释放情况。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
实施例1
确定PTPN2基因的有效靶序列，并在其5'端和3'端加上限制性内切酶Eco I、BspEI位点，合成需要的DNA双链片段，得到3个候选序列，分别命名为sh-PTPN2-1、sh-PTPN2-2、sh-PTPN2-3，各组DNA互补序列如下所示：
sh-PTPN2-1：
5'-CCGGC GCTATTACTACCTTTAATTCA TTTTTTG-3'；
5'-AATTCAAAAAA AATTAAAGGTAGTAATAGCCC-3'；
sh-PTPN2-2：
5'-CCGGC CGTATCAACAATGTTCGATGTTTTTTTG-3'；
5'-AATTCAAAAAAATCGAACATTGTTGATACGAA-3'；
sh-PTPN2-3：
5'-CCGGC CCAGGATCAATCATGTCATTCTTTTTTG-3'；
5'-AATTCAAAAAA ATGACATGATTGATCCTGGAT-3'。
培养293T细胞呈对数生长期，转染前2天接种1.3-1.5×106个细胞进10cm培养皿，添加含10％FBS的10mL DMEM培养基(胎牛血清提前进行热灭活)，待细胞长至70-80％汇合度。
取一个无菌1.5mL EP管(标记管①)，加入200uL培养基(Opti-MEMP)+2.5uglentiviral shRNA表达质粒+5.0uL(0.5ug/uL)lenti-Pac HIV mix；另取一个无菌1.5mLEP管(标记管②)，加入200uL培养基(Opti-MEMP)+15uL EndoFectin Lenti。将管②轻柔的逐滴加入到管①中，室温孵育10-25min。转染包装细胞。将管中DNA-EndoFectin Lenticomplex加入培养皿中，轻柔地混匀，细胞培养箱中孵育包装细胞8-14小时(<16h)，更换培养基，DMEM+2.5％热灭活FBS+1％双抗+1/500总体积的TiterBoost reagent，继续放于细胞培养箱孵育。
24h-48h后利用实时定量RT-PCR检测。结果如图1中A所示。
蛋白质印记法(western-blot)检测293T细胞中PTPN2的表达情况。结果如图1中B所示，1号干扰片段sh-PTPN2-1的干扰效果最优。
制备sh-PTPN2-1的阴性对照命名为sh-NC，其DNA序列如下所示：
sh-NC：
5'-CCGGTTCTCCGAACGTGTCACGTCTCGATTTTTTTG-3'；
5'-AATTCAAAAAATTCTCCGAACGTGTCACGTCTGAA-3'。
用10×T4连接酶分别将干扰片段和对照片段插入线性载体基因中，进行PRC扩增。对上述序列进行0.8％琼脂糖凝胶电泳检测，得到分子量约为500kb左右的条带，与预期结果相符，如图2所示。用琼脂糖凝胶回收试剂盒回收该片段。
在Trop2/PD-L1 CAR(质粒的制备方法参见专利201910163481.0)序列的hCMVp与CAR载体的接合处用限制性内切酶Eco I、BspEI切开(具体位置如图3所示)，用10×T4连接酶分别将sh-PTPN2、sh-NC干扰片段插入到线性载体基因中。回收的片段与酶切后的载体通过In-Fusion PCR进行连接。连接体系如下：目的基因酶切产物100ng、载体酶切产物300ng、In-Fusion 1uL，加水补足至20uL体系，37℃15min，50℃15min。
取10uL产物加入DH5感受态细胞中，37℃培养过夜，挑取单个菌落，扩大培养，用质粒提取试剂盒(Axygene公司)提取阳性克隆质粒，经酶切和测序检测，筛选正确的单克隆菌，命名为shPTPN2-T/P-CAR，结果如图4所示。
实施例2
1.Western-blot检测CAR-T细胞中CD3ζ和PTPN2的表达情况
提取双特异性嵌合抗原受体逆转录病毒质粒，用PI转染试剂转染到人胚肾细胞293T细胞中，48h后，用PBS洗一遍，用细胞蛋白提取试剂RIPA裂解细胞，提取转染后的293T细胞的蛋白经10％的SDS-PAGE进行凝胶电泳，转膜，分别用鼠抗人CD3ζ抗体和PTPN2抗体孵育4℃过夜，再用辣根过氧酶标记的抗小鼠的二抗孵育1h，ECL显色。
结果如图5所示，抗人CD3ζ抗体能检测到CAR分子表达，蛋白分子大小与理论相符，为75kDa，PTPN2位置在46kDa左右。
2.流式检测CAR-T细胞中PTPN2的表达分子的表达
将制备好的CAR-T细胞用PE标记的抗人PTPN2抗体37℃孵育1h后，进行流式检测。
检测结果如图6所示，结果表明sh-PTPN2 CAR-T细胞能成功敲减T细胞中的PTPN2的表达。
3.荧光条形码标记单分子检测(Nanostring nCounter)检测T细胞耗竭因子表达情况
分别制备不同T细胞中的mRNA，进行杂交、纯化和计数。数字化成像分析仪进行mRNA分子计数，本次实验计数方式以FOV(Fields of View视野)来表示，并采用280FOV进行计数。采用nCounter GX Human Kinase Kit(NanoString Technologies)进行基因表达分析。样品加样量为100ng，选取三个管家基因(CLTC，GAPDH，β-tubulin)表达的均值校正样品表达量水平。
