WO2018176844A1 - Car-cik转基因细胞及其制备方法和应用 - Google Patents
Car-cik转基因细胞及其制备方法和应用 Download PDFInfo
- Publication number
- WO2018176844A1 WO2018176844A1 PCT/CN2017/110659 CN2017110659W WO2018176844A1 WO 2018176844 A1 WO2018176844 A1 WO 2018176844A1 CN 2017110659 W CN2017110659 W CN 2017110659W WO 2018176844 A1 WO2018176844 A1 WO 2018176844A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cell
- cells
- seq
- car
- cik
- Prior art date
Links
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 204
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims abstract description 24
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims abstract description 24
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 15
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 claims abstract description 11
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims abstract description 11
- 101150058049 car gene Proteins 0.000 claims abstract description 11
- 238000010363 gene targeting Methods 0.000 claims abstract description 3
- 210000004405 cytokine-induced killer cell Anatomy 0.000 claims description 29
- 239000000203 mixture Substances 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 28
- 238000010361 transduction Methods 0.000 claims description 27
- 230000026683 transduction Effects 0.000 claims description 27
- 239000006228 supernatant Substances 0.000 claims description 24
- 206010028980 Neoplasm Diseases 0.000 claims description 23
- 239000013598 vector Substances 0.000 claims description 19
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 15
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 14
- 239000008188 pellet Substances 0.000 claims description 14
- 239000006285 cell suspension Substances 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 11
- 108700010039 chimeric receptor Proteins 0.000 claims description 11
- 238000005119 centrifugation Methods 0.000 claims description 10
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 10
- 238000000338 in vitro Methods 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 9
- 239000002504 physiological saline solution Substances 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- 230000008685 targeting Effects 0.000 claims description 8
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 7
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 7
- 239000012895 dilution Substances 0.000 claims description 7
- 210000000601 blood cell Anatomy 0.000 claims description 6
- 238000004113 cell culture Methods 0.000 claims description 6
- 238000004806 packaging method and process Methods 0.000 claims description 6
- 210000005259 peripheral blood Anatomy 0.000 claims description 6
- 239000011886 peripheral blood Substances 0.000 claims description 6
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 5
- 210000004698 lymphocyte Anatomy 0.000 claims description 5
- 108010056030 retronectin Proteins 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 4
- 230000037396 body weight Effects 0.000 claims description 4
- 230000003833 cell viability Effects 0.000 claims description 4
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 4
- 238000001802 infusion Methods 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 238000013518 transcription Methods 0.000 claims description 4
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 3
- 230000006044 T cell activation Effects 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 230000000139 costimulatory effect Effects 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 claims description 3
- 230000035897 transcription Effects 0.000 claims description 3
- 230000003612 virological effect Effects 0.000 claims description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 2
- 101001052462 Homo sapiens Homeobox protein MIXL1 Proteins 0.000 claims description 2
- 108700019146 Transgenes Proteins 0.000 claims description 2
- 241001492404 Woodchuck hepatitis virus Species 0.000 claims 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 claims description 2
- 229960000723 ampicillin Drugs 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 230000002708 enhancing effect Effects 0.000 claims description 2
- 239000005090 green fluorescent protein Substances 0.000 claims description 2
- 102000044386 human MIXL1 Human genes 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 230000000977 initiatory effect Effects 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 230000010076 replication Effects 0.000 claims description 2
- 210000003705 ribosome Anatomy 0.000 claims description 2
- 229940126585 therapeutic drug Drugs 0.000 claims description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 claims 1
- 230000001124 posttranscriptional effect Effects 0.000 claims 1
- 210000004881 tumor cell Anatomy 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract 1
- 229940079593 drug Drugs 0.000 abstract 1
- 230000002147 killing effect Effects 0.000 description 16
- 239000002158 endotoxin Substances 0.000 description 12
- 238000009169 immunotherapy Methods 0.000 description 12
- 108090000695 Cytokines Proteins 0.000 description 9
- 102000004127 Cytokines Human genes 0.000 description 9
- 210000001744 T-lymphocyte Anatomy 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 229920001917 Ficoll Polymers 0.000 description 8
- 238000002659 cell therapy Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 239000000872 buffer Substances 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 239000012636 effector Substances 0.000 description 7
- 239000000427 antigen Substances 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 241000204031 Mycoplasma Species 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000013394 immunophenotyping Methods 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000011324 bead Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000012467 final product Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 102100037850 Interferon gamma Human genes 0.000 description 3
- 108010074328 Interferon-gamma Proteins 0.000 description 3
- 108010004729 Phycoerythrin Proteins 0.000 description 3
- 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 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000004005 microsphere Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 2
- 238000011357 CAR T-cell therapy Methods 0.000 description 2
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 2
- 102000001301 EGF receptor Human genes 0.000 description 2
- 108060006698 EGF receptor Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000020411 cell activation Effects 0.000 description 2
- 230000022534 cell killing Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- OOIBFPKQHULHSQ-UHFFFAOYSA-N (3-hydroxy-1-adamantyl) 2-methylprop-2-enoate Chemical compound C1C(C2)CC3CC2(O)CC1(OC(=O)C(=C)C)C3 OOIBFPKQHULHSQ-UHFFFAOYSA-N 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 101001002634 Homo sapiens Interleukin-1 alpha Proteins 0.000 description 1
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 108091006004 biotinylated proteins Proteins 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000008004 cell lysis buffer Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 210000003162 effector t lymphocyte Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000002625 monoclonal antibody therapy Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000009258 tissue cross reactivity Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4613—Natural-killer cells [NK or NK-T]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4631—Chimeric Antigen Receptors [CAR]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
- A61K39/464411—Immunoglobulin superfamily
- A61K39/464412—CD19 or B4
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- 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
- 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
- C12N15/867—Retroviral 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
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/24—Interferons [IFN]
-
- 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
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
-
- 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
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/515—CD3, T-cell receptor complex
-
- 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
Definitions
- the invention belongs to the field of medical biology, and particularly relates to a cell, in particular to a CAR-CIK transgenic cell for tumor immunotherapy. Furthermore, the invention relates to methods and applications for the construction of such cell-associated vectors.
