CN109680004A - 联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在卵巢癌中的应用 - Google Patents
联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在卵巢癌中的应用 Download PDFInfo
- Publication number
- CN109680004A CN109680004A CN201910019807.2A CN201910019807A CN109680004A CN 109680004 A CN109680004 A CN 109680004A CN 201910019807 A CN201910019807 A CN 201910019807A CN 109680004 A CN109680004 A CN 109680004A
- Authority
- CN
- China
- Prior art keywords
- epcam
- chain antibody
- cell
- msln
- target spot
- 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
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 title claims abstract description 67
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 title claims abstract description 67
- 101000576802 Homo sapiens Mesothelin Proteins 0.000 title claims abstract description 64
- 102100025096 Mesothelin Human genes 0.000 title claims abstract description 64
- 239000000969 carrier Substances 0.000 title claims abstract description 30
- 238000010276 construction Methods 0.000 title claims abstract description 10
- 210000004027 cell Anatomy 0.000 claims abstract description 121
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims abstract description 56
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 23
- 239000000427 antigen Substances 0.000 claims abstract description 20
- 108091007433 antigens Proteins 0.000 claims abstract description 20
- 102000036639 antigens Human genes 0.000 claims abstract description 20
- 206010033128 Ovarian cancer Diseases 0.000 claims abstract description 13
- 206010061535 Ovarian neoplasm Diseases 0.000 claims abstract description 13
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 239000013612 plasmid Substances 0.000 claims description 54
- 241000700605 Viruses Species 0.000 claims description 41
- 239000002773 nucleotide Substances 0.000 claims description 24
- 125000003729 nucleotide group Chemical group 0.000 claims description 24
- 230000029087 digestion Effects 0.000 claims description 16
- 238000004806 packaging method and process Methods 0.000 claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 13
- 239000012634 fragment Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 6
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 230000008685 targeting Effects 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 230000004940 costimulation Effects 0.000 claims description 3
- 230000003834 intracellular effect Effects 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 3
- 230000019491 signal transduction Effects 0.000 claims description 3
- 241000894007 species Species 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 25
- 230000009385 viral infection Effects 0.000 abstract description 7
- 238000002560 therapeutic procedure Methods 0.000 abstract description 5
- 239000013598 vector Substances 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 description 32
- 239000000243 solution Substances 0.000 description 15
- 239000012636 effector Substances 0.000 description 14