结果如图7所示，对比其他组T细胞，增殖细胞抗原Ki67在sh-PTPN2-T/P CAR-T细胞中表达最高，并且sh-PTPN2-T/P CAR-T细胞能分泌较多的细胞因子(IFN-γ，TNF-a，IL-2)。由于耗竭T细胞耗竭早期IL-2和TNF产生缺陷，而在衰竭期，IFN-γ产生缺陷；Ki67是一种增殖细胞的相关抗原，其功能与丝分裂密切相关，在细胞增殖中是不可缺少的。因此，敲减了PTPN2的CAR-T细胞促进IFN-γ，TNF-a，IL-2等细胞因子的释放，sh-PTPN2-T/P CAR-T细胞的增殖能力较强，能够发挥较好的杀伤作用。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
序列表
<110> 南京市第一医院
<120> 重组表达载体、耗竭减少的嵌合抗原受体T细胞及其应用
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccggcgctat tactaccttt aattcatttt ttg 33
<210> 2
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aattcaaaaa aaattaaagg tagtaatagc cc 32
<210> 3
<211> 84
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ala Thr Gly Gly Cys Cys Thr Thr Ala Cys Cys Ala Gly Thr Gly Ala
1 5 10 15
Cys Cys Gly Cys Cys Thr Thr Gly Cys Thr Cys Cys Thr Gly Cys Cys
20 25 30
Gly Cys Thr Gly Gly Cys Cys Thr Thr Gly Cys Thr Gly Cys Thr Cys
35 40 45
Cys Ala Cys Gly Cys Cys Gly Cys Cys Ala Gly Gly Cys Cys Gly Met
50 55 60
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
65 70 75 80
Ala Ala Arg Pro
<210> 4
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Pro Gly Trp Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 5
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Glu Leu 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 Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105
<210> 6
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 7
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
85 90 95
Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu
100 105 110
<210> 8
<211> 68
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
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 Arg Ser Lys Arg Ser
20 25 30
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
35 40 45
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
50 55 60
Ala Tyr Arg Ser
65
<210> 9
<211> 69
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
35 40 45
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
50 55 60
Ile Thr Leu Tyr Cys
65
<210> 10
<211> 42
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 11
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110