- the hCAR19 lentiviral transgenic vector targeting the CD19 antigen comprises:
- a chimeric antigen receptor for a three generation CAR that constitutes a collection recognition, delivery, initiation, comprises: a CD8 leader chimeric receptor signal as set forth in SEQ ID NO. a peptide, a CD19 single-chain antibody light chain VL as shown in SEQ ID NO. 17, an Optimal Linker C as shown in SEQ ID NO. 19, a CD19 single-chain antibody heavy chain VH as shown in SEQ ID NO. 16, such as CD8 Hinge chimeric receptor hinge set forth in SEQ ID NO.
- the CAR-CIK technology used in the present invention is a novel targeted therapy technology which combines the advantages of immunotherapy of tumor monoclonal antibodies and adoptive immunotherapy of tumors.
- CAR-CIK cells have Beyond the killing ability of CAR-T cells, the future can be used in cell therapy.
- FIG. 2 is a flow chart showing the steps of treatment and reinfusion of CIK cells according to the background of the present invention, including cell collection, cell culture, cell expansion, cell recovery, and the like;
- Figure 9 is a schematic diagram showing the comparison of disease-free survival of 1, 3, and 5 years in the control group, the 3 course of treatment CIK group, and the 6 course of treatment CIK group in Example 4 of the present invention.
- SEQ ID NO. 17 light chain VL of CD19 single-chain antibody
- SEQ ID NO. 16 CD19 single-chain antibody heavy chain VH
- the refrigerated centrifuge was purchased from Thermo Scientific, USA;
- the construction method of the CAR-CIK cells of the present invention is as follows:
- RetroNectin 1 ⁇ 10 3 ug/L to 1 ⁇ 10 4 ug/L RetroNectin was coated in a 24-well plate one day in advance, and the sealing film was sealed and coated overnight at 4°C.
- CIK cells and T cells were collected 72 hours after virus transduction, and the cells were resuspended in a D-PBS(-) solution containing 1 to 4% human albumin and adjusted to 1 ⁇ 10 6 /ml.
- step 14 The corrected values obtained in step 14 are taken to the following formula to calculate the percentage of cytotoxicity produced by each of the target ratios. The results are shown in Figure 7. The killing efficiency of CAR-CIK cells was significantly higher than that of CAR-T cells under different target-to-target ratios.
