CN117924507A - CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof - Google Patents
CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof Download PDFInfo
- Publication number
- CN117924507A CN117924507A CN202211315740.5A CN202211315740A CN117924507A CN 117924507 A CN117924507 A CN 117924507A CN 202211315740 A CN202211315740 A CN 202211315740A CN 117924507 A CN117924507 A CN 117924507A
- Authority
- CN
- China
- Prior art keywords
- αcd3
- αgpc3
- cell
- nitrogen
- gpc3
- 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
Links
- 102100032530 Glypican-3 Human genes 0.000 title claims abstract description 33
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 title claims abstract description 33
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 32
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 56
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 28
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 26
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 26
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 3
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 45
- 239000013612 plasmid Substances 0.000 claims description 26
- 239000002773 nucleotide Substances 0.000 claims description 19
- 125000003729 nucleotide group Chemical group 0.000 claims description 19
- 206010028980 Neoplasm Diseases 0.000 claims description 18
- 239000013604 expression vector Substances 0.000 claims description 12
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 9
- 239000013598 vector Substances 0.000 claims description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 238000001890 transfection Methods 0.000 claims description 7
- 241000700605 Viruses Species 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 150000007523 nucleic acids Chemical class 0.000 claims description 6
- 229950010131 puromycin Drugs 0.000 claims description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 5
- 201000007270 liver cancer Diseases 0.000 claims description 5
- 208000014018 liver neoplasm Diseases 0.000 claims description 5
- 230000010473 stable expression Effects 0.000 claims description 5
- 239000013613 expression plasmid Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000010367 cloning Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- 210000003205 muscle Anatomy 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000003248 secreting effect Effects 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 9
- 108010075254 C-Peptide Proteins 0.000 abstract description 4
- 108020004705 Codon Proteins 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000013519 translation Methods 0.000 abstract description 2
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 1
- 230000008707 rearrangement Effects 0.000 abstract 1
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 21
- 241000699670 Mus sp. Species 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 210000004881 tumor cell Anatomy 0.000 description 11
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 9
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 8
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 229930182555 Penicillin Natural products 0.000 description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011065 in-situ storage Methods 0.000 description 4
- 230000002147 killing effect Effects 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 229940049954 penicillin Drugs 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 229960005322 streptomycin Drugs 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 3
- 102100037850 Interferon gamma Human genes 0.000 description 3
- 108010074328 Interferon-gamma Proteins 0.000 description 3
- 101710160107 Outer membrane protein A Proteins 0.000 description 3
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 230000022534 cell killing Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 2
- 229920002971 Heparan sulfate Polymers 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 101100337463 Mus musculus Gpc3 gene Proteins 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 108090000054 Syndecan-2 Proteins 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000004068 intracellular signaling Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 210000002027 skeletal muscle Anatomy 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 108700031361 Brachyury Proteins 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 1
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000010956 Glypican Human genes 0.000 description 1
- 108050001154 Glypican Proteins 0.000 description 1
- 108050007237 Glypican-3 Proteins 0.000 description 1
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000006911 nucleation Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002363 skeletal muscle cell Anatomy 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
- 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
- C07K16/303—Liver or Pancreas
-
- 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
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- 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
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/80—Vaccine for a specifically defined cancer
- A61K2039/844—Liver
-
- 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
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Reproductive Health (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof, wherein a connecting peptide is sequentially connected with VL αGPC3、VHαGPC3,VHαCD3 and VL αCD3,VLαGPC3 nitrogen ends and is connected with VH αGPC3 nitrogen ends, VH αGPC3 carbon end is connected with VH αCD3 carbon end, VH αCD3 nitrogen end is connected with VL αCD3 nitrogen end or VL αGPC3 carbon end is connected with VH αGPC3 carbon end, VH αGPC3 nitrogen end is connected with VH αCD3 nitrogen end, VH αCD3 carbon end is connected with VL αCD3 carbon end, and different from traditional BiTE, the light/heavy chain amino acid sequence of scFv is the same as the original sequence but opposite in translation direction through genetic engineering rearrangement of protein coding gene codon sequence, and the formed BiTE structure is more similar to a natural antibody molecular structure and is used for preparing antitumor drugs.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof.
