CN114107378A - 一种通用型car-t细胞的制备方法 - Google Patents
一种通用型car-t细胞的制备方法 Download PDFInfo
- Publication number
- CN114107378A CN114107378A CN202111067059.9A CN202111067059A CN114107378A CN 114107378 A CN114107378 A CN 114107378A CN 202111067059 A CN202111067059 A CN 202111067059A CN 114107378 A CN114107378 A CN 114107378A
- Authority
- CN
- China
- Prior art keywords
- cells
- car
- cell
- ipsc
- hlai
- 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.)
- Withdrawn
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 210000004027 cell Anatomy 0.000 claims abstract description 123
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 33
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 claims abstract description 16
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 claims abstract description 16
- 108091033409 CRISPR Proteins 0.000 claims abstract description 13
- 239000013598 vector Substances 0.000 claims abstract description 7
- 238000003209 gene knockout Methods 0.000 claims abstract description 6
- 101150076800 B2M gene Proteins 0.000 claims abstract description 5
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 5
- 241000713666 Lentivirus Species 0.000 claims description 20
- 239000013612 plasmid Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 15
- 238000004806 packaging method and process Methods 0.000 claims description 10
- -1 EGFRVIIII Proteins 0.000 claims description 9
- 239000012634 fragment Substances 0.000 claims description 6
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 4
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 3
- 101150084532 CD47 gene Proteins 0.000 claims description 2
- 102000003735 Mesothelin Human genes 0.000 claims description 2
- 108090000015 Mesothelin Proteins 0.000 claims description 2
- 241000711408 Murine respirovirus Species 0.000 claims description 2
- 108091023040 Transcription factor Proteins 0.000 claims description 2
- 102000040945 Transcription factor Human genes 0.000 claims description 2
- 210000002950 fibroblast Anatomy 0.000 claims description 2
- 230000000735 allogeneic effect Effects 0.000 abstract description 10
- 230000004069 differentiation Effects 0.000 abstract description 10
- 210000005259 peripheral blood Anatomy 0.000 abstract description 5