- CIK cell therapy has a certain effect on the immunotherapy of tumors.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
提供了一种CAR-CIK转基因细胞,其包含靶向CD19抗原的嵌合抗原受体CAR基因、CD56细胞以及CD3细胞,CAR基因、CD56细胞、CD3细胞均表达阳性。还提供了该CAR-CIK转基因细胞的制备方法,及其在制备针对肿瘤的细胞治疗药物中的应用。
Description
本发明属于医学生物领域,具体涉及一种细胞,尤其涉及一种针对肿瘤免疫治疗的的CAR-CIK转基因细胞。此外,本发明还涉及该细胞相关的载体的构建方法和应用。
肿瘤免疫治疗的理论基础是免疫系统具有识别肿瘤相关抗原、调控机体攻击肿瘤细胞(高度特异性的细胞溶解)的能力。1950年代,Burnet和Thomas提出了“免疫监视”理论,认为机体中经常会出现的突变的肿瘤细胞可被免疫系统所识别而清除,为肿瘤免疫治疗奠定了理论基础[Burnet FM.Immunological aspects of malignant disease.Lancet,1967;1:1171-4]。随后,各种肿瘤免疫疗法包括细胞因子疗法、单克隆抗体疗法、过继免疫疗法、疫苗疗法等相继应用于临床。
2013年一种更先进的肿瘤免疫疗法---CAR-T疗法成功用于临床,并表现了前所未有的临床疗效。CAR-T,全称是Chimeric Antigen Receptor T-Cell Immunotherapy,嵌合抗原受体T细胞免疫疗法。该疗法是通过转基因的手段,将启动子、抗原识别区、共刺激因子、效应区等共同组成的嵌合分子,导入T细胞基因组内,从而使T细胞对靶细胞的识别、信号转导、杀伤等功能融为一体,实现了对靶细胞的特异性杀伤[Eleanor J.Cheadle,et al.CAR T cells:driving the road from the laboratory to the clinic.Immμnological Reviews2014.Vol.257:91–106]。CAR-T疗法在临床上最领先的有诺华的CLT019,采用CLT019治疗复发难治急性淋巴细胞白血病患者,六个月的肿瘤无进展生存率达到67%,其中最长的应答时间达到两年多。总部位于中国上海的上海优卡迪生物医药科技有限公司与医院合作,截止到2017年2月,共治疗复发难治急性淋巴细胞白血病患者36例,其中完全24例,缓解比例达到66.6%。这是抗癌研究的颠覆性突破。CAR-T细胞疗法可能是最有可能治愈癌症的手段之一,并被《Science》杂志评为2013年度十大科技突破之首。CAR-T目前在治疗血液肿瘤方面疗效显著,但是在骨髓瘤、淋巴瘤或者实体瘤方面,目前治疗效果不是很好,其中的原因有肿瘤微环境、免疫逃脱、细胞增殖等原因,T细胞杀伤能力不足也是一个重要原因。
CIK(cytokine-induced killer)细胞,最初在指正常人体外周血中只占1~5%的CD3+CD56+的T淋巴细胞,目前用于CIK过继性免疫治疗(如图2所示,CIK细胞治疗和回输的步骤,包含细胞采集、细胞培养、细胞扩增、细胞回输等阶段)[YANFENG LIU,et al.Dendritic cell-activated cytokine-induced killer cell-mediated immunotherapy is safe and effective for cancer patients>65years old.ONCOLOGY LETTERS.12:5205-5210,2016]的CIK细胞实际上是体外扩增出的以CD3+CD56+、CD3+CD8+为主的异质性细胞群。该细胞群在IFN-γ、重组人IL1α、CD3单克隆抗体、重组人IL-2刺激下产生的,CIK细胞是多样化的效应T细胞集群,这群细胞中CD3+CD56+细胞占了很大比例,CIK细胞具有很强的细胞杀伤活性。但是,缺乏抗原识别特异性限制了它的应用(REN X,MA W,LU H,et al.Modification of cytokine-induced killer cells with chimeric antigen receptors(CARs)enhances antitumor immunity to epidermal growth factor receptor(EGFR)-positive malignancies[J].Cancer Immunol Immunother,2015,64(12):1517-29.)。
发明内容
本发明要解决的技术问题之一是提供一种CAR-CIK转基因细胞,该CAR-CIK转基因细胞有着超越CAR-T细胞的杀伤能力,未来可以在细胞治疗方面大显身手。