Background
Chimeric antigen receptor T cells (CHIMERIC ANTIGEN receptor-Tcell, CAR-T) are prepared by chimeric single-chain variable domain fragments (scFv) with T cell surface receptors on T cells, thereby allowing T cells to recognize tumor cells independent of major histocompatibility complex I (major histocompatibility complexI, mhc I), and then purifying, amplifying and activating in vitro, and infusing back CAR-T cells into patients, thereby allowing them to specifically exert the function of killing tumor cells. The CAR vector is mainly composed of 3 parts: an antigen binding domain, a transmembrane region, and an intracellular signaling region.
In CAR-T molecules, the intracellular signaling domain plays an important role. The effect of co-stimulation of CARs in CAR engineering is of great concern, with the goal of generating CAR constructs with optimal intracellular domains. Both of the two most common FDA-approved co-stimulatory domains CD28 and 4-1BB (CD 137) are associated with higher patient response rates. There was a difference in CD28 and 4-1BB functions. CD28 mainly promotes CAR-T proliferation, enhancing effector functions, especially cytokine production. 4-1BB promotes survival and increases retention time in CAR-T. Clinical results indicate that the CAR-T cells have great advantages in treating hematological malignancies.
The selection of tumor targets is a key factor in determining success or failure of CAR-T treatment, and the selected tumor-specific antigens need to be highly expressed in tumor tissues but not expressed or at very low expression levels in important tissues.
Glypican-3 (GPC 3) is a member of the family of heparan sulfate glycoproteins (heparan sulfate proteoglycans, HSPG), the GPC3 protein consists of 580 amino acids and has a relative molecular mass of about 70kDa. GPC3 is expressed abundantly in fetuses, whereas it is not expressed or expressed in very low amounts in most tissues of healthy adults. In contrast, immunohistochemical staining of tumor tissue in 147 HCC patients found that up to 87% (128/147) samples were GPC3 positive. Numerous documents indicate that GPC3 is an important index for HCC diagnosis and prognosis effect evaluation, and is also an ideal target for HCC treatment.
Bispecific T cell adaptors (bispecific T-CELL ENGAGER, biTE) represent a class of bispecific antibodies with remarkable anti-tumor effects, which are capable of targeted activation of self T cells to kill tumor cells. BiTE consists of two single-chain variable fragments (scFv) connected in series by a flexible linker. One scFv recognizes the T cell surface protein CD3 epsilon, while the other scFv recognizes a specific tumor cell surface antigen. This structure of BiTE and the ability to specifically bind proteins allows it to physically bridge T cells to tumor cells to form T cell-BiTE-tumor cell complexes, induce immune synapse formation, stimulate T cell activation, and produce cytokines that kill tumors. In recent years, biTE has been significantly advanced in anti-tumor studies, and clinically, an ideal therapeutic effect has been achieved.
Conventional scFv are generally designed according to the following principles:
1. Extracting a variable region fragment Fv of the IgG antibody according to the sequence, wherein the variable region fragment Fv comprises two peptide chains, namely a heavy chain variable region VH and a light chain variable region VL;
2. Linking VH and VL together through a linker to form a single peptide chain (VH-VL or VL-VH);
3. (Gly 4Ser)3 is one of the most commonly used connecting peptides at present, which is soft but the prior art has the following defects that 1) (G 4S)3 connecting peptide is too short, so that the spatial conformation of the connected scFv is far away from that of a natural antibody structure, and 2) the existing scFv targeting GPC3 has limited killing effect on tumor cells.
Disclosure of Invention
The present invention aims to overcome the disadvantages of the prior art and to provide a CD3 epsilon and GPC3 bispecific T cell adaptor and uses thereof.
The aim of the invention is achieved by the following technical scheme: a CD3 epsilon and GPC3 bispecific T cell adaptor comprising an alpha GPC3 light chain variable region VL, an alpha GPC3 heavy chain variable region VH, an alpha CD3 light chain variable region VL connected in sequence by a Linker peptide Linker, the nitrogen end of VL αGPC3 connected to the nitrogen end of VH αGPC3, the carbon end of VH αGPC3 connected to the carbon end of VH αCD3, the nitrogen end of VH αCD3 connected to the nitrogen end of VL αCD3.