- 238000001514 detection method Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 210000004881 tumor cell Anatomy 0.000 description 10
- 239000002609 medium Substances 0.000 description 8
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 8
- 101000699762 Homo sapiens RNA 3'-terminal phosphate cyclase Proteins 0.000 description 7
- 102100029143 RNA 3'-terminal phosphate cyclase Human genes 0.000 description 7
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 7
- 239000011248 coating agent Substances 0.000 description 7
- 238000000576 coating method Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 239000002699 waste material Substances 0.000 description 7
- 238000007792 addition Methods 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 102000000588 Interleukin-2 Human genes 0.000 description 5
- 108010002350 Interleukin-2 Proteins 0.000 description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 5
- 229960000723 ampicillin Drugs 0.000 description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 239000013613 expression plasmid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 229920001917 Ficoll Polymers 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000012097 Lipofectamine 2000 Substances 0.000 description 3
- 230000022534 cell killing Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- -1 1640 culture mediums Substances 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000011357 CAR T-cell therapy Methods 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 102000003735 Mesothelin Human genes 0.000 description 2
- 108090000015 Mesothelin Proteins 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004820 blood count Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 210000003162 effector t lymphocyte Anatomy 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000011888 foil Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002045 lasting effect Effects 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 230000000873 masking effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000003595 mist Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 229910001868 water Inorganic materials 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 108010083359 Antigen Receptors Proteins 0.000 description 1
- 102000006306 Antigen Receptors Human genes 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- 206010041047 Slow virus infection Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 108091093126 WHP Posttrascriptional Response Element Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000004405 cytokine-induced killer cell Anatomy 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 201000003373 familial cold autoinflammatory syndrome 3 Diseases 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000004882 non-tumor cell Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000004999 sex organ Anatomy 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 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