本发明要解决的技术问题之二是提供该CAR-CIK转基因细胞的制备方法,该能够明显增强CIK细胞的增殖能力,显著提高CD3+CD56+细胞在最终产品中的比例,能高效地将CAR基因转导进入CIK细胞,对比CAR-T细胞的转染效率,有效缩短了CAR-CIK细胞的培养周期,节约了生产成本,降低了患者病情进展的风险。
本发明要解决的技术问题之三是提供该CAR-CIK转基因细胞的应用。
嵌合抗原受体(CAR)是CAR-CIK的核心部件(如图1所示),赋予CIK细胞HLA非依赖的方式识别肿瘤抗原的能力,这使得经过CAR改造的CIK相较于天然CIK细胞表面受体TCR能够识别更广泛的目标。CAR的基础设计中包括一个肿瘤相关抗原(tμmor-associated antigen,TAA)结合区(通常来源于单克隆抗体抗原结合区域的scFv段),一个胞外铰链区,一个跨膜区和一个胞内信号转导区。scFv段的设计对于CAR的特异性、有效性以及基因改造T细胞自身的安全性来是关键的决定因素。
为解决上述技术问题,本发明采用如下技术方案:
在本发明的第一方面,提供一种CAR-CIK转基因细胞,其包含靶向CD19抗原的嵌合抗原受体CAR基因、CD56细胞以及CD3细胞,所述CAR基因表达阳性,所述CD56细胞表达阳性,所述CD3细胞表达阳性。
在本发明的第二方面,提供一种所述的CAR-CIK转基因细胞的制备方法,包括如下步骤:
步骤1,分离外周血单个核细胞PBMC;
步骤2,激活CIK细胞;
步骤3,加入靶向CD19抗原的hCAR19慢病毒转基因载体转导及CAR-CIK细胞诱导培养;
步骤4,CAR-CIK细胞体外扩增,得到CAR-CIK转基因细胞。
作为本发明优选的技术方案,步骤1中,所述分离外周血单个核细胞PBMC的具体步骤包括:抽取健康供者新鲜外周血,加入淋巴细胞分离液及血细胞稀释液,离心,吸取离心后的白膜层细胞,加入人血白蛋白重悬细胞沉淀。
作为本发明优选的技术方案,步骤2中,所述激活CIK细胞的具体步骤包括:
(1)使用50-500ug/L CD3单克隆抗体加入24孔板,封口膜封口,4℃过夜包被;
(2)将分离得到的外周血单个核细胞PBMC按1×109/L密度,置于37℃、5%CO2培养箱中培养,RPMI 1640培养基中含有1×106~1×107U/L的rIFN-γ;
(3)24h后,将细胞加入包被CD3单克隆抗体的24孔板中,置于37℃、5%CO2培养箱中培养。
作为本发明优选的技术方案,步骤3中,所述加入靶向CD19抗原的hCAR19慢病毒转基因载体转导及CAR-CIK细胞培养的具体步骤包括:
(1)提前一天包被1×103ug/L~1×104ug/L RetroNectin于24孔板内,封口膜封口,4℃过夜包被;
(2)往24孔板中,根据每孔5×105细胞量,按MOI=5~20的量,加入hCAR19慢病毒转基因载体,同时添加3×105~3×106U/L rIL-2,500-2000ug/L rIL-1a,37℃、5%CO2继续培养。
作为本发明优选的技术方案,步骤3中,所述靶向CD19抗原的hCAR19慢病毒转基因载体包括:
用于质粒复制的原核复制子pUC Ori序列,如SEQ ID NO.2所示;用于目的菌株大量扩增的含氨苄青霉素抗性基因AmpR序列,如SEQ ID NO.1所示;用于增强真核细胞内的复制的病毒复制子SV40Ori序列,如SEQ ID NO.3所示;用于真核细胞表达绿色荧光的ZsGreen1绿色荧光蛋白,如SEQ ID NO.11所示;用于共同转录表达蛋白质的IRES核糖体结合序列,如SEQ ID NO.12所示;用于嵌合抗原受体基因的真核转录的人EF1α启动子,如SEQ ID NO.14所示;用于增强转基因的表达效率的eWPRE增强型土拨鼠乙肝病毒转录后
调控元件,如SEQ ID NO.13所示;用于组成集识别、传递、启动于一体的三代CAR的嵌合抗原受体包括:如SEQ ID NO.15所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.17所示的CD19单链抗体轻链VL、如SEQ ID NO.19所示的Optimal Linker C、如SEQ ID NO.16所示的CD19单链抗体重链VH、如SEQ ID NO.21所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.22所示的CD8 Transmembrane嵌合受体跨膜区、CD28、如SEQ ID NO.23所示的CD137嵌合受体共刺激因子、如SEQ ID NO.24所示的TCR嵌合受体T细胞激活域、以及如SEQ ID NO.20所示的CD28嵌合受体共刺激因子;用于慢病毒包装的慢病毒包装顺式元件,采用第三代慢病毒载体包括:如SEQ ID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件所述慢病毒包装顺式元件,以及如SEQ ID NO.4所示的RSV启动子;