Further, the nucleotide sequence of the Linker is shown as SEQ ID No.1, the nucleotide sequence of the light chain variable region VL of alpha GPC3 is shown as SEQ ID No.2, the nucleotide sequence of the light chain variable region VH of alpha GPC3 is shown as SEQ ID No.3, the nucleotide sequence of the heavy chain variable region VH of alpha CD3 is shown as SEQ ID No.4, and the nucleotide sequence of the light chain variable region VL of alpha CD3 is shown as SEQ ID No. 5.
A nucleic acid molecule comprising the CD3 epsilon and GPC3 bispecific T cell adaptors described above.
An expression vector comprising the nucleic acid molecule described above.
Further, the expression vector is a secretory expression vector, the expression plasmid is an enhanced muscle specific expression plasmid pEMS, the promoter of pEMS is EMS, and the nucleotide sequence of the pEMS is shown as SEQ ID No. 6.
Further, it also comprises a mouse Ig kappa signal peptide, and the nucleotide sequence of the mouse Ig kappa signal peptide is shown as SEQ ID No. 7.
A cell line comprising the expression vector described above.
A method of constructing a cell line comprising the steps of:
s1, synthesizing target gene fragments of CD3 epsilon and GPC3 dual-specificity T cell adaptors;
S2, cloning and connecting the target gene fragment constructed in the step S1 to pLVX-Puro vectors to form transfection plasmids;
s3, co-transferring psPAX plasmids, pMD2.G and the transfection plasmids into HEK293T cells to obtain virus liquid;
s4, infecting CHO-K1 cells by using virus liquid, and screening cell lines with stable expression by using puromycin to obtain CHO-K1 cell lines with stable expression transfection plasmid.
The application of the CD3 epsilon and GPC3 dual-specificity T cell adapter, the nucleic acid molecule, the expression vector and the cell strain in preparing antitumor drugs.
Further, the tumor is liver cancer.
The invention has the following advantages: the CD3 epsilon and GPC3 dual-specificity T cell adapter changes the connection mode of the traditional BiTE, rearranges the codon sequence of the protein coding gene by a genetic engineering technology, ensures that the light/heavy chain amino acid sequence of scFv is completely the same as the original sequence but has opposite translation directions, the formed BiTE structure is more similar to the conformation of a natural antibody molecule, the expression of therapeutic protein can be detected 7-14 days after plasmid injection is carried out on skeletal muscle, the expressed protein can activate and maintain the activity of the T cell and promote the proliferation of the T cell, and the CD3 epsilon and GPC3 dual-specificity T cell adapter can simultaneously identify and link tumor cells and T cells, guide the T cells to kill the liver cancer cells and has good killing effect on tumors; the invention utilizes skeletal muscle to secrete therapeutic protein, modifies T cells of a patient in vivo, avoids graft versus host reaction (graft versus host reaction, GVHR) and host versus graft reaction (host versus graft reaction, HVGR) caused by allogeneic CAR-T therapy, and reduces the anti-tumor treatment cost.
Drawings
FIG. 1 shows a pcDNA3.1 (+) vector map.
FIG. 2 is a schematic diagram of pEMS-mαGPC3 xαCD3 plasmids.
FIG. 3 is a block diagram of pEMS-mαGPC3 xαCD3-Ctrl and pEMS-mαGPC3 xαCD3-Exp1, wherein A is the nitrogen terminal of VL αGPC3 linked to the nitrogen terminal of VH αGPC3, the carbon terminal of VH αGPC3 linked to the carbon terminal of VH αCD3, and the nitrogen terminal of VH αCD3 linked to the nitrogen terminal of VL αCD3; the carbon terminal of VL αGPC3 is connected to the carbon terminal of VH αGPC3, the nitrogen terminal of VH αGPC3 is connected to the nitrogen terminal of VH αCD3, and the carbon terminal of VH αCD3 is connected to the carbon terminal of VL αCD3.