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000008280 toxic mechanism Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- C07K16/30—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
- C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
-
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
- 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
- C07K2319/70—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)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Epidemiology (AREA)
- Developmental Biology & Embryology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gynecology & Obstetrics (AREA)
- Pregnancy & Childbirth (AREA)
- Reproductive Health (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于医药生物领域,具体涉及联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在卵巢癌中的应用。所述的双靶点CAR载体通过联合EpCAM单链抗体和MSLN单链抗体,CAR的具体组成结构为CD8leader‑EpCAM scFv‑(G4S)5‑MSLN scFv‑CD8α‑CD28‑CD137‑CD3ζ。以病毒感染方式获得含有该治疗载体的CAR‑T细胞(Chimeric Antigen Receptor T‑Cell),通过表达CAR结构,可靶向识别和杀伤高表达EpCAM和MSLN两种肿瘤相关抗原的卵巢癌细胞。
Description
技术领域
本发明属于医药生物领域,具体涉及联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在卵巢癌中的应用。
背景技术
卵巢癌是女性生殖器官常见的恶性肿瘤之一,其死亡率占女性恶性肿瘤的第一位,是女性癌症病人死亡的首要死因。目前卵巢癌的治疗方法有手术治疗、化疗、中医治疗、免疫治疗等。近年来,随着基础科学研究的不断发展,免疫治疗越来越受到人们的重视。
LAK细胞、DC、CIK细胞都曾作为肿瘤的免疫治疗手段,但是以上各种治疗方法均缺乏特异性识别肿瘤相关抗原(tumor associated antigen,TAA)和杀伤特定肿瘤靶细胞的能力;而肿瘤免疫编辑的过程又会使靶细胞表面的主要组织相容性复合物(mainhistocompatibility complex,MHC)在肿瘤细胞表面表达下降,形成免疫逃逸,使肿瘤细胞可成功躲避T细胞攻击。
嵌合抗原受体(CAR)-T细胞疗法是通过基因工程技术在T细胞表面人为过表达能识别特定的肿瘤表面抗原的单链抗体可变区基因片段,从而使T细胞识别特定抗原、杀伤表达该抗原的靶细胞。同时,由于CAR-T细胞是采用抗体识别抗原模式,因此不受MHC的限制。
上皮特异性粘附分子(EpCAM,Epithelial cell adhesion molecule)是一种上皮细胞跨膜糖蛋白,参与Wnt信号转导通路,调控靶基因转录,与细胞的粘附、迁移、增殖、分化等有关。最初是以一种肿瘤相关抗原而被发现的,由于EpCAM在多种肿瘤组织如卵巢癌、胃癌、结直肠腺癌、乳腺癌等中的表达升高而备受关注。研究发现,EpCAM的表达与卵巢肿瘤的进展及预后相关,可作为卵巢癌靶向治疗的靶点。
间皮素(mesothelin,MSLN)是一种存在于细胞表面的锚定糖蛋白,因其在正常组织中不表达或间皮组织中低表达,但在30%的肿瘤中高表达,如卵巢癌、胃癌、结肠癌等实体瘤,而成为备受关注的肿瘤细胞特异性靶向抗原。
脱靶效应是CAR-T细胞疗法副作用的主要来源,脱靶效应是指未能达到预先设定的目标,有所偏移的现象。CAR-T细胞胞外识别结构域绝大多数为肿瘤相关抗原(Tumorassociated antigen,TAA),而肿瘤相关抗原并非肿瘤细胞所特有,其在健康组织上也存在不同程度的表达。这种CAR-T细胞在杀伤肿瘤细胞的同时也会攻击正常组织,这种毒性即非肿瘤靶向毒性,该毒性机制造成的毒性效应称为脱靶效应。
因此,如何有效的避免降低脱靶效应的发生,增强T淋巴细胞对肿瘤细胞的杀伤,从而提高CAR-T免疫疗法抗卵巢癌的效果,成为CAR-T治疗的一个技术难题。
目前,尚未见有关能靶向EpCAM和MSLN的双靶点的治疗方案,以降低脱靶效应发生的CAR载体的报道。
发明内容
本发明目的是为了解决上述问题,本发明提供了能靶向EpCAM和MSLN并减少脱靶效应发生的CAR载体。本发明所采用的针对EpCAM和MSLN的CAR-T技术,降低CAR-T细胞疗法的脱靶效应的发生,增强T淋巴细胞对肿瘤细胞的杀伤,从而提高CAR-T免疫疗法抗卵巢癌的效果。此外,还包括联合EpCAM和MSLN单链抗体的双靶点CAR载体的构建方法及其应用。
本发明提供的联合EpCAM和MSLN单链抗体的双靶点CAR载体具体技术方案如下:
联合EpCAM和MSLN单链抗体的双靶点CAR载体,包括嵌合抗原受体和载体,所述嵌合抗原受体连接在所述载体上,所述嵌合抗原受体包括两种特异性结构分别为:EpCAM单链抗体和MSLN单链抗体,所述EpCAM单链抗体的核苷酸序列如SEQ ID NO.2所示,所述MSLN单链抗体的核苷酸序列如SEQ ID NO.4所示,
所述嵌合抗原受体的结构组成为CD8leader-EpCAM scFv-(G4S)5-MSLN scFv-CD8α-CD28-CD137-CD3ζ。
在某些实施方式中,其特征在于,所述CD8leader可表达引导新合成的蛋白质进行跨膜转移的引导肽,所述CD8leader的核苷酸序列SEQ ID NO.1所示,所述EpCAM scFv表示EpCAM单链抗体,所述(G4S)5为单链抗体间铰链Inner-Linker,且核苷酸序列如SEQ ID NO.3所示,所述EpCAM单链抗体和MSLN单链抗体通过所述(G4S)5进行连接,所述MSLN scFv表示MSLN单链抗体,所述CD8α为跨膜区,连接胞外抗原结合域和胞内信号域,且核苷酸序列如SEQ ID NO.5所示,所述CD28-CD137为共刺激结构域,所述CD28核苷酸序列如SEQ ID NO.6所示,所述CD137核苷酸序列如SEQ ID NO.7所示,所述CD3ζ为信号转导域,核苷酸序列如SEQ ID NO.8所示。
在某些实施方式中,所述载体包括PUC19质粒、慢病毒载体。
在某些实施方式中,所述慢病毒载体为pCDH-CMV-MCS-EF1-Puro。