Claims (10)

1.重组表达载体，其特征在于，所述重组表达载体共表达sh-PTPN2-1序列与Trop2/PD-L1双特异性嵌合抗原受体的核苷酸序列，其中，所述sh-PTPN2-1序列能够敲除人源PTPN2基因。
2.根据权利要求1所述的重组表达载体，其特征在于，所述sh-PTPN2-1序列的核苷酸序列如SEQ ID No.1所示。
3.根据权利要求1所述的重组表达载体，其特征在于，所述Trop2/PD-L1双特异性嵌合抗原受体由CD8、人源化抗Trop2、抗PD-L1单链抗体、跨膜区CD8TM、胞内区CD28、CD137和CD3ζ串联构成。
4.根据权利要求1所述的重组表达载体，其特征在于，所述sh-PTPN2-1序列的上游还含有hU6p启动子，以驱动sh-PTPN2-1序列的表达。
5.权利要求1～4任一项所述的重组表达载体在制备嵌合抗原受体T细胞中的应用。
6.耗竭减少的嵌合抗原受体T细胞，其特征在于，所述嵌合抗原受体T细胞含有能够共表达sh-PTPN2-1序列与Trop2/PD-L1双特异性嵌合抗原受体的核苷酸序列的重组表达载体，其中，sh-PTPN2-1序列能够敲除人源PTPN2基因。
7.根据权利要求6所述的耗竭减少的嵌合抗原受体T细胞，其特征在于，所述sh-PTPN2-1序列的核苷酸序列如SEQ ID No.1所示。
8.根据权利要求6所述的耗竭减少的嵌合抗原受体T细胞，其特征在于，所述Trop2/PD-L1双特异性嵌合抗原受体由CD8、人源化抗Trop2、抗PD-L1单链抗体、跨膜区CD8TM、胞内区CD28、CD137和CD3ζ串联构成。
9.权利要求6所述的耗竭减少的嵌合抗原受体T细胞的制备方法，其特征在于，所述方法包括以下步骤：提取含有重组表达载体的慢病毒，并感染T细胞，使T细胞表达sh-PTPN2-1和Trop2/PD-L1双特异性嵌合抗原受体。
10.权利要求6所述的耗竭减少的嵌合抗原受体T细胞在制备抗肿瘤药物和/或制备抗肿瘤药物增效剂方面的应用。
CN202010973707.6A 2020-09-16 2020-09-16 重组表达载体、耗竭减少的嵌合抗原受体t细胞及其应用 Pending CN112080527A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010973707.6A CN112080527A (zh) 2020-09-16 2020-09-16 重组表达载体、耗竭减少的嵌合抗原受体t细胞及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010973707.6A CN112080527A (zh) 2020-09-16 2020-09-16 重组表达载体、耗竭减少的嵌合抗原受体t细胞及其应用

Publications (1)

Publication Number Publication Date
CN112080527A true CN112080527A (zh) 2020-12-15

Family

ID=73736888

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010973707.6A Pending CN112080527A (zh) 2020-09-16 2020-09-16 重组表达载体、耗竭减少的嵌合抗原受体t细胞及其应用

Country Status (1)

Country Link
CN (1) CN112080527A (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118562802A (zh) * 2024-06-20 2024-08-30 深圳细胞谷生物医药有限公司 靶向抑制PTPN2的shRNA及包括其的CAR-T细胞、药物组合物

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105431524A (zh) * 2013-06-10 2016-03-23 达娜-法勃肿瘤研究所公司 用于降低通过肿瘤细胞的免疫抑制的方法和组合物
CN107109368A (zh) * 2014-10-07 2017-08-29 塞勒克提斯公司 用于调节car诱导的免疫细胞活性的方法
CN109748975A (zh) * 2019-03-05 2019-05-14 南京市第一医院 一种双特异性嵌合抗原受体及其应用
CN110257429A (zh) * 2019-05-15 2019-09-20 中国人民解放军总医院 重组表达载体、靶向性的t细胞及它们的应用
WO2020072126A2 (en) * 2018-08-07 2020-04-09 Dana-Farber Cancer Institute, Inc. Modulating ptpn2 to increase immune responses and perturbing gene expression in hematopoietic stem cell lineages