- 239000011886 peripheral blood Substances 0.000 abstract description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 4
- 206010057249 Phagocytosis Diseases 0.000 abstract description 4
- 208000021601 lentivirus infection Diseases 0.000 abstract description 4
- 210000000822 natural killer cell Anatomy 0.000 abstract description 4
- 230000008782 phagocytosis Effects 0.000 abstract description 4
- 101001000998 Homo sapiens Protein phosphatase 1 regulatory subunit 12C Proteins 0.000 abstract description 3
- 102100035620 Protein phosphatase 1 regulatory subunit 12C Human genes 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 239000002609 medium Substances 0.000 description 36
- 241000700605 Viruses Species 0.000 description 17
- 238000001890 transfection Methods 0.000 description 17
- 238000003306 harvesting Methods 0.000 description 10
- 239000000203 mixture Substances 0.000 description 8
- 239000004698 Polyethylene Substances 0.000 description 7
- 108091027544 Subgenomic mRNA Proteins 0.000 description 7
- 229920000573 polyethylene Polymers 0.000 description 7
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- 239000011435 rock Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 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 4
- 239000006228 supernatant Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 239000012124 Opti-MEM Substances 0.000 description 3
- 102100035140 Vitronectin Human genes 0.000 description 3
- 108010031318 Vitronectin Proteins 0.000 description 3
- 108010076089 accutase Proteins 0.000 description 3
- 230000002354 daily effect Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000010899 nucleation Methods 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- 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 2
- 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 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 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 description 2
- 230000008672 reprogramming Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 102100020715 Fms-related tyrosine kinase 3 ligand protein Human genes 0.000 description 1