所述靶向CD19抗原的hCAR19慢病毒转基因载体的核苷酸序列如SEQ ID NO.18所示。
作为本发明优选的技术方案,步骤4中,所述CAR-CIK细胞体外扩增的具体为:每2天等量补加含1×105~1×106U/L rIL-2RPMI 1640培养基,使PH值维持在6.5~7.5之间,细胞密度维持在5×105~2×106/ml之间,37℃、5%CO2继续培养14-21天。
作为本发明优选的技术方案,在步骤4之后可以增加如下步骤:
(1)用含1×105~1×106U/L rIL-2RPMI 1640培养基调整细胞密度至2×106~4×106/mL之间;
(2)按照细胞的转导效率、回输剂量和患者体重,用下列公式计算需要回输的细胞总量:
total cell=(回输剂量X患者体重)/转导效率;
(3)离心,弃上清,加生理盐水吹打混匀;重复该步骤;并过筛网,计数并计算细胞活率;
(4)按照计数结果,取适量细胞悬液到离心管内,补加生理盐水,离心后弃上清,细胞沉淀用生理盐水重悬,控制细胞密度在1×107~4×107/mL之间,吹打混匀之后,加入10%人血白蛋白,混匀;
(5)将细胞悬液注入回输袋中,封口,贴好标签后,-80℃保存。
在本发明的第三方面,提供上述CAR-CIK转基因细胞在制备肿瘤的细胞治疗药物中的应用。
与现有技术相比,本发明具有如下有益效果:
本发明所采用的CAR-CIK技术,是一种综合了肿瘤单克隆抗体的免疫治疗和肿瘤的过继免疫治疗优点的靶向治疗新技术,通过一系列的临床前实验表明,CAR-CIK细胞有着超越CAR-T细胞的杀伤能力,未来可以在细胞治疗方面大显身手。
本发明所采用的CIK细胞培养技术,能够明显增强CIK细胞的增殖能力,显著提高CD3+CD56+细胞在最终产品中的比例。
本发明所采用的CAR-CIK细胞转基因技术,能高效的将CAR基因转导进入CIK细胞,对比CAR-T细胞的转染效率,有效缩短了CAR-CIK细胞的培养周期,节约了生产成本,降低了患者病情进展的风险。
本发明所述的CD19单链抗体轻链VL(如SEQ ID NO.17所示)、CD19单链抗体重链VH(如SEQ ID NO.16所示)经过人源化优化,避免了进入人体后产生HAMA,增加CAR-CIK细胞的存在时间。
本发明提供的hCAR19-CIK细胞和hCAR19-T细胞的制备方案,经过本公司长时间的摸索和优化,能够制备出存活时间长、杀伤效果好、副作用小的用于临床级别的治疗性细胞。
hCAR19-CIK细胞与hCAR19-T细胞相比,对CD19-K562靶细胞具有较高的特异性杀伤效率。同时hCAR19-CIK细胞对比hCAR19-T细胞,对K562细胞也有较高的非特异性杀伤效率。两者叠加,hCAR19-CIK细胞比hCAR19-T细胞杀伤效率更高,杀伤范围更广,显示了较高的
应用价值,有助于回输体内后,较为彻底的清除肿瘤细胞群,防止由于肿瘤表面靶标分子表达的多样性而导致清除不彻底。
本发明通过将CAR元件转导进入CIK细胞,使得CIK细胞表面增加了具有靶向功能的嵌合抗原受体,给CIK细胞增加了特异性杀伤的功能,使得CAR-CIK应用价值大大提高。
本发明中使用的共刺激因子的一种或若干种组合,能够增加转导后细胞的存活时间、杀伤效率、免疫记忆等特性。
可见,本发明所述的CAR-CIK细胞将给肿瘤细胞治疗提供可靠的保障。
图1为本发明所述的CAR的示意图,其中,图1A是CAR的基本结构图,图1B是CAR的代次改进示意图;
图2为本发明背景技术中所述的CIK细胞治疗和回输的步骤流程图,包含细胞采集、细胞培养、细胞扩增、细胞回输等阶段;
图3为本发明实施例1中所述的CAR-CIK细胞治疗和回输的步骤流程图,包含细胞采集、细胞激活、基因转导、体外培养、细胞扩增、细胞回输等阶段;
图4为本发明实施例2中所述的CAR-T细胞治疗和回输的步骤流程图,包含细胞采集、细胞分选、细胞激活、基因转导、体外培养、细胞扩增、细胞回输等阶段;
图5为本发明实施例3中重组慢病毒载体的不同纯化方式的支原体检测结果,lane1为DL2000marker,从上到下条带条带从上到下依次为:2kb、1kb、750bp、500bp、250bp、100bp;
lane2为阳性对照;lane3为阴性对照;lane4为PBS;lane5为裂解液;lane6为CAR-CIK细胞;lane7为CAR-T病毒;
图6为本发明实施例3中流式检测CAR-CIK细胞和CAR-T细胞的转导效率以及免疫分型结果;其中,图6A表示CAR-CIK细胞的转导效率结果;图6B表示CAR-CIK细胞的免疫分型结果;图6C表示CAR-T细胞的转导效率结果;图6D表示CAR-T细胞的免疫分型结果;
图7为本发明实施例4中不同效靶比条件下CAR-CIK细胞和CAR-T细胞的杀伤效率折线图;
图8为本发明实施例4中10:1效靶比条件下,CAR-CIK细胞和CAR-T细胞的因子分泌情况示意图;
图9为本发明实施例4中对照组、3疗程CIK组、6疗程CIK组的1、3、5年无病生存期对比示意图。