FIG. 4 is a pLVX-Puro vector map.
FIG. 5 is a graph showing the results of in vitro killing ability of 2 BiTEs against Hepa 1-6 cells.
FIG. 6 is a graph showing the results of 2 BiTE-induced release of IFN-gamma from PBMC.
FIG. 7 is a graph showing the results of 2 BiTE-induced PBMC release of TNF- α.
FIG. 8 is a schematic of in situ plasmid injection in mice.
FIG. 9 is a graph showing tumor growth after in situ injection of tumor-bearing mouse plasmids.
FIG. 10 is a graph showing survival after in situ injection of tumor-bearing mouse plasmids.
Detailed Description
The invention will be further described with reference to the accompanying drawings and examples, to which the scope of the invention is not limited: example 1: a CD3 epsilon and GPC3 bispecific T cell adaptor comprising an alpha GPC3 light chain variable region VL, an alpha GPC3 heavy chain variable region VH, an alpha CD3 light chain variable region VL connected in sequence by a Linker peptide Linker, the nitrogen terminus of VL αGPC3 being connected to the nitrogen terminus of VH αGPC3, the carbon terminus of VH αGPC3 being connected to the carbon terminus of VH αCD3, the nitrogen terminus of VH αCD3 being connected to the nitrogen terminus of VL αCD3, as shown as a in fig. 3; or the carbon terminal of VL αGPC3 is linked to the carbon terminal of VH αGPC3, the nitrogen terminal of VH αGPC3 is linked to the nitrogen terminal of VH αCD3, and the carbon terminal of VH αCD3 is linked to the carbon terminal of VL αCD3, as shown in B in fig. 3.
The nucleotide sequence of the connecting peptide Linker is shown as SEQ ID No. 1. The nucleotide sequence of the alpha GPC3 light chain variable region VL is shown as SEQ ID No.2, the nucleotide sequence of the alpha GPC3 light chain variable region VH is shown as SEQ ID No.3, the nucleotide sequence of the alpha CD3 heavy chain variable region VH is shown as SEQ ID No.4, and the nucleotide sequence of the alpha CD3 light chain variable region VL is shown as SEQ ID No. 5.
The method comprises the following steps:
1. linker peptide Linker: (Gly 4Ser)3 total 45bp, sequence as follows (SEQ ID No. 1):
5’-GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC-3’
2. Light chain variable region (VL αGPC3) of the targeted mouse GPC3 antibody: the total length is 336bp, the sequence is as follows (SEQ ID No. 2):
5'-GACGTGGTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGGTGCACAGCAACGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGAGCAACAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCAGAACACCCACGTGCCCCCCACCTTCGGCAGCGGCACCAAGCTGGAGATCAAG-3'
3. The heavy chain variable region (VH αGPC3) of the targeted mouse GPC3 antibody was 345bp in total length, with the following sequence (SEQ ID No. 3):
5'-CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGGCCCGGCGCCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGACTACGAGATGCACTGGGTGAAGCAGACCCCCGTGCACGGCCTGAAGTGGATCGGCGCCCTGGACCCCAAGACCGGCGACACCGCCTACAGCCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCACCAGGTTCTACAGCTACACCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC-3'
4. the heavy chain variable region (VH αCD3) of the targeted mouse CD3 antibody is 348bp in total, and the sequence is as follows (SEQ ID No. 4):
5'-GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCAAGAGCCTGAAGCTGAGCTGCGAGGCCAGCGGCTTCACCTTCAGCGGCTACGGCATGCACTGGGTGAGGCAGGCCCCCGGCAGGGGCCTGGAGAGCGTGGCCTACATCACCAGCAGCAGCATCAACATCAAGTACGCCGACGCCGTGAAGGGCAGGTTCACCGTGAGCAGGGACAACGCCAAGAACCTGCTGTTCCTGCAGATGAACATCCTGAAGAGCGAGGACACCGCCATGTACTACTGCGCCAGGTTCGACTGGGACAAGAACTACTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC-3'
5. The total length of the light chain variable region (VL αCD3) of the targeted mouse CD3 antibody was 321bp, and the sequence was as follows (SEQ ID No. 5):
5'-GACATCCAGATGACCCAGAGCCCCAGCAGCCTGCCCGCCAGCCTGGGCGACAGGGTGACCATCAACTGCCAGGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCAACAAGCTGGCCGACGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCAGGGACAGCAGCTTCACCATCAGCAGCCTGGAGAGCGAGGACATCGGCAGCTACTACTGCCAGCAGTACTACAACTACCCCTGGACCTTCGGCCCCGGCACCAAGCTGGAGATCAAG-3'
EMS is an enhanced skeletal muscle cell-specific promoter (Enhanced Muscle Specific Promoter) and is named EMS, in order to be able to help the plasmid expressed proteins secrete out of the cell, we have added a mouse Ig kappa signal peptide after the promoter and before the multiple cloning site region to direct the secretion of the expressed proteins out of the cell.