- 101710162577 Fms-related tyrosine kinase 3 ligand protein Proteins 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 239000004695 Polyether sulfone Substances 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000012499 inoculation medium Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229920006393 polyether sulfone Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000011536 re-plating Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
-
- 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
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
-
- 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
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/604—Klf-4
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/606—Transcription factors c-Myc
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18811—Sendai virus
- C12N2760/18841—Use of virus, viral particle or viral elements as a vector
- C12N2760/18843—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)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Hematology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于生物医药技术领域,公开了种通用型CAR‑T细胞的制备方法,CAR‑T的DNA序列及CD47分子的DNA序列插入到iPS细胞的AAVS1位点,B2M基因敲除位点对应的CRISPR/Cas9载体转入iPS细胞,得到的CAR‑iPS细胞分化获得通用型CAR‑T细胞。本发明制备的通用型CAR‑T细胞由CD47+/HLAI‑,CAR‑iPS分化获得,CAR‑T细胞高表达CD47,不表达HLAI。相对于慢病毒感染获得的CAR‑T细胞,CAR‑iPS分化获得的iCAR‑T纯度更高,可以接近于100%。且由于iPS可近乎无限增殖,因此由CAR‑iPS获得的iCAR‑T也可近乎无限获得,不受外周血分离影响。且CD47+/HLAI‑,CAR‑T细胞不但可逃避异体T细胞的杀伤,且不会发生“丢失自我”反应,能够避免异体NK细胞的吞噬,是一种真正意义上的通用型CAR‑T细胞。
Description
技术领域
本发明涉及生物医药技术领域,更具体地,涉及一种通用型CAR-T细胞的制备方法。
背景技术
嵌合抗原受体T细胞(CART)治疗癌症患者已显示出令人振奋的结果。但是,目前大多数CAR-T临床试验使用的是自体T细胞,因此可能会受到T细胞质量和数量不佳(如接受放化疗的患者)以及制造自体CAR-T细胞产品的时间和费用的限制。
异体来源的T细胞目前主要有:外周血来源T细胞,脐带血来源的T细胞,ips细胞诱导分化的T细胞。使用来自异体ips细胞诱导分化的T细胞可以克服这些缺陷。(传代次数多(100代),大量生产,降低成本,冷冻保存以备随时使用)。但是异体IPSC来源的T细胞存在的最大问题是会引起免疫排斥反应。因此如何消除异体IPSC来源的T细胞引起的免疫排斥是亟待解决的问题。
发明内容
本发明所要解决的技术问题是克服现有技术所描述的上述缺陷,首先提供一种通用型CAR-T细胞的制备方法。
本发明的目的通过以下技术方案实现:
一种通用型CAR-T的制备方法,包括以下步骤:
S1、制备IPSC:将携带三种转录因子(KOS,hc-Myc,hKlf4)的仙台病毒感染T细胞,获得T细胞来源的IPSC;
S2、利用三质粒系统或者四质粒系统包装单链可变片段基因序列及CD47基因序列的慢病毒;
S3、利用S2得到的慢病毒感染S1得到的IPSC,所述IPSC的细胞表面表达含有针对CD19的单链可变片段及CD47分子;
S4、构建B2M基因敲除位点对应的CRISPR/Cas9载体并电转入CD47+CAR-iPSC,获得HLAI-CD47+CAR-iPSC;
S5、将HLAI-CD47+CAR-iPSC诱导成T细胞,获得HLAI-CD47+CAR-T细胞。