下面结合具体实施例进一步阐述此发明。应理解的是,在此描述的特定实施方式通过举例的方式来表示,并不作为对本发明的限制。在不偏离本发明范围的情况下,本发明的主要特征可以用于各种实施方式。
材料
1、hCAR19慢病毒转基因载体(SEQ ID NO.1)、支原体检测试剂盒、内毒素检测试剂盒、CD19+K562细胞购自世翱(上海)生物医药科技有限公司;hCAR19慢病毒转基因载体的制备方法参见发明名称为“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”的发明专利申请(申请号:201610008360.5)说明书的实施例1,本发明除了CD19单链抗体的轻链VL、CD19单链抗体重链VH的序列因经过人源化优化与该专利申请记载的不同,其它都相同。
2、人新鲜外周血由健康供者提供;
3、SEQ ID NO.17(CD19单链抗体的轻链VL)、SEQ ID NO.16(CD19单链抗体重链VH)所示的DNA序列由上海捷瑞生物公司根据本发明人提供的序列合成,并以寡核苷酸干粉或者质粒
形式保存;
4、0.22μm-0.8μm PES滤器购自millipore公司;
5、D-PBS(-)、0.4%台盼蓝、筛网、各类型细胞培养皿、培养袋、培养板均购自corning公司;
6、FBS、AIM-V、RPMI 1640、DMEM、lipofectamine 3000购自invitrogen公司;
7、Biotinylated protein L购自GeneScript公司;
8、LDH检测试剂盒购自promega公司;
9、Ficoll淋巴细胞分离液购自GE公司;
10、20%人血白蛋白注射液购自杰特贝林公司;
11、CryoPremium冻存液、分选缓冲液来自上海优卡迪生物医药科技有限公司;
12、rIL-2、rIL-1a、rIFN-γ,rIL-7,,rIL-15,rIL-21购自peprotech公司;
13、CD3单克隆抗体,CD28单克隆抗体,CD3/CD28磁珠CD4/CD8磁珠购自德国Miltenyi公司;
14、冷冻离心机购自美国ThermoScientific公司;
15、FACS流式细胞仪购自Thermo公司;
16、荧光倒置显微镜购自Olympus公司;
17、CD3-FITC、CD56-AF647购自BioLegend公司;
18、0.9%生理盐水购自今迈公司;
19、RetroNectin购自Takara公司;
20、phycoerythrin(PE)-conjugated streptavidin购自BD Bioscience公司。
实施例1 CAR-CIK细胞构建。
参见图3,本发明所述CAR-CIK细胞的构建方法如下:
1、分离PBMC。
(1)抽取健康供者新鲜外周血50ml;
(2)将采血袋喷拭酒精两遍,并擦干。
(3)用50ml注射器将袋中的血细胞吸出来移至新50ml管中。
(4)400g,20℃离心10min。
(5)将上层血浆移到新的50ml离心管中,56℃,30min灭活血浆,恢复至室温,2000g,离心30min,取上清到50ml离心管中待用。
(6)用D-PBS(-)补至50ml,拧紧盖子,颠倒混匀。
(7)取2个新50ml离心管,每管加入15ml Ficoll淋巴细胞分离液。
(8)向每管Ficoll上小心加入血细胞稀释液25ml。800g,20℃离心20min。
(9)离心管中液体分为四层,从上至下分别为:黄色的血浆层(回收待用)、白膜层、无色透明的Ficoll层、红黑色的混合细胞层。
(10)小心吸取白膜层到新50ml离心管中,补加D-PBS(-)至50ml,颠倒混匀后500g,20℃离心10min。
(11)加入25ml 5%人血白蛋白并重悬细胞,400g,20℃离心10min。
(12)弃上清,加入25ml 5%人血白蛋白重悬细胞沉淀,并过70um筛网,计数。
(13)取1份含1.25x108cells用于激活;剩余细胞悬液400g,20℃离心10min,加CryoPremium并冻存。
2、CIK细胞激活。
(1)使用50-500ug/L CD3单克隆抗体加入24孔板,封口膜封口,4℃过夜包被。
(2)将Ficoll分离得到的PBMC按1×109/L密度,置于37℃、5%CO2培养箱中培养,RPMI
1640培养基中含有1×106~1×107U/L的rIFN-γ。
(3)24h后,将细胞加入包被CD3单克隆抗体的24孔板中,置于37℃、5%CO2培养箱中培养。
3、CAR基因转导及CAR-CIK细胞诱导培养。
(1)提前一天包被1×103ug/L~1×104ug/L RetroNectin于24孔板内,封口膜封口,4℃过夜包被。
(2)往24孔板中,根据每孔5×105细胞量,按MOI=5~20的量,加入hCAR19慢病毒转基因载体,同时添加3×105~3×106U/L rIL-2,500-2000ug/L rIL-1a,37℃、5%CO2继续培养。
4、CAR-CIK细胞体外扩增。