本发明还提供了联合EpCAM和MSLN单链抗体的双靶点CAR载体的构建方法,包括步骤如下:
(1)将所述嵌合抗原受体保存在所述PUC19质粒上;
(2)将含有步骤(1)中PUC19质粒和所述慢病毒载体进行双酶切,将酶切产物分别经过琼脂糖凝胶电泳分离,获得双酶切后的含有所述嵌合抗原受体结构的目的片段和双酶切后慢病毒载体,
(3)将步骤(2)中的含有所述嵌合抗原受体结构的目的片段和步骤(2)中的慢病毒载体进行连接,将连接产物进行转化,提取质粒,获得含有嵌合抗原受体结构的重组质粒,所述重组质粒为联合EpCAM和MSLN单链抗体的双靶点CAR载体。
在某些实施方式中,所述步骤(2)中的双酶切的位点为EcoRI和NotI,所述(3)中含有所述嵌合抗原受体结构的目的片段和慢病毒载体连接的摩尔比为5:1。
本发明还提供了联合EpCAM和MSLN单链抗体的双靶点CAR载体的应用,联合EpCAM和MSLN单链抗体的双靶点CAR载体进行慢病毒包装,经离心浓缩后获得高滴度的慢病毒悬液,所述慢病毒悬液感染用于T细胞,获得CAR-T细胞,所述CAR-T细胞通过识别特异性蛋白EpCAM和MSLN靶向杀伤卵巢癌细胞。
在某些实施方式中,其特征在于,所述慢病毒包装采用三质粒包装系统,所述三质粒包装系统包括PSPAX2质粒、pMD2G质粒和所述联合EpCAM和MSLN单链抗体的双靶点CAR载体,所述PSPAX2质粒、所述pMD2G质粒、所述联合EpCAM和MSLN单链抗体的双靶点CAR载体的比例为27:3:20,所述慢病毒包装采用293T细胞作为慢病毒的包装细胞。
本发明具有以下有益效果:本发明针对治疗载体的CAR结构包括EpCAM单链抗体、MSLN单链抗体两个特异性结构;EpCAM单链抗体、MSLN单链抗体是根据多种卵巢癌细胞表面会表达肿瘤相关抗原EpCAM、MSLN确定的特异性结构,该结构表达的Anti-EpCAM、Anti-MSLN两种特异性抗体负责识别TAA,一方面可靶向性杀伤肿瘤细胞;另一方面,赋予T细胞新的抗原特异性,可有效避免肿瘤细胞MHC表达下调这一免疫逃逸机制。另外,双靶点的设计可以有效减少CAR-T治疗过程中产生的脱靶效应,使CAR-T细胞可以更好的识别双阳性靶点的卵巢癌细胞,提高杀伤效率的同时,减少CAR-T治疗中带来的风险。当T细胞通过病毒感染方式获得该载体并表达该CAR结构后,T细胞即可靶向性识别并杀伤卵巢癌细胞。
附图说明
图1是本发明的慢病毒表达载体pCDH-CMV-MCS-EF1-Puro结构示意图;
图2是本发明的CAR结构示意图;
图3是本发明的慢病毒表达载体pCDH-CMV-MCS-EF1-Puro EcoRI与NotI双酶切产物琼脂糖凝胶电泳图;
图4是本发明的联合EpCAM和MSLN单链抗体的双靶点CAR载体EcoRI与NotI双酶切产物琼脂糖凝胶电泳图;
图5是本发明的CAR-T细胞的流式细胞检测图;
图6是本发明的RTCA检测CAR-T细胞对SK-OV-3的杀伤效率曲线图;
图7是本发明的CAR-T细胞对SK-OV-3杀伤24h后IFNgamma因子的ELISA检测柱状图。
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明白,以下结合具体实施例,并参照附图,对本发明进一步详细说明。
本发明提供了联合EpCAM和MSLN单链抗体的双靶点CAR载体,具体方案如下:包括嵌合抗原受体和载体,嵌合抗原受体连接在载体上,所述嵌合抗原受体包括两种特异性结构分别为:EpCAM单链抗体和MSLN单链抗体,EpCAM单链抗体的核苷酸序列如SEQ ID NO.2所示,MSLN单链抗体的核苷酸序列如SEQ ID NO.4所示。嵌合抗原受体的结构组成为CD8leader-EpCAM scFv-(G4S)5-MSLN scFv-CD8α-CD28-CD137-CD3ζ。
嵌合抗原受体的结构组成为CD8leader-EpCAM scFv-(G4S)5-MSLN scFv-CD8α-CD28-CD137-CD3ζ(如图2所示)。CD8leader可表达引导新合成的蛋白质进行跨膜转移的引导肽,CD8leader的核苷酸序列SEQ ID NO.1所示,EpCAM scFv表示EpCAM单链抗体,(G4S)5为单链抗体间铰链Inner-Linker,且核苷酸序列如SEQ ID NO.3所示,EpCAM单链抗体和MSLN单链抗体通过(G4S)5进行连接,MSLN scFv表示MSLN单链抗体,CD8α为跨膜区,连接胞外抗原结合域和胞内信号域,且核苷酸序列如SEQ ID NO.5所示,CD28-CD137为共刺激结构域,CD28核苷酸序列如SEQ ID NO.6所示,CD137核苷酸序列如SEQ ID NO.7所示,CD3ζ为信号转导域,核苷酸序列如SEQ ID NO.8所示。表达针对卵巢癌细胞表面的肿瘤相关抗原EpCAM、MSLN的抗体Anti-EpCAM、Anti-MSLN,使得其能够更精准的识别并杀伤同时表达上述两种肿瘤相关抗原的卵巢癌细胞,大大增加了CAR-T细胞的靶向性,降低脱靶效应。本发明中提到的CAR结构赋予了T细胞更强的增殖性,持久的生命力,使其表现出更强的肿瘤细胞杀伤能力。
载体包括PUC19质粒、慢病毒载体。慢病毒载体为pCDH-CMV-MCS-EF1-Puro。通过Snap Gene软件分析该载体并查找相关文献可知,pCDH-CMV-MCS-EF1-Puro用EcoRI与NotI双酶切插入片段。该表达载体包括:CMV启动子-是哺乳动物细胞特异性启动子,驱动能力较强;多克隆位点(MCS)-包含多个限制性酶切位点(restriction site),是外源基因插入的位置;WPRE元件-可提高mRNA的polyA加尾效率,改进转移基因的表达效率;SV40polyA序列-能有效终止转录以及为转录的mRNA添加PolyA尾;杂交型RSV/5’LTR-含有启动子和增强子等调控元件,使其在293T细胞中高水平的表达全长病毒转录物;遗传要素(cPPT,gag,env,LTRs)-用于包装、转导和稳定地将病毒表达结构整合到宿主的基因组DNA中;SV40 origin-使质粒在包装细胞中稳定增殖。
该慢病毒表达载体可作为使目的基因在几乎所有哺乳动物细胞包括非分裂细胞和分裂细胞中表达的最有效载体,其可容载外源基因片段大,转染效率较高,对T细胞也能达到满意的转染效果。
一、实验材料
1.慢病毒表达质粒pCDH-CMV-MCS-EF1-Puro,慢病毒包装质粒pMD2G,载体质粒PSPAX2购自SBI;慢病毒表达质粒pCDH-CMV-MCS-EF1-Puro结构如图1所示;
2.CAR结构序列由上海美丽人生医疗科技有限公司设计,由生工生物工程(上海)股份有限公司合成,以PUC19质粒形式保存;
3.限制性核酸内切酶EcoRI、NotI购自NEB;
4.T4 DNA ligase、Free H2O购自宝生物;
5.感受态细胞购自Trans;
6.胶回收试剂盒,质粒小提试剂盒购自天根生化科技有限公司;
7.293T细胞、SK-OV-3细胞购自中科院细胞库;
8.FBS,DMEM、1640培养基,PBS,Opti-MEM,lipofectamine 2000购自Gibco;