本发明通过CAR-T的DNA序列及CD47分子的DNA序列插入到iPS细胞的AAVS1位点,并将B2M基因敲除位点对应的CRISPR/Cas9载体转入iPS细胞,最后将得到的CAR-iPS细胞分化成T细胞获得通用型CAR-T细胞。本发明制备的通用型CAR-T细胞由CD47+/HLAI-,CAR-iPS分化获得,CAR-T细胞高表达CD47,不表达HLAI。相对于慢病毒感染获得的CAR-T细胞,CAR-iPSC分化获得的CAR-T细胞纯度更高,可以接近于100%。且由于iPS可近乎无限增殖,因此由CAR-iPSC获得的CAR-T细胞也可近乎无限获得,不受外周血分离影响。且CD47+/HLAI-,CAR-T细胞不但可逃避异体T细胞的杀伤,且不会发生“丢失自我”反应,能够避免异体NK细胞的吞噬,是一种真正意义上的通用型CAR-T细胞。
优选的,所述的IPSC细胞的来源包括但不限于T细胞、PBMC、成纤维细胞、造血干细胞。
优选的,所述单链可变片段选自CD19、EGFRVIII、间皮素、CD133、CEA的scFv。
优选的,所述电转的参数为:电压1000V、时间30ms,电击次数为1-2次。
之前的一些研究表明来自CAR-IPSC分化成的CAR-T细胞、CAR-NK细胞或CAR-巨噬细胞,对特定的肿瘤抗原具有良好的靶向性和杀伤能力。因此,优选地,所述HLAI-CD47+CAR-IPSC分化的免疫细胞包括但不限于CAR-T细胞、CAR-NK细胞或CAR-巨噬细胞。
与现有技术相比,本发明具有以下有益效果:
(1)本发明通过在CAR-IPSC上过表达CD47并将其诱导分化成CAR-T细胞,克服了使用异体CAR-T治疗肿瘤时可能出现的免疫排斥反应及“丢失自我”反应引起的NK细胞吞噬。
(2)相对于慢病毒感染获得的CD47+CAR-T细胞,本发明通过CAR-IPSC分化获得的CAR-T纯度更高,可以接近于100%。
(3)相对于Crispr/Cas9基因敲除系统敲除CAR-T细胞获得的HLAI-CAR-T细胞,本发明通过HLAI-CAR-IPSC分化获得的HLAI-CAR-T纯度更高,可以接近于100%。
(4)本发明制备的HLAI-CD47+CAR-T细胞由HLAI-CD47+CAR-iPS分化获得,iPS细胞可增殖周期长,更便于进行基因修饰。
(5)HLAI-CD47+CAR-IPSC诱导的的HLAI-CD47+CAR-T细胞可近乎无限获得,不受外周血分离影响。
(6)使用HLAI-CD47+CAR-IPSC诱导得到HLAI-CD47+CAR-T细胞,来源于同一株单克隆,DNA遗传信息均一稳定。
(7)相对于现有的Crispr/Cas9基因敲除系统敲除CAR-T细胞获得的通用型HLAI-CAR-T细胞,HLAI-CD47+CAR-IPSC诱导得到HLAI-CD47+CAR-T细胞不但可逃避异体T细胞的杀伤,且可避免“丢失自我”反应,从而逃避NK细胞的吞噬。
附图说明
图1为T细胞诱导的IPSC;
图2为慢病毒感染表达CAR的CAR-IPSC;
图3为敲除HLAI的T细胞。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实验例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1 T细胞来源的IPS制备
采用Ficoll密度梯度离心法分离人外周血PBMC,置于无血清培养基中静置4小时获取上清中的细胞并用磁珠分选上清中的T细胞。
第-4天:将T细胞以5×105cells/mL的浓度铺至24孔板的中部,在完全T细胞培养基中培养。
第-3天至第-1天:用0.5mL新鲜的完整T细胞培养基替换一半的培养基。
第0天:在适当的MOI下使用CytoTuneTM2.0仙台重编程载体感染细胞。孵育细胞过夜。
第1天:用新鲜的完整T细胞培养基替换培养基,以移除CytoTune2.0仙台重新编程载体。
第3天:将转导细胞接种于不含细胞因子的全StemproTM-34培养基中,vitronectin包被的培养皿中。
第4-6天:每隔一天更换一次用过的不含细胞因子的完整stemproTM-34培养基。
第7天:开始过渡到Essential 8TM培养基,将不含细胞因子的一半StemproTM-34培养基换成Essential 8TM培养基。
第8天:将整个培养基更换为Essential 8TM培养基以结束过渡,并继续在vitronectin包被的培养皿上培养细胞。
第9~28天:每天用新鲜的Essential 8TM培养基更换废培养基,并监测培养容器中是否有IPSC集落的出现。当IPSC集落准备好转移时,进行活体染色,然后将未分化的IPSC挑选并转移到新鲜的vitronectin涂层的培养皿中进行扩增。
实施例2过表达CD47的CAR-IPSC细胞的制备
利用三质粒系统包装慢病毒用于后续的实验。三质粒系统分别是含有CD47-CD19-scFv的pCDH-CMV-MCS-EF1-copGFP的质粒、psPAX2质粒和pMD2.G质粒。本发明中是利用293T细胞包装慢病毒。收集293T培养基上清中的病毒,然后通过超高速离心、重悬,以及后续的慢病毒滴度滴定,即可用于靶细胞的感染。感染的靶细胞是293T细胞:将293T细胞铺于6孔板中,待其长至70%-80%的密度。
本实施例中慢病毒的包装具体操作如下:
(1)将293T包装细胞以1.3-1.5X 105细胞/ml(每板6ml)的浓度接种到培养板中的接种培养基(不含青霉素/链霉素的DMEM+10%FBS)中。孵育细胞24小时(37℃,5%CO2),或直到第二天下午。约24小时后,细胞应约70%汇合。