(1)每2天等量补加含1×105~1×106U/L rIL-2RPMI 1640培养基,使PH值维持在6.5~7.5之间,细胞密度维持在5×105~2×106/ml之间,37℃、5%CO2继续培养14-21天。
(2)第7天左右,可取部分细胞用于各类检测。
5、CAR-CIK细胞终产品制剂。
(1)14-21天后,收货细胞,用含1×105~1×106U/L rIL-2RPMI 1640培养基调整细胞密度至2×106~4×106/mL之间。
(2)按照细胞的转导效率,回输剂量和患者体重,用下列公式计算需要回输的细胞总量:total cell=(回输剂量X患者体重)/转导效率。
(3)930g,离心10min。
(4)弃上清,加0.9%生理盐水45ml吹打混匀,并转移到新50ml离心管内。
(5)350g,离心5min。
(6)弃上清,用0.9%生理盐水45ml吹打混匀。
(7)重复步骤(6)1次。
(8)弃上清,用0.9%生理盐水45mL吹打混匀,并过70um筛网,使用台盼蓝计数并计算细胞活率。
(9)按照计数结果,取适量细胞悬液到新的50ml离心管内,补加0.9%生理盐水到45ml。
(10)350g,离心5min。
(11)离心后弃上清,细胞沉淀用一定体积0.9%生理盐水重悬,控制细胞密度在1×107~4×107/mL之间,吹打混匀之后,加入10%人血白蛋白,混匀。
(12)将细胞悬液注入回输袋中,热合机封口,贴好标签后,-80℃保存。
实施例2 CAR-T细胞构建。
参见图4,本发明所述CAR-T细胞的构建方法如下:
1、分离PBMC。
(1)抽取健康供者新鲜外周血50ml;
(2)将采血袋喷拭酒精两遍,并擦干。
(3)用50ml注射器将袋中的血细胞吸出来移至新50ml管中。
(4)400g,20℃离心10min。
(5)将上层血浆移到新的50ml离心管中,56℃,30min灭活血浆,恢复至室温,2000g,离心30min,取上清到50ml离心管中待用。
(6)用D-PBS(-)补至50ml,拧紧盖子,颠倒混匀。
(7)取2个新50ml离心管,每管加入15ml Ficoll淋巴细胞分离液。
(8)向每管Ficoll上小心加入血细胞稀释液25ml。800g,20℃离心20min。
(9)离心管中液体分为四层,从上至下分别为:黄色的血浆层(回收待用)、白膜层、无色
透明的Ficoll层、红黑色的混合细胞层。
(10)小心吸取白膜层到新50ml离心管中,补加D-PBS(-)至50ml,颠倒混匀后500g,20℃离心10min。
(11)加入25ml 5%人血白蛋白并重悬细胞,400g,20℃离心10min。
(12)弃上清,加入25ml 5%人血白蛋白重悬细胞沉淀,并过70um筛网,计数。
(13)取1份含1.25x108cells用于激活;剩余细胞悬液400g,20℃离心10min,加CryoPremium并冻存。
2、CD4/CD8阳性T细胞分选。
(1)将获得的PBMC计数,以80ul/107cells的比例加入分选缓冲液,重悬细胞沉淀。
(2)再以20ul/107cells的比例加入CD4/CD8磁珠,吹打混匀后放入4℃中孵育15min。
(3)取出磁珠-细胞混合液,以2ml/107cells的比例加入分选缓冲液,颠倒混匀后,250g,4℃离心10min。
(4)以500ul/108cells的比例加入分选缓冲液,重悬细胞沉淀。
(5)用镊子夹取LS分离柱到磁力架上。
(6)同时准备2个15ml离心管,分别标记:CD4-/CD8-细胞液(A管)、CD4+/CD8+细胞液(B管)。
(7)用3ml分离缓冲液润洗LS,并用A管接缓冲液。
(8)加入细胞-磁珠混合液,滴完后加入3ml缓冲液冲洗柱子(每次无液体残留时再加入新的液体),总共三次,收集得到CD4/CD8-细胞。
(9)LS分离柱与磁力架分离,用B管接细胞悬液,加入5ml缓冲液,将并用柱子内塞稍用力冲洗,收集为CD4+/CD8+细胞,取样计数。
(10)按1x106/ml-4x106/ml的细胞密度用AIM-V培养基重悬细胞沉淀,并加入2×105~1×106U/L IFN-γ因子。
3、T细胞激活。
(1)提前一天将1×103ug/L~1×104ug/L CD3单克隆抗体和1×103ug/L~1×104ug/L CD28单克隆抗体加入24孔板,封口膜封口,4℃过夜包被。
(2)取出包被的T75瓶,倒掉包被液,用D-PBS(-)洗涤一次,并将分选得到的细胞悬液接种到T75瓶中,摇匀,放入37℃、5%CO2培养箱中培养。
4、CAR基因转导及CAR-T细胞诱导培养。
(1)提前一天包被1×103ug/L~1×104ug/L RetroNectin于24孔板内,封口膜封口,4℃过夜包被。
(2)往24孔板中,根据每孔5×105细胞量,按MOI=5~20的量,加入hCAR19慢病毒转基因载体,同时添加含2×105~5×105U/L rIL-2,5×103ng/L~1×104ng/L rIL-7,5×103ng/L~1×104ng/L rIL-15,5×103ng/L~1×104ng/L rIL-21和含10%自体血清的AIM-V培养基37℃、5%CO2继续培养。