8.FBS、DMEM、1640培养基,PBS,Opti-MEM,lipofectamine 2000购自Gibco;
9.CD3单抗、CD28单抗、CH38蛋白、IL-2购自于上海近岸蛋白质科技有限公司;
10.Multiskan GO酶标仪+uDrop超微量板、流式细胞仪购自ThermoFisher;
11.HE120水平电泳槽、Tanon凝胶成像仪购自Tanon;
12.隔水式恒温培养箱、恒温培养摇床购自上海一恒科技有限公司;
13.Bio-Rad Mini-PROTEAN Tetra Cell小型电泳系统购自Bio-Rad;
14.奥林巴斯显微镜购自奥林巴斯;
15.接种环、涂布棒购自洁特生物过滤股份有限公司;
16.注射器,0.45μm滤膜、各规格的培养皿,培养瓶,多孔培养板,各种规格离心管购自Corning。
二、联合EpCAM和MSLN单链抗体的双靶点CAR载体的构建方法
(一)质粒提取
LB液体培养基的配制方法:电子天平称取5g液体培养基干粉于500mL锥形瓶,加100mL超纯水,锡箔纸封口后,于高压蒸汽灭菌锅中进行灭菌,冷却至40℃-50℃时,以1000:1加入0.2%氨苄青霉素(AMP),小心混匀后,转入到干净的500mL试剂瓶中备用,储存条件为4℃。
LB固体培养基的配制方法:电子天平称取5g固体培养基干粉于500mL锥形瓶,加100mL超纯水,锡箔纸封口后,于高压蒸汽灭菌锅中进行灭菌,冷却至40℃-50℃时,以1000:1加入0.2%氨苄青霉素,小心混匀后,倒板,凝固后,贴封口膜储存于4℃。
从-80℃冰箱分别取出含有慢病毒表达质粒pCDH-CMV-MCS-EF1-Puro和PUC19质粒的甘油菌,分别取5μL接种于5mL LB液体培养基(AMP抗性)中,于恒温摇床振荡培养12-16h,条件为37℃,250rmp。
按照购自天根生化科技有限公司的质粒小提试剂盒(货号:DP103-03)的说明书进行质粒提取,获得慢病毒表达质粒pCDH-CMV-MCS-EF1-Puro和含有双靶点(EpCAM单链抗体和MSLN单链抗体)CAR结构的PUC19质粒。
将已提取的慢病毒表达质粒pCDH-CMV-MCS-EF1-Puro、和含有双靶点(E EpCAM单链抗体和MSLN单链抗体)CAR结构的PUC19质粒同时进行EcoRI、NotI双酶切,将酶切产物分别进行琼脂糖凝胶电泳,用凝胶成像仪观察并记录结果,检测结果见附图3。
用天根琼脂糖凝胶回收试剂盒(货号:DP209-02)说明书进行目的条带回收,将回收片段按CAR结构:pCDH-CMV-MCS-EF1-Puro=5:1的摩尔比进行连接,并进行转化至感受态细胞,将转化后的感受态细胞滴于预热的固体培养基上,做好标记,在3 7℃孵箱过夜。
(三)提质粒、酶切验证、测序
挑取部分菌落于5mL LB液体培养基(AMP抗性)中,于恒温摇床进行增菌培养并进行质粒抽提,质粒抽提之前取出500μL于1.5mL Ep管中,用作菌种保存,储存于-80℃;将抽提产物采用EcoRI、NotI双酶切验证,取条带正确的质粒1μg送生工生物工程(上海)有限公司进行测序(如图4所示,图中目的片段为含有CAR结构的PUC19质料),条带异常的质粒以及留存的菌液均丢弃。将测序结果正确的质粒进行抽提,测序结果错误的质粒及其菌液均丢弃。
三、联合EpCAM和MSLN单链抗体的双靶点CAR载体的应用
(一)浓缩病毒液的制备
采用三质粒包装系统进行慢病毒包装。三质粒分别为含有CAR结构的慢病毒表达质粒pCDH-CMV-MCS-EF1-Puro,慢病毒包装质粒pMD2G,载体质粒PSPAX2。细胞为293T细胞。
具体实施步骤如下:
(1)转染前24h内进行铺板:一般选择传代次数在3代以内的细胞,根据细胞生长密度和状态调整细胞密度,生长密度达到80%的293T细胞进行铺板;
(2)待生长密度达60-90%,细胞状态良好,即可进行病毒包装;
(3)按照PSPAX2质粒、pMD2G质粒、所述联合EpCAM和MSLN单链抗体的双靶点CAR载体(重组质粒)的比例为27:3:20进行病毒包装,以6cm皿为例,所需的三质粒混合液配比如下:按照各质粒浓度确定质粒加入量。
重组质粒 3μg
pMD2G 0.5μg
PSPAX2 4μg
(4)转染试剂选择lipofectamine 2000(4℃保存),加入量为2μL/μg质粒;
(5)将(3)中质粒混合物与(4)中转染试剂混合物混于一管中,室温静止20min后,加入换液细胞中,继续培养;
(6)分别收集48h、72h后培养上清,通过0.45μm滤膜过滤;
(7)将收集的病毒液采用PEG8000浓缩法进行浓缩,并测定病毒滴度,储存于﹣80℃备用。
(二)病毒液感染T细胞
1、PBMC分离
1)用含有肝素的真空采血管收集约6mL人外周新鲜血液;
2)稀释:室温下加入等体积的PBS,轻轻吹打混匀;
3)加样:取50mL离心管,吸取6mL Ficoll(淋巴细胞分离液)于离心管中(Ficoll与稀释前血液的体积比为1:1),离心管倾斜45°,将稀释后的血液在Ficoll液面上方约1cm处沿管壁缓慢加至Ficoll上面;
4)离心:18-20℃,2000rpm,30min,离心后从管底至液面分四层,依次为红细胞和粒细胞层、分层液层、单个核细胞层、血浆层;
5)回收:将移液管直接插入云雾层(或者先吸去上层的血浆),轻轻吸出云雾层,放入新的离心管中;
6)洗涤:加入至少于PBMC(外周血单个核细胞)体积3倍的PBS,18-20℃,2000rpm,10min,两次;
7)细胞计数:弃上清,加1mL淋巴细胞培养基,吹打混匀,制备成PBMC细胞悬液。采用血细胞计数板计数:取一滴PBMC悬液与一滴2%台盼蓝染液混匀后加于血细胞计数板中,在显微镜下计数4大格内细胞总数。细胞数/mL=4个大方格细胞总数/4×104×2(稀释倍数)。
2、T细胞的激活与慢病毒感染
(1)实验前准备:
1)Anti-CD3单抗液配制:CD3单抗50μg溶于5mLPBS溶液中,配制成浓度为10μg/mL的溶液,溶解后按照每EP管400μL进行分装,并置于-80℃冰箱保存。(此浓度下每24孔板内加入175μL抗体溶液);
2)Anti-CD28单抗液配制:CD28单抗50μg溶于5mLPBS溶液中,配制成浓度为10μg/mL的溶液,溶解后按照每EP管400μL进行分装,并置于-80℃冰箱保存。(此浓度下每24孔板内加入175μL抗体溶液);
3)CH-38蛋白液配制:CH38蛋白500μg溶于10mL PBS溶液中,配制成50μg/mL溶液,溶解后按照每EP管400μL进行分装,并置于-80℃冰箱保存。(此浓度下每24孔板内加入175μL抗体溶液);
4)IL-2因子配制:IL-2蛋白固体按1×107U/mg进行配制,每50μg IL-2蛋白加入500μL的PBS溶液,配制成103U/μL的浓度,配制后按照每EP中加入32μL进行分装,并置于-80℃冰箱保存;
5)淋巴细胞培养基配制:Takara-551h3淋巴细胞培养基每50mL加入30μL已分装的IL-2溶液,0.5mL双抗,250μL自体血清。
(2)实验流程:
Day﹣1:24孔板包被:取Corning 24孔板,以2孔为例,在2孔中各加入CD3单抗液175μL,CD28单抗液175μL,CH-38蛋白液175μL。加入后轻震荡混匀后,用封口膜将孔板封闭,放入4℃冰箱内过夜。细胞复苏:取液氮中的PBMC细胞,复苏;
Day0:清洗包被板:取出昨日包被的24孔板,弃去上清,PBS清洗2次后,加入PBS待用;
PBMC铺板:收集PBMC细胞,计数并将浓度最终调整到0.7×106cells/mL,每孔中加入400μL细胞悬液,即每孔中加入2.8×105个cells;
病毒感染:以MOI=30进行感染,配制1mL病毒培养基悬液,加入24孔板中,1000g离心30min,将离心机温度调节至32℃;
Day1-Day2:观察细胞状态;
Day3:将24孔板中的所有细胞转移至加入10mL培养基的25cm2培养瓶中,观察细胞状态;
Day4-Day7:观察细胞状态以及细胞数量,若细胞开始明显扩增,并局部区域细胞密度较多,加入10mL培养基;
Day8:此时25cm2培养瓶中的细胞已经长满,转移到加入20mL培养基的75cm2培养瓶中继续培养;
Day9-Day10:观察细胞状态,当细胞在75cm2培养瓶中处于充满状态时,停止继续生长,富集细胞并计算扩增比例,流式检测检测细胞分型,进行细胞杀伤检测或细胞冻存等后续实验。
四、CAR-T细胞的鉴定与检测
(一)流式细胞术检测CAR结构阳性表达率
1、检测细胞CD3、CD4、CD8阳性率
1)将取得的NC组细胞(未进行病毒感染)和样本组细胞(已进行病毒感染)使用PBS+2%BSA温和洗涤2次,离心条件为1500rpm、3min;
2)NC管中加入1000μLPBS+2%BSA,分装成5管,分别为NC、NC-CD3、NC-CD4、NC-CD8、NC-CD3/4/8,样本管中加入200μLPBS,标记为样本-CD3/4/8,分别加入CD3/4/8抗体5μL/20μL/20μL,温和混匀;
3)室温避光孵育30min后,1500rpm,3min,弃去废液;
4)加入200μLPBS+2%BSA,温和混匀重悬,1500rpm/3min,弃去废液;
5)加入100μLPBS+2%BSA,温和混匀重悬后即可上机检测;
如图5所示,CD3+细胞占所检测细胞总量的98.18%,CD4-CD8+细胞占CD3+细胞总量的60.40%,CD4+CD8-细胞占CD3+细胞总量的32.56%,符合T细胞的表型。
2、检测CAR结构阳性表达率
1)将取得的NC组细胞(未进行病毒感染)和样本组细胞(已进行病毒感染),使用PBS+2%BSA温和洗涤2次,1500rpm/6min,弃去废液;
2)NC管中加入200μLPBS,重悬;样本管中加入100μLPBS,重悬,再加入100μL一抗工作液(3μg/mL),混匀;
3)室温孵育1h,1500rpm/3min,弃去废液;
4)加入200μLPBS,温和混匀重悬,1500rpm/3min,弃去废液;
5)NC管中加入200μLPBS,重悬;样本管中加入200μLPBS,重悬,再加入5μL二抗工作液,混匀;
6)室温避光孵育1h后,1500rpm/3min,弃去废液,使用PBS+2%BSA温和洗涤3次,离心条件为1500rpm、3min,弃去废液;
7)加入100μLPBS,温和混匀重悬后上机检测;
如图5所示,CAR-T细胞占T细胞总量的49.58%
(二)RTCA实时细胞杀伤检测
1)以卵巢癌细胞SK-OV-3为例,消化后制备成细胞悬液,吹打混匀后进行细胞计数;
2)将细胞悬液稀释成5×104cells/mL浓度,放置在冰上备用;
3)将RTCA检测板取出,加入50μL培养基;
4)在RTCA检测仪程序中编选本次试验的试验程序;
5)将RTCA检测板置入检测仪中,观测程序中Messege项是否正常,待正常后启动实验程序;
6)程序1运行完毕后取出检测板,在对应孔中加入肿瘤细胞悬液100μL,加入之前混匀每管细胞悬液;
7)细胞悬液加入后,将检测板置入培养箱中静置30min,使细胞自然沉降;
8)30min后,将检测板放入检测仪中,运行程序2;
9)24h后观察细胞生长曲线,待细胞处于对数生长期时,准备加入效应T细胞;
10)培养瓶中取出效应T细胞,离心,清洗,计数,按不同效靶比配制效应组细胞浓度;
11)暂停程序,取出检测板,对应位置加入效应细胞50μL,放回检测仪中,继续程序,每天观察。
图6为RTCA检测双靶点(EpCAM单链抗体和MSLN单链抗体)CAR-T细胞对SK-OV-3细胞的杀伤效率,图中5:1表示:效应细胞:靶细胞=5:1;2.5:1表示:效应细胞:靶细胞=2.5:1;1.25:1表示:效应细胞:靶细胞=1.25:1;NCT5:1表示:效应细胞为未感染的T细胞,效应细胞:靶细胞=5:1;NCT2.5:1表示:效应细胞为未感染的T细胞,效应细胞:靶细胞=2.5:1;NCT1.25:1表示:效应细胞为未感染的T细胞,效应细胞:靶细胞=1.25:1。如图所示,CAR-T细胞的杀伤效果明显强于未感染的T细胞,且其杀伤效果具有浓度依赖性,效应细胞比例越高,杀伤效果越好。
(三)ELISA检测细胞因子分泌
1)用ddH2O稀释10×包被液buffer,按比例配制250×包被蛋白,例如2mL包被液buffer中加入8μL 250×包被蛋白;
2)向Corning 9018 Elisa高亲和96孔板中加入100μL/孔1)中配制完成的包被液,密封放入4℃冰箱中,过夜;
3)使用PBST(0.05%Tween 20)对已包被的96孔板进行清洗,3次;
4)用ddH2O配制5×封闭液,200μL/孔加入,室温下封闭1h;
5)按瓶装标准品要求加入1×封闭液进行配制,进行7次倍比稀释,同时对样品(取RTCA检测实验中的CAR-T细胞对SK-OV-3杀伤24h后的上清液)进行5倍稀释;
6)PBST清洗封闭后的板子5次,加入标准品和稀释后的样品液,室温孵育2h或4℃过夜;
7)PBST清洗4次;
8)使用1×封闭液稀释250×检测抗体,100μL/孔中加入,室温孵育1h;
9)PBST清洗4次,使用1×封闭液稀释250×HRP,100μL/孔中加入,室温孵育30min;
10)PBST清洗5次,每孔中加入1×TMB试剂100μL,室温孵育15min;
11)加入50μL/孔终止液终止显色;
12)酶标仪450nm检测OD值。
图7为CAR-T细胞对SK-OV-3杀伤24h后IFNgamma因子的ELISA检测,图中5:1表示:效应细胞:靶细胞=5:1;2.5:1表示:效应细胞:靶细胞=2.5:1;1.25:1表示:效应细胞:靶细胞=1.25:1。CAR-T细胞与SK-OV-3细胞共培养24h后,有明显的细胞因子IFNgamma释放,效应细胞:靶细胞为5:1时,细胞因子IFNgamma释放最多。
综上所述,本发明提供的联合EpCAM和MSLN单链抗体的双靶点CAR载体应用于感染T细胞,获得CAR-T细胞可同时表达针对卵巢癌细胞表面的肿瘤相关抗原EpCAM、MSLN的抗体Anti-EpCAM、Anti-MSLN,使得其能够更精准的识别并杀伤同时表达上述两种肿瘤相关抗原的卵巢癌细胞,大大增加了CAR-T细胞的靶向性,降低脱靶效应;另外,本发明中提到的CAR结构赋予了T细胞更强的增殖性,持久的生命力,使其表现出更强的肿瘤细胞杀伤能力。