(2)转染包装细胞:准备3种转染质粒的混合物:250ng pMD2.G质粒、750ng psPAX2质粒、1μg重组质粒;10-30μlOptiMEM培养基。
(3)用OptiMEM稀释Lipofectamine 2,000:10μl Lipofectamine+90μl OptiMEM。滴加Lipofectamine试剂,并通过旋转尖端或轻拂试管进行混合(勿通过移液或涡旋混合);在室温下孵育5分钟。
(4)将3种质粒混合物逐滴添加到稀释的Lipofectamine试剂中,并通过旋转尖端或轻弹试管进行混合。
(5)在室温下将转染混合物孵育20-30分钟。
(6)小心地将转染混合物转移到接种培养基中的包装细胞中,包装细胞可能对扰动敏感;注意不要将细胞从培养皿中移出。每块板的转染混合物总体积应为100至125μl。
(7)孵育细胞18小时(37℃,5%CO2),或直到第二天早晨,更换培养基以除去转染试剂,并用6ml收获培养基(DMEMD+30%FBS+1x青霉素/链霉素)进行病毒收获。
(8)孵育细胞24小时(37℃,5%CO2)。
(9)转染后约40小时收获含有慢病毒的培养基。将介质转移到存储管中。更换为6ml Harvest培养基。
(10)每12-24小时重复一次病毒收获,并用6ml收获培养基替换,以后的收获期病毒滴度趋于降低;通常收集总共2-3个时间点;最后一次收获后,丢弃包装细胞;病毒收获物可以根据需要合并。
(11)以1,250rpm的转速将含有病毒的培养基旋转5分钟,以沉淀收获期间收集的所有包装细胞。使上清液通过45μm过滤器,并转移到无菌存储管中。
(12)病毒可以在4℃下短时间(数小时至数天)存储,但应在-20℃或-80℃下冷冻以进行长期存储。为了减少冷冻/解冻循环的次数,请在长期储存之前将大规模病毒制剂分装到较小的储存管中。
通过上述的慢病毒包装,我们获得了含有目的基因的慢病毒。由于不同的细胞器感染复数不同,因此有必要进一步确定慢病毒载体的滴度。由于慢病毒带有荧光标记可以使用显微镜来确定荧光细胞的百分比,进而确定慢病毒滴度。本实施例中慢病毒滴度滴定的具体操作如下:
(1)将75,000个细胞接种到6孔培养皿的每个孔中。
(2)将细胞孵育过夜。
(3)如果使用新鲜收集的病毒,可通过0.45μm聚醚砜过滤器进行过滤,以除去细胞和碎片。
(4)在冻融循环中,慢病毒滴度会降低。如果要冷冻并分装病毒,必须从冷冻原种确定滴度,以解决与冻融相关的滴度损失。
(5)如果使用冷冻病毒,须在温水中搅拌,在37℃下快速融化慢病毒等分试样。
(6)将慢病毒稀释液制备到含有10μg/mL聚乙烯的DMEM中。充分混合稀释液。
(7)从细胞中轻轻吸出培养基。
(8)向每孔中加入1.5mL病毒稀释液(每孔稀释一孔,剩余一孔)。
(9)计数剩余孔中的细胞,计算滴度需要细胞计数。
(10)孵育48-72小时。
(11)轻轻吸出培养基,并用1mLPBS代替。
(12)计算每个孔中荧光阳性细胞的百分比。
计算滴度时,仅考虑荧光阳性细胞少于40%的孔。滴定方法假设每个单元有1个积分事件。当百分比超过40%时,可能会对带有多个整合事件的细胞进行计数,这会导致低估真正的效价。
使用稀释因子(方法1)或病毒体积(方法2)计算每mL的转导单位(TU/mL):
方法1:TU/mL=(转导的细胞数x荧光百分率x稀释因子)/(转导体积(mL))。
方法1的示例:如果1:100的孔中有25%的荧光细胞并且最初转导了150,000个细胞,则存在(150,000x0.25x100)/(1.5mL)=2.5x106TU/mL。
方法2:TU/mL=(转导的细胞数x荧光百分比)/(病毒体积(mL))。
方法2的示例:如果将15μL病毒添加到150,000个细胞中导致产生25%的荧光细胞,则存在(150,000x0.25)/(0.015mL)=2.5x106TU/mL。
通过上述步骤确定好慢病毒的滴度后,即可使用慢病毒感染IPSC细胞。慢病毒感染IPSC的具体步骤如下:
(1)通过将20μL的10mg/mL的聚乙烯稀释到20mL的介质中,制备一批Essential 8TM培养基+10μg/mL的聚乙烯。
(2)在37℃下快速融化慢病毒等分试样。
(3)在Essential 8TM培养基+10μg/mL聚乙烯中准备一系列慢病毒稀释液。
(4)充分混合稀释液。
(5)向每个孔中添加0.5mL单一病毒稀释液。
(6)通过将50,000个细胞接种到6孔培养皿的每个孔中来执行“反向转导”,将这些细胞添加到已经含有0.5mL各种稀释度的病毒溶液的孔中,确保在此步骤中使用含聚乙烯的介质制作细胞溶液。
(7)用移液管或倒置管充分混合。
(8)将1mL细胞悬液(即50,000个细胞)等分到6孔培养皿的每个孔中。这样可使每个孔的总体积达到1.5mL。由于这些孔中的所有培养基均使用DMEM完全溶液+10μg/mL聚乙烯制成,因此每个孔中的聚乙烯终浓度应为10μg/mL。
(9)用病毒孵育细胞48-72小时。
(10)轻轻吸出细胞中的培养基。
(11)加入1.5mL含有适当抗生素的Essential 8TM培养基,这是选择过程的开始,它将开始选择稳定的细胞池。
(12)每天观察培养皿,以确保未转导的孔(上述0μL慢病毒)中的细胞即将死亡。定期进行液体更换,监测细胞的生长。