(6) EMS total length 701bp, sequence as follows (SEQ ID No. 6):
5'-GGTACCTTGATGTACTGCCAAGTTGGAAAGTCCCGTTAGTGCCCATTGACGTCAATAATATATGGCGACGGCCGGGCCCCTCCCTGGGGACAGCCCCGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGACTATATAAAAAACCTGACCCGATATGCCTGGCCAGCCAATAGCGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGACACCCAAATATGGCGACGGGTGAGGAATGGTGACCAAGTCAGCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCACCAACACCTGCTGCCTGCCCGCTCTAAAAATAACTCCCGGCTTCAGGTTTCCCTAGGGCCCCTCCCTGGGGACAGCCCCATATGGCGACGGCCCCCCATTGACGTCAATGGGACGGTAAATGGCCCGCCTGGCGCCCATTGACGTCAATAATCCAGCCAATAGCACCCGATATGCCTGGGGACTATATAAAAAACCTGGGACACCCGAGATGCCTGGTTACAAGGCCTGGGGACACGCTCTAAAAATAACTCCCCCAACACCTGCTGCCTGCCGGCTTCAGGTTTCCCTACTCGAG-3'
(7) The total length of the mouse Ig kappa signal peptide is 60bp, and the sequence is as follows (SEQ ID No. 7):
5’-GAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC-3’
The 7 parts are synthesized by Shanghai, constructed on a pcDNA3.1 (+) vector, the vector map is shown in figure 1, and the enzyme cutting sites are BamH I and EcoR I; the connection sequence is performed in the sequence shown in fig. 2. The above genes were cloned in pEMS for expression of secreted proteins.
Example 2: construction of plasmids expressing double-targeting adapter antibodies recognizing T cells and tumor cells
The BiTE comprises an antibody alpha CD3 for recognizing a mouse T cell surface antigen CD3 and an antibody alpha GPC3 for recognizing a mouse liver cancer cell surface antigen GPC3, which are connected through peptide segments. The BiTE acts to simultaneously recognize and link two cells to direct T cells to kill tumor cells. The plasmids expressing the BiTE were designated pEMS-mαGPC3 xαCD3-Ctrl, pEMS-mαGPC3 xαCD3-Exp1, pEMS-mαGPC3 xαCD3-Ctrl and pEMS-mαGPC3 xαCD3-Exp1 structures as shown in FIG. 3.
To construct CHO-K1 cell lines capable of stably expressing pEMS-mαGPC3 xαCD3-Ctrl and pEMS-mαGPC3 xαCD3-Exp 1. The present invention is characterized in that mαGPC3 xαCD3-Ctrl and mαGPC3 xαCD3-Exp1 target gene fragments are connected to pLVX-Puro vector by molecular cloning, and pLVX-Puro vector map is shown in FIG. 4.