上述仅本发明较佳可行实施例,并非是对本发明的限制,本发明也并不限于上述举例,本技术领域的技术人员,在本发明的实质范围内,所作出的变化、改型、添加或替换,也应属于本发明的保护范围。
Claims (8)
1.联合EpCAM和MSLN单链抗体的双靶点CAR载体,其特征在于,包括嵌合抗原受体和载体,所述嵌合抗原受体连接在所述载体上,
所述嵌合抗原受体包括EpCAM单链抗体和MSLN单链抗体两种特异性结构,所述EpCAM单链抗体的核苷酸序列如SEQ ID NO.2所示,所述MSLN单链抗体的核苷酸序列如SEQ ID NO.4所示,
所述嵌合抗原受体的结构组成为CD8leader-EpCAM scFv-(G4S)5-MSLNscFv-CD8α-CD28-CD137-CD3ζ。
2.根据权利要求1所述的联合EpCAM和MSLN单链抗体的双靶点CAR载体,其特征在于,所述CD8leader可表达引导新合成的蛋白质进行跨膜转移的引导肽,所述CD8leader的核苷酸序列SEQ ID NO.1所示,
所述EpCAM scFv表示EpCAM单链抗体,
所述(G4S)5为单链抗体间铰链Inner-Linker,且核苷酸序列如SEQ ID NO.3所示,所述EpCAM单链抗体和MSLN单链抗体通过所述(G4S)5进行连接,
所述MSLN scFv表示MSLN单链抗体,
所述CD8α为跨膜区,连接胞外抗原结合域和胞内信号域,且核苷酸序列如SEQ ID NO.5所示,
所述CD28-CD137为共刺激结构域,所述CD28核苷酸序列如SEQ ID NO.6所示,所述CD137核苷酸序列如SEQ ID NO.7所示,
所述CD3ζ为为信号转导域,核苷酸序列如SEQ ID NO.8所示。
3.根据权利要求1所述的联合EpCAM和MSLN单链抗体的双靶点CAR载体,其特征在于,所述载体包括PUC19质粒、慢病毒载体。
4.根据权利要求4所述的联合EpCAM和MSLN单链抗体的双靶点CAR载体,其特征在于,所述慢病毒载体为pCDH-CMV-MCS-EF1-Puro。
5.联合EpCAM和MSLN单链抗体的双靶点CAR载体的构建方法,用于构建如权利要求1-4任一项所述的联合EpCAM和MSLN单链抗体的双靶点CAR载体,其特征在于,所述构建方法的步骤如下:
(1)将所述嵌合抗原受体保存在所述PUC19质粒上;
(2)将含有步骤(1)中PUC19质粒和所述慢病毒载体进行双酶切,将酶切产物分别经过琼脂糖凝胶电泳分离,获得双酶切后的含有所述嵌合抗原受体结构的目的片段和双酶切后慢病毒载体,
(3)将步骤(2)中的目的片段和步骤(2)中的慢病毒载体进行连接,将连接产物进行转化,提取质粒,获得含有嵌合抗原受体结构的重组质粒,所述重组质粒为联合EpCAM和MSLN单链抗体的双靶点CAR载体。
6.根据权利要求5所述的联合EpCAM和MSLN单链抗体的双靶点CAR载体的构建方法,其特征在于,所述步骤(2)中的双酶切位点为EcoRI和NotI,所述(3)的含有所述嵌合抗原受体结构的目的片段和慢病毒载体连接的摩尔比为5:1。
7.联合EpCAM和MSLN单链抗体的双靶点CAR载体的应用,基于权利要求1-4任一项所述的联合EpCAM和MSLN单链抗体的双靶点CAR载体,其特征在于,联合EpCAM和MSLN单链抗体的双靶点CAR载体进行慢病毒包装,经离心浓缩后获得高滴度的慢病毒悬液,所述慢病毒悬液用于感染T细胞,获得CAR-T细胞,所述CAR-T细胞通过识别特异性蛋白EpCAM和MSLN靶向杀伤卵巢癌细胞。
8.根据权利要求7所述的联合EpCAM和MSLN单链抗体的双靶点CAR载体的应用,其特征在于,所述慢病毒包装采用三质粒包装系统,所述三质粒包装系统包括PSPAX2质粒、pMD2G质粒和所述联合EpCAM和MSLN单链抗体的双靶点CAR载体,所述PSPAX2质粒、所述pMD2G质粒、所述联合EpCAM和MSLN单链抗体的双靶点CAR载体的比例为27:3:20,所述慢病毒包装采用293T细胞作为慢病毒的包装细胞。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910019807.2A CN109680004A (zh) | 2019-01-09 | 2019-01-09 | 联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在卵巢癌中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910019807.2A CN109680004A (zh) | 2019-01-09 | 2019-01-09 | 联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在卵巢癌中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN109680004A true CN109680004A (zh) | 2019-04-26 |
Family
ID=66192734
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910019807.2A Pending CN109680004A (zh) | 2019-01-09 | 2019-01-09 | 联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在卵巢癌中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109680004A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115491358A (zh) * | 2021-06-17 | 2022-12-20 | 复星凯特生物科技有限公司 | 一种靶向b7-h3和folr1双打靶点car t的制备及应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2996060A1 (en) * | 2015-08-21 | 2017-03-02 | Carsgen Therapeutics, Ltd | Fully human anti-mesothelin antibodies and immune effector cells targeting mesothelin |
CN107325185A (zh) * | 2017-06-06 | 2017-11-07 | 上海优卡迪生物医药科技有限公司 | 基于octs‑car的抗psca及pdl1双靶向嵌合抗原受体、编码基因及表达载体 |
WO2017219936A1 (zh) * | 2016-06-20 | 2017-12-28 | 上海细胞治疗研究院 | 一种高效稳定表达激活型抗体的car-t细胞及其用途 |