(13)随着耐药细胞的多克隆种群开始通过并且各个孔汇合,可扩展成更大的容器。6孔培养皿的融合孔可以扩展为10厘米培养皿。可以将融合的10厘米培养皿扩展到两个75厘米的烧瓶中。
(14)一旦多克隆种群生长良好并充分扩展,准备细胞储备和/或收获以测试蛋白质表达。
实施例3敲除HLAI的CD47+CAR-IPSC细胞的制备
1、设计针对B2M基因的SgRNA(5′-CGTGAGTAAACCTGAATCTT-3′)通过酶切酶连将sgRNA寡核苷酸克隆到pSpCas9(BB)中。
2、转化:根据细胞随附的规程,将PlasmidSafe处理的质粒转化进感受态大肠杆菌菌株。我们建议使用Stbl3菌株进行快速转化。简而言之,将2μl步骤1中连接好的质粒添加到20μl遇冷的化学感受态的Stbl3细胞中,将混合物在冰上孵育10分钟,在42℃加热震荡30s,然后立即返回冰上2分钟。加入100μl SOC培养基,并将其铺在含有100μg ml-1氨苄青霉素的LB平板上。将其在37℃下孵育过夜。请注意,当转化氨苄青霉素抗性质粒时,不必在热休克后将感受态细胞孵育一段时间。
3、检查培养皿中菌落的生长情况。通常,在阴性对照板上没有菌落(仅BbsI消化的pSpCas9(BB)的连接而没有退火的sgRNA oligo插入片段),在pSpCas9(sgRNA)克隆板上有数十到数百个菌落(将sgRNA插入pSpCas9(BB))。
4、从每个平板中,选择两个或三个菌落,以检查sgRNA的正确插入。使用无菌移液器吸头将单个菌落接种到含有100μg ml-1氨苄青霉素的LB培养基的3ml培养物中。37℃摇动过夜孵育。
5、提质粒后CRISPR质粒的序列验证:通过使用U6-Fwd引物从U6启动子测序来验证每个菌落的序列。将序列结果与pSpCas9(BB)克隆载体序列进行对照,以检查U6之间是否插入了20nt向导序列启动子和sgRNA支架的其余部分。
6、sgRNA的功能验证:IPSC细胞培养和转染
培养IPS细胞。我们通常使用Essential 8TM培养基将IPS细胞维持在无饲养层的条件下。准备一份10ml的Essential 8TM等分试样,并进一步添加10μM ROCK抑制剂。
涂覆组织培养板。将冷的GelTrex 1:100在冷的DMEM中稀释,并覆盖100mm组织培养板的整个表面。
将板置于培养箱中于37℃放置至少30分钟。
在37℃解冻一小瓶细胞,将细胞转移到15ml Falcon管中,加入5ml Essential8TM,在室温下以200g离心5分钟。
吸出GelTrex包被液,并用10ml含有的ROCK抑制剂的mTeSR1培养基接种1×106细胞。24小时后换成不含ROCK抑制剂的Essential 8TM,每天换液。
传代细胞。在细胞达到70%汇合之前先传代细胞。
吸出Essential 8TM,并用DPBS洗涤细胞一次。
加入2ml Accutase将细胞解离,并在37℃下孵育3–5分钟。
向分离的细胞中加入10ml Essential 8TM,将混合物转移至15ml Falcon管中并轻轻重悬。
将细胞重新铺板在含有10μM ROCK抑制剂的mTeSR1培养基中的GelTrex包被的板上。
接种后24小时用普通的Essential 8TM更换。
转染:转染前2h,用新鲜培养基给对数期生长的细胞(50-70%汇合)换液。
使用Accutase和温和的重悬液解离单个细胞或不超过十个细胞的小簇(在显微镜下观察)。
计算核转染所需的细胞数(每次转染200,000个细胞),并在室温下以200g离心5分钟。
每1×105细胞,吸弃培养基,并将其重悬于10μl转染缓冲液R中。
根据推荐的程序,将添加了DNA的重悬细胞移液到电穿孔比色杯中并进行电穿孔。对于1×105细胞,我们通常使用1μg总DNA。
轻轻将电穿孔的细胞铺板到涂有10μM ROCK抑制剂的24孔板涂层板上。
检查转染是否成功,并在核转染后24小时开始每天用常规的Essential 8TM换液。嘌呤霉素的选择浓度可以为0.5μg ml-1(可能因细胞系而异)。通常,我们观察到Amaxa核转染的转染效率>70%。
转染后48-72小时,将细胞用Accutase分离,然后将其轻轻重悬于5倍体积的mTeSR1中。在此阶段保留一部分重悬的细胞用于重铺,用于下游应用或克隆分离,并将剩余的细胞用于基因分析。
实施例4 HLAI-CD47+CAR-IPSC向T细胞的分化
包括以下步骤:
1.在T细胞分化开始的前一天,用FC-DLL4包被平皿。
2.除去涂层溶液,并分配适当体积的含有细胞因子(50ng/ml SCF,100ng/ml TPO,50ng/ml Flt3L,50ng/ml IL-7)的分化培养基。
3.用流式细胞仪分选表达CD34的造血细胞。
4.将细胞放入具有适当细胞密度的预备孔中(48孔板中每孔2000个细胞)。
5.在37℃、5%CO2湿度培养箱中培养细胞。
6.每2天更换一次培养基,每周将分化细胞转移到新制备的FC-DL4包被板内。
7.培养3周后,取CD4、CD8均表达的细胞,刺激其成熟为成熟T细胞。
8.流式验证T细胞的HLAI表达。
以上对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
Claims (4)