Example 3: the lentivirus packaging steps are as follows:
1) Inoculating HEK293T cells in a 10cm dish until the cell confluency reaches 70% -90%;
2) HEK293T cell medium (10% FBS, DMEM medium containing 100IU/mL penicillin and 100. Mu.g/mL streptomycin) was replaced with 10mL fresh Quan Pei (10% FBS, 100IU/mL penicillin and 100. Mu.g/mL streptomycin DMEM medium) 1h in advance;
3) Viral packaging plasmid (psPAX and pMD2. G), transfection plasmid (pLVX-mαGPC3 αCD3-Ctrl, pLVX-mαGPC3 αCD3-Exp 1) were prepared as shown in Table 1, added to 750. Mu.L Opti-MEM, mixed, and left to stand for 5min;
Table 1: preparation of lentivirus packaging plasmid
4) Adding 24 μL of Lipo8000 into 750 μL of Opti-MEM, mixing, and standing for 5min;
5) Mixing the solutions obtained in the steps 3 and 4, and incubating for 5min at room temperature;
6) Uniformly dispersing the mixed solution obtained in the step 5 into HEK293T cells, uniformly shaking in a cross manner, and putting into an incubator;
7) After 12h, 10mL of complete medium (DMEM medium containing 10% FBS, 100IU/mL penicillin and 100. Mu.g/mL streptomycin) was changed, and calculation was started from this point. After 48 hours, collecting the cell culture solution (namely virus solution) into a 15mL centrifuge tube, and centrifuging at 1000rpm for 5 minutes;
8) The supernatant was aspirated by syringe, filtered through 0.45 μm microporous filter membrane, dispensed into 1.5mL EP tubes, 500. Mu.L per tube, sealed with sealing membrane and stored at-80 ℃.
Example 4: CHO-K1 cell lentiviral infection:
1) And (3) paving: spreading a 6-hole plate to be infected with cells CHO-K1 until the cells grow to 60-80%;
2) Changing 600 μl of complete culture medium, sequentially adding 500 μl of virus solution, and polybrene (final concentration 10 μg/mL); incubating the incubator overnight;
3) The following day, the complete medium was changed and after 48h, screened with puromycin (final concentration 2. Mu.g/mL);
example 5: stable expression cell strain selection:
1) After 72h, adding puromycin with a final concentration of 2 mug/mL for culturing for 24h, changing to normal culture medium if the cell confluency is lower than 30%, culturing until the cell confluency is higher than 30%, changing to fresh culture medium with a concentration of 8 mug/mL puromycin (10% FBS, 100IU/mL penicillin, 100 mug/mL streptomycin and 8 mug/mL DMEM culture medium) for culturing and screening for 2 weeks; if the cell confluency is not less than 30%, 2 mug/mL puromycin fresh medium is used for continuous culture screening for 2 weeks;
2) The obtained CHO-K1 cell lines stably expressing pLVX-mαGPC3 xαCD3-Ctrl and pLVX-mαGPC3 xαCD3-Exp1 were frozen at-80℃for use.
Example 6: the effect of expressed antibodies on T cell killing capacity was studied:
mice Hepa 1-6 were labeled with 1 μ L CELL TRACKER Violet and tumor cells after labeling were incubated overnight at 37 ℃. After obtaining peripheral blood mononuclear cells (PERIPHERAL BLOOD MONOCYTE CELL, PBMC) from mice, enriched lymphocytes were incubated overnight at 37℃at a concentration of 1X 10 6/mL, and adherent cells were removed the next day, at which time the cells were ready for use in subsequent co-culture experiments. Target cells were co-incubated with adherent cell depleted PBMCs [ effector cells: target (E: T) cell ratio, 1:1,4:1, 10:1]. After the co-culture is completed, the upper layer of suspended lymphocytes are discarded, PBS is used for washing, and the attached target cells are digested by pancreatin and then subjected to flow cytometry analysis, as shown in FIG. 5, it can be seen from FIG. 5 that mαGPC3 xαCD3-Exp1 shows a tumor cell killing effect similar to mαGPC3 xαCD3-Ctrl.