CN108864289A (zh) * | 2018-04-26 | 2018-11-23 | 上海怡豪生物科技有限公司 | 胃癌的双靶点car-t治疗载体及其构建方法和应用 |
CN108864288A (zh) * | 2018-04-26 | 2018-11-23 | 上海怡豪生物科技有限公司 | 乳腺癌的双靶点car-t治疗载体及其构建方法和应用 |
CN108913721A (zh) * | 2018-07-23 | 2018-11-30 | 安徽古生物科技有限公司 | 表达cd40抗体的慢病毒载体、car-t细胞的构建方法及应用 |
-
2019
- 2019-01-09 CN CN201910019807.2A patent/CN109680004A/zh active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2996060A1 (en) * | 2015-08-21 | 2017-03-02 | Carsgen Therapeutics, Ltd | Fully human anti-mesothelin antibodies and immune effector cells targeting mesothelin |
WO2017219936A1 (zh) * | 2016-06-20 | 2017-12-28 | 上海细胞治疗研究院 | 一种高效稳定表达激活型抗体的car-t细胞及其用途 |
CN107523549A (zh) * | 2016-06-20 | 2017-12-29 | 上海细胞治疗研究院 | 一种高效稳定表达激活型抗体的car‑t细胞及其用途 |
CN107325185A (zh) * | 2017-06-06 | 2017-11-07 | 上海优卡迪生物医药科技有限公司 | 基于octs‑car的抗psca及pdl1双靶向嵌合抗原受体、编码基因及表达载体 |
CN108864289A (zh) * | 2018-04-26 | 2018-11-23 | 上海怡豪生物科技有限公司 | 胃癌的双靶点car-t治疗载体及其构建方法和应用 |
CN108864288A (zh) * | 2018-04-26 | 2018-11-23 | 上海怡豪生物科技有限公司 | 乳腺癌的双靶点car-t治疗载体及其构建方法和应用 |
CN108913721A (zh) * | 2018-07-23 | 2018-11-30 | 安徽古生物科技有限公司 | 表达cd40抗体的慢病毒载体、car-t细胞的构建方法及应用 |
Non-Patent Citations (1)
Title |
---|
CARLTON L SCHWAB ET AL.: "Past, present and future targets for immunotherapy in ovarian cancer", 《IMMUNOTHERAPY》, vol. 6, no. 12, pages 1280 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115491358A (zh) * | 2021-06-17 | 2022-12-20 | 复星凯特生物科技有限公司 | 一种靶向b7-h3和folr1双打靶点car t的制备及应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109593786A (zh) | 联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在乳腺癌应用 | |
CN105602992B (zh) | 一种基于复制缺陷性重组慢病毒的car-t转基因载体及其构建方法和应用 | |
CN104910279B (zh) | 靶向癌胚抗原的嵌合抗原受体、慢病毒表达载体及其制备方法和应用 | |
CN109485732A (zh) | 基因工程修饰的双靶点嵌合抗原受体及其用途 | |
CN105296431B (zh) | 肿瘤结合特异性γδTCR基因修饰的αβT细胞及其抑癌用途 | |
US11932872B2 (en) | Dual chimeric antigen receptor-t cell which can be regulated, construction method therefor and use thereof | |
CN105950664B (zh) | 一种靶向cd123的复制缺陷性重组慢病毒car-t转基因载体及其构建方法和应用 | |
CN105949316B (zh) | 抗EGFRvIII嵌合抗原受体、编码基因、重组表达载体及其构建方法和应用 | |
CN106317228A (zh) | 一种嵌合抗原受体分子及其应用 | |
CN108641000A (zh) | 肝癌的双靶点car-t治疗载体及其构建方法和应用 | |
CN106279432B (zh) | 一种vc-car分子及在清除hiv-1感染细胞中的应用 | |
CN106047817A (zh) | Car‑t细胞及其制备方法与应用 | |
CN109628492A (zh) | 联合MSLN单链抗体和PD-1mAb杀伤胃癌细胞的CAR载体及其构建方法和应用 | |
CN109680003A (zh) | 靶向卵巢癌细胞特异性高表达蛋白fshr的car载体及其构建方法和应用 | |
CN109680002A (zh) | 联合msln单链抗体和ccl19杀伤胃癌细胞的car载体及其构建方法和应用 | |
CN114957484A (zh) | 靶向实体肿瘤细胞b7-h3蛋白的car载体、car-t细胞及其构建方法和应用 | |
CN105384826A (zh) | 表达嵌合抗原受体的脐血有核细胞及其应用 | |
CN105950662B (zh) | 一种靶向cd22的复制缺陷性重组慢病毒car-t转基因载体及其构建方法和应用 | |
CN113604507A (zh) | 靶向胃癌细胞特异性高表达蛋白msln的car载体及其构建方法和应用 | |
CN108659133A (zh) | 肺癌的双靶点car-t治疗载体及其构建方法和应用 | |
CN108864289A (zh) | 胃癌的双靶点car-t治疗载体及其构建方法和应用 | |
CN109762843A (zh) | 一种利用脐血来源的cd3阳性t细胞制备通用car-t细胞的方法 | |
CN111606996B (zh) | 一种靶向4d5的鼠源单克隆抗体及其制备方法和应用 | |
CN113652452A (zh) | 靶向卵巢癌细胞特异性高表达蛋白b7-h3的car载体及其构建方法和应用 | |
CN109680004A (zh) | 联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在卵巢癌中的应用 |
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 |