1.一种通用型CAR-T的制备方法,包括以下步骤:
S1、制备IPSC:将携带三种转录因子KOS,hc-Myc和hKlf4的仙台病毒感染T细胞,获得T细胞来源的IPSC;
S2、利用三质粒系统或者四质粒系统包装单链可变片段基因序列及CD47基因序列的慢病毒;
S3、利用S2得到的慢病毒感染S1得到的IPSC,所述IPSC的细胞表面表达含有针对CD19的单链可变片段及CD47分子;
S4、构建B2M基因敲除位点对应的CRISPR/Cas9载体并电转入CD47+CAR-iPSC,获得HLAI-CD47+CAR-iPSC;
S5、将HLAI-CD47+CAR-iPSC诱导成T细胞,获得HLAI-CD47+CAR-T细胞。
2.根据权利要求1所述的制备方法,其特征在于,所述的IPSC细胞的来源包括但不限于T细胞、PBMC、成纤维细胞、造血干细胞。
3.根据权利要求1所述的制备方法,其特征在于,所述单链可变片段选自CD19、EGFRVIII、间皮素、CD133、CEA的scFv。
4.根据权利要求1所述的制备方法,其特征在于,所述电转的参数为:电压1000V、时间30ms,电击次数为1-2次。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111067059.9A CN114107378A (zh) | 2021-09-13 | 2021-09-13 | 一种通用型car-t细胞的制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111067059.9A CN114107378A (zh) | 2021-09-13 | 2021-09-13 | 一种通用型car-t细胞的制备方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114107378A true CN114107378A (zh) | 2022-03-01 |
Family
ID=80441300
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111067059.9A Withdrawn CN114107378A (zh) | 2021-09-13 | 2021-09-13 | 一种通用型car-t细胞的制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114107378A (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120135525A1 (en) * | 2009-06-05 | 2012-05-31 | Matthew Brown | Reprogramming t cells and hematopoietic cells |
CN108642083A (zh) * | 2018-04-28 | 2018-10-12 | 安徽中盛溯源生物科技有限公司 | 一种将t细胞高效诱导成多能干细胞的重编程方法 |
CN109234317A (zh) * | 2018-10-22 | 2019-01-18 | 北京呈诺医学科技有限公司 | 一种iCAR-T细胞的制备方法 |
US20200297763A1 (en) * | 2018-10-18 | 2020-09-24 | Zhejiang University | Pluripotent stem cell-derived macrophage capable of targeting tumor cells and preparation method thereof |
US20210015859A1 (en) * | 2017-12-08 | 2021-01-21 | Fate Therapeutics, Inc. | IMMUNOTHERAPIES USING ENHANCED iPSC DERIVED EFFECTOR CELLS |
-
2021
- 2021-09-13 CN CN202111067059.9A patent/CN114107378A/zh not_active Withdrawn
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120135525A1 (en) * | 2009-06-05 | 2012-05-31 | Matthew Brown | Reprogramming t cells and hematopoietic cells |
US20210015859A1 (en) * | 2017-12-08 | 2021-01-21 | Fate Therapeutics, Inc. | IMMUNOTHERAPIES USING ENHANCED iPSC DERIVED EFFECTOR CELLS |
CN108642083A (zh) * | 2018-04-28 | 2018-10-12 | 安徽中盛溯源生物科技有限公司 | 一种将t细胞高效诱导成多能干细胞的重编程方法 |