The IFN-gamma content released by lymphocytes in PBMC is detected by ELISA kit, the operation steps are described by referring to the Eboltag kit (Cat: RK 00019), the TNF-alpha content released by lymphocytes in PBMC is detected by ELISA kit, the operation steps are described by referring to the Eboltag kit (Cat: RK 00027), and after mαGPC3 xαCD3-Exp1 treatment, more IFN-gamma is released by PBMC than after mαGPC3 xαCD3-Ctrl treatment as shown in FIG. 6; after mαGPC3 xαCD3-Exp1 treatment, the PBMC released TNF- α content was comparable to that of the mαGPC3 xαCD3-Ctrl treated group as shown in FIG. 7. From the cellular level, it was verified that mαGPC3 xαCD3-Exp1 exhibited cell killing ability comparable to that of conventional mαGPC3 xαCD3-Ctrl.
Example 7: treatment effect detection
1. Mice weight detection:
In situ plasmid injection is schematically shown in FIG. 8 (cell inoculation 6X 10 5/mouse, plasmid injection (40. Mu.g/mouse), time to kill mice, etc.); day 0 each mouse was inoculated with 6 x 10 5 Hepa 1-6 liver cancer cells, day 2 mice were injected with 40 μg plasmid, and 1h post-plasmid injection was shocked with a Datura-brand electric needle for 3min to assist plasmid nucleation (continuous wave, 5Hz, intensity 3).
2. Tumor volume detection in mice:
Tumor volume was measured every two days during animal experiments, and tumor growth curves of tumor-bearing mice were plotted, and it can be seen from fig. 9 that tumor growth of tumor-bearing mice was inhibited after mαgpc3×αcd3-Exp1 treatment, and the therapeutic effect was not significantly different from mαgpc3×αcd3-Ctrl.
3. Median survival of mice:
Counting the death time of the mice, and judging whether each treatment scheme prolongs the service life of the mice; tumor-bearing mice, which had tumor volumes exceeding 1500mm3 or mice died, were considered the experimental endpoint and tumor-bearing mice were plotted for survival, as shown in fig. 10, showing comparable survival rates after treatment with mαgpc3×αcd3-Exp1 plasmid as after treatment with mαgpc3×αcd3-Ctrl plasmid.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art who is skilled in the art to which the present invention pertains will appreciate that the technical scheme and the inventive concept according to the present invention are equally substituted or changed within the scope of the present invention.
Claims (10)
1. A CD3 epsilon and GPC3 bispecific T cell adaptor comprising an alpha GPC3 light chain variable region VL, an alpha GPC3 heavy chain variable region VH, an alpha CD3 light chain variable region VL connected in sequence by a Linker peptide Linker, the nitrogen terminal of VL αGPC3 being connected to the nitrogen terminal of VH αGPC3, the carbon terminal of VH αGPC3 being connected to the carbon terminal of VH αCD3, the nitrogen terminal of VH αCD3 being connected to the nitrogen terminal of VL αCD3;
Or the carbon terminal of VL αGPC3 is linked to the carbon terminal of VH αGPC3, the nitrogen terminal of VH αGPC3 is linked to the nitrogen terminal of VH αCD3, and the carbon terminal of VH αCD3 is linked to the carbon terminal of VL αCD3.
2. The CD3 epsilon and GPC3 bispecific T cell adapter of claim 1, wherein the Linker peptide Linker has a nucleotide sequence shown in SEQ ID No. 1, the light chain variable region VL of αgpc3 has a nucleotide sequence shown in SEQ ID No.2, the light chain variable region VH of αgpc3 has a nucleotide sequence shown in SEQ ID No. 3, the heavy chain variable region VH of αcd3 has a nucleotide sequence shown in SEQ ID No. 4, and the light chain variable region VL of αcd3 has a nucleotide sequence shown in SEQ ID No. 5.
3. A nucleic acid molecule comprising the CD3 epsilon and GPC3 bispecific T cell engager of claim 1 or 2.
4. An expression vector comprising the nucleic acid molecule of claim 3.
5. The expression vector of claim 4, wherein the expression vector is a secretory expression vector, the expression plasmid is an enhanced muscle-specific expression plasmid pEMS, the promoter of pEMS is EMS, and the nucleotide sequence of pEMS is shown as SEQ ID No. 6.
6. The expression vector of claim 5, further comprising a mouse Ig kappa signal peptide having the nucleotide sequence set forth in SEQ ID No. 7.