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105431524A (zh) * 2013-06-10 2016-03-23 达娜-法勃肿瘤研究所公司 用于降低通过肿瘤细胞的免疫抑制的方法和组合物
CN107109368A (zh) * 2014-10-07 2017-08-29 塞勒克提斯公司 用于调节car诱导的免疫细胞活性的方法
WO2020072126A2 (en) * 2018-08-07 2020-04-09 Dana-Farber Cancer Institute, Inc. Modulating ptpn2 to increase immune responses and perturbing gene expression in hematopoietic stem cell lineages
CN109748975A (zh) * 2019-03-05 2019-05-14 南京市第一医院 一种双特异性嵌合抗原受体及其应用
CN110257429A (zh) * 2019-05-15 2019-09-20 中国人民解放军总医院 重组表达载体、靶向性的t细胞及它们的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FLORIAN WIEDE等: ""PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours"", 《THE EMBO JOURNAL》 *
ROBERT T.MANGUSO等: ""In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target"", 《NATURE》 *
郭葆玉著: "《基因工程药学》", 31 March 2010, 人民卫生出版社 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118562802A (zh) * 2024-06-20 2024-08-30 深圳细胞谷生物医药有限公司 靶向抑制PTPN2的shRNA及包括其的CAR-T细胞、药物组合物

Similar Documents

Publication Publication Date Title
CN110684739B (zh) 一种嵌合抗原受体t细胞及其应用
CN108018299B (zh) 靶向bcma的嵌合抗原受体及其用途
CN108384760B (zh) 携带cd20/cd19双特异性嵌合抗原受体的人t淋巴细胞及制备方法和应用
CN111511911B (zh) 表达特异性地识别人间皮素的细胞表面分子、il-7和ccl19的免疫活性细胞
CN111748044B (zh) Cd19和pd-l1双靶点嵌合抗原受体及其应用
WO2021249549A1 (zh) 一种嵌合抗原受体及其用途
CN113913379A (zh) T淋巴细胞及其应用
CN113896801B (zh) 靶向人Claudin18.2和NKG2DL的嵌合抗原受体细胞及其制备方法和应用
US20210122831A1 (en) Chimeric antigen receptor
CN113692441A (zh) 一种包含肿瘤抗原识别受体的免疫细胞及其应用
CN106117367B (zh) 一种her-3特异性嵌合抗原受体及其应用
CN114517185B (zh) 嵌合抗原受体nk细胞及其制备方法和应用
CN107624119B (zh) 一种分子、表达其的细胞及其制备方法和用途
CN110358735B (zh) 一种ctl细胞的制备方法及应用
Greenshpan et al. Synthetic promoters to induce immune-effectors into the tumor microenvironment
CN110194803B (zh) 一种靶向EpCAM的嵌合抗原受体及其应用
CN117510647A (zh) 携带pd-1单链抗体和ccr6趋化因子受体的car-t免疫细胞的制备及其用途
CN111378046B (zh) 一种免疫效应细胞转换受体
CN111875712A (zh) 一种增强型靶向muc1的嵌合抗原受体及其应用
CN112080527A (zh) 重组表达载体、耗竭减少的嵌合抗原受体t细胞及其应用
WO2022007796A1 (zh) 共表达IL-21和hrCD16嵌合受体的免疫细胞及其应用
CN106008721B (zh) 一种c-Met特异性嵌合抗原受体及其应用
KR20210150446A (ko) 종양 항원 인식 수용체를 함유하는 면역 세포 및 이의 적용
CN112469734A (zh) 表达嵌合抗原受体的t细胞、该嵌合抗原相关表达载体以及它们的应用
CN114249807B (zh) 缺氧触发的人工转录因子、转录控制系统及其应用

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
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20201215