US20200297763A1 (en) * | 2018-10-18 | 2020-09-24 | Zhejiang University | Pluripotent stem cell-derived macrophage capable of targeting tumor cells and preparation method thereof |
CN109234317A (zh) * | 2018-10-22 | 2019-01-18 | 北京呈诺医学科技有限公司 | 一种iCAR-T细胞的制备方法 |
Non-Patent Citations (3)
Title |
---|
CHAD C MACARTHUR等: "Generation and comprehensive characterization of induced pluripotent stem cells for translational research", 《METHODOLOGY》, vol. 14, no. 6, pages 505 - 524 * |
SPECIFIC HU等: "Abstract LB144: Overexpression of CD47protects hypoimmune CAR T cells frominnate immune cell killing", 《CANCER RESEARCH》, vol. 81, no. 13, pages 1 - 5 * |
张明英等: "不同转染方法包装慢病毒感染人白血病细胞的比较研究", 《生物技术进展》, no. 3, pages 52 - 60 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7193886B2 (ja) | キメラ抗原受容体で修飾されたγδ T細胞を生産する方法 | |
CN106636090B (zh) | 人源白细胞介素6的siRNA、重组表达CAR-T载体及其构建方法和应用 | |
US20210040448A1 (en) | Closed-system manufacturing process for car-t cells | |
CN111518773A (zh) | 一种抗新型冠状病毒s蛋白的car-t细胞、制备方法及其应用 | |
CN106978442B (zh) | 一种嵌合抗原受体t细胞的制备方法 | |
CN114921416B (zh) | 一种nk细胞及其制备方法 | |
CN113881707B (zh) | 调控脐带间充质干细胞免疫抑制作用的产品、方法及用途 | |
US20060013803A1 (en) | Modified dendritic cells | |
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 | |
CN107286246B (zh) | 治疗脑胶质瘤的嵌合抗原受体修饰的树突状细胞及其制备方法 | |
CN114107378A (zh) | 一种通用型car-t细胞的制备方法 | |
CN105200059B (zh) | 靶向抑制小鼠UCP2基因表达的siRNA及其表达载体的构建 | |
Li et al. | Preclinical development and clinical-scale manufacturing of HIV Gag-specific, lentivirusmodified CD4 T cells for HIV functional cure | |
CN109266683B (zh) | 一种包含e4bp4基因的慢病毒重组载体及其制备方法和应用 | |
CN113025660A (zh) | 一种慢病毒体外转染人造血干细胞的试剂盒及方法 | |
JP5778147B2 (ja) | 遺伝子導入方法 | |
CN110951695A (zh) | 通用型car-t细胞、其制备方法及其应用 | |
US20190010467A1 (en) | Method for preparing cultured cells or tissues for transplantation | |
CN110484507B (zh) | 一种靶向肿瘤Her2的新型嵌合抗原受体T细胞的制备技术 | |
CN108103027B (zh) | 高效率血细胞重编程同时实现基因编辑的方法 | |
CN110904032A (zh) | 提高慢病毒转染人多能干细胞的方法 | |
WO2012086702A1 (ja) | 遺伝子導入方法 | |
WO2023140349A1 (ja) | 細胞シート | |
CN113584085B (zh) | 一种针对悬浮细胞的慢病毒载体及其应用 | |
CN116656616B (zh) | 一种制备外泌体的方法及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WW01 | Invention patent application withdrawn after publication |
Application publication date: 20220301 |
|
WW01 | Invention patent application withdrawn after publication |