7. A cell line comprising the expression vector of claim 4.
8. The method for constructing a cell line according to claim 7, comprising the steps of:
s1, synthesizing the target gene fragment of claim 1;
s2, cloning and connecting the target gene fragment constructed in the step S1 to pLVX-Puro vectors to form transfection plasmids;
s3, co-transferring psPAX plasmids, pMD2.G and the transfection plasmids into HEK293T cells to obtain virus liquid;
S4, infecting CHO-K1 cells by using virus liquid, and screening cell strains with stable expression by using puromycin to obtain CHO-K1 cell strains with stable expression transfection plasmid.
9. Use of CD3 epsilon and GPC3 bispecific T cell adaptors according to claim 1 or 2, a nucleic acid molecule according to claim 3, an expression vector according to claim 4 or 5 or 6, a cell strain according to claim 7 for the preparation of an antitumor drug.
10. The use according to claim 9, wherein the tumor is liver cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211315740.5A CN117924507A (en) | 2022-10-26 | 2022-10-26 | CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211315740.5A CN117924507A (en) | 2022-10-26 | 2022-10-26 | CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117924507A true CN117924507A (en) | 2024-04-26 |
Family
ID=90761521
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211315740.5A Pending CN117924507A (en) | 2022-10-26 | 2022-10-26 | CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117924507A (en) |
-
2022
- 2022-10-26 CN CN202211315740.5A patent/CN117924507A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108341881B (en) | Chimeric antigen receptor with safety switch, expression gene thereof, NK cell modified by chimeric antigen receptor and application of chimeric antigen receptor | |
| CN111944054B (en) | anti-BCMA CAR and expression vector and application thereof | |
| CN109320615A (en) | Target the Chimeric antigen receptor and application thereof of novel B CMA | |
| CN104910278B (en) | A kind of slow virus with high-efficiency transfection ability and biological activity for being used to prepare CART cells | |
| CN118792252A (en) | T lymphocyte and application thereof | |
| CN107475275B (en) | Chimeric antigen receptor and expression gene thereof, double-antigen-regulated chimeric antigen receptor modified T cell and application thereof | |
| CN116041542A (en) | NK cell preparation method for reversing tumor microenvironment inhibitory signals and application | |
| CN111139222B (en) | Recombinant mesenchymal stem cell and preparation method and application thereof | |
| CN111875712A (en) | Enhanced MUC 1-targeted chimeric antigen receptor and application thereof | |
| CN109517798B (en) | NK (natural killer) cell of chimeric CEA antigen receptor as well as preparation method and application of NK cell | |
| CN109666074B (en) | Application of chemokine receptor CXCR5 | |
| CN111732665B (en) | Chimeric antigen receptor of cells for targeted expression of carcinoembryonic antigen | |
| CN108728460A (en) | Target the Chimeric antigen receptor and application thereof of GD2 | |
| WO2018205343A1 (en) | Car.il-33-t and preparation and application thereof | |
| CN110699371A (en) | Fc gamma RIIa-based chimeric gene and application thereof | |
| CN117736335A (en) | Double-targeting CAR-T cell targeting mesothelin and NKG2D ligand and application thereof | |
| CN107502596A (en) | Express T cell and its application of the specificity TCRs of NY ESO 1 | |
| CN117924507A (en) | CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof | |
| CN115960256A (en) | Long-acting chimeric antigen receptor, long-acting chimeric antigen vector, and construction method and application thereof | |
| CN114957483A (en) | CD 155-targeted CAR (CAR-based protein) structural design and application thereof | |
| CN115141806A (en) | Chimeric antigen receptor T cell targeting Her2 and expressing PD-L1 antibody, and preparation method and application thereof | |
| CN108165568A (en) | A kind of culture CD19CAR-iNKT cellular processes and purposes | |
| CN111763264A (en) | PSCA (phosphosilicate antigen) -targeted chimeric antigen receptor and application thereof | |
| CN107557338A (en) | Specific recognition NY ESO 1 T cell and its united application with cell factor | |
| CN107630005A (en) | Express T cell and its application of PLAC1 specificity TCRs |
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 |