5、CAR-T细胞体外扩增。
(1)每2天等量补加含2×105~5×105U/L rIL-2,5×103ng/L~1×104ng/L rIL-7,5×103ng/L~1×104ng/L rIL-15,5×103ng/L~1×104ng/L rIL-21和含10%自体血清的AIM-V培养基,使PH值维持在6.5~7.5之间,细胞密度维持在5×105~2×106/ml之间,37℃、5%CO2继续培养10-14天。
(2)第7天左右,可取部分细胞用于各类检测。
6、CAR-T细胞终产品制剂。
(1)10-14天后,收货细胞,用含2×105~5×105U/L rIL-2AIM-V培养基调整细胞密度至2×106~4×106/mL之间。
(2)按照细胞的转导效率,回输剂量和患者体重,用下列公式计算需要回输的细胞总量:total cell=(回输剂量X患者体重)/转导效率。
(3)930g,离心10min。
(4)弃上清,加0.9%生理盐水45ml吹打混匀,并转移到新50ml离心管内。
(5)350g,离心5min。
(6)弃上清,用0.9%生理盐水45ml吹打混匀。
(7)重复步骤(6)1次。
(8)弃上清,用0.9%生理盐水45mL吹打混匀,并过70um筛网,使用台盼蓝计数并计算细胞活率。
(9)按照计数结果,取适量细胞悬液到新的50ml离心管内,补加0.9%生理盐水到45ml。
(10)350g,离心5min。
(11)离心后弃上清,细胞沉淀用一定体积0.9%生理盐水重悬,控制细胞密度在1×107~4×107/mL之间,吹打混匀之后,加入10%人血白蛋白,混匀。
(12)将细胞悬液注入回输袋中,热合机封口,贴好标签后,-80℃保存。
实施例3 CAR-CIK细胞和CAR-T细胞病原检测和表达检测。
一、内毒素检测;
(1)、内毒素工作标准品为15EU/支;
(2)、鲎试剂灵敏度λ=0.25EU/ml,0.5ml/管
(3)、内毒素标准品稀释:取内毒素标准品一支,分别用BET水按比例稀释成4λ和2λ的溶解,封口膜封口,震荡溶解15min;稀释时每稀释一步均应在漩涡混合器上混匀30s;
(4)、加样:取鲎试剂若干支,每支加入BET水0.5ml溶解,分装至若干支无内毒素试管中,每管0.1ml。其中2支为阴性对照管,加入BET水0.1ml;
2支为阳性对照管,加入2λ浓度的内毒素工作标准品溶液0.1ml;
2支为样品阳性对照管,加入0.1ml含2λ内毒素标准品的样品溶液(稀释20倍的待测样品1ml+4λ的内毒素标准品溶液1ml=2ml含2λ内毒素标准品的稀释40倍样品)。
样品管中加入0.1ml样品,稀释比例见表1,37±1℃水浴(或培养箱)保温60±1min;
(5)、CAR-CIK细胞和CAR-T细胞的内毒素检测结果(如表1所示),两种细胞的内毒素含量均在在0.25~1.25EU/ml之间,符合《中华人民共和国药典》中小于10EU/ml的标准;
表1 CAR-CIK细胞和CAR-T细胞的内毒素检测结果
二、支原体检测;
(1)在实验前三日,细胞样品用无抗生素培养基进行培养;
(2)收集1ml细胞悬浮液(细胞数大于1*105),置于1.5ml离心管中;
(3)13000g离心1min,收集沉淀,弃去培养基;
(4)加入500ul PBS用枪头吹吸或涡旋振荡,重悬沉淀。13000g离心5min;
(5)步骤4重复一次;
(6)加入50μl Cell Lysis Buffer,用枪头吹吸,充分混匀后,在55℃水浴中孵育20min;
(7)将样品置于95℃中加热5min;
(8)13000g离心5min后,取5μl上清作为模板,25μlPCR反应体系为:ddH20 6.5μl、Myco Mix 1μl、2x Taq Plus Mix Master(Dye Plus)12.5μl、模板5μl;PCR循环条件为:95℃30sec,(95℃30sec,56℃30sec,72℃30sec)*30cycle,72℃5min。
(9)支原体检测结果显示(如图5所示),CAR-CIK细胞和CAR-T细胞中均不含支原体。
三、CAR基因转导效率检测及免疫分型检测;
(1)收集经病毒转导72h后CIK细胞和T细胞,用含1~4%人血白蛋白的D-PBS(-)溶液重悬细胞并调整为1×106/ml。
(2)向离心管中加入含1~4%人血白蛋白的D-PBS(-)溶液1ml并混匀,350g离心5min,弃上清。
(3)重复步骤2一次。
(4)用0.2ml的含1~4%人血白蛋白的D-PBS(-)溶液重悬细胞,并向离心管中加入1ul的1mg/ul protein L,5ul CD3-FITC,5ul CD56-AF647,混匀,4℃孵育45min。
(5)向离心管中加入1ml含1~4%人血白蛋白的D-PBS(-)溶液并混匀,350g离心5min,弃上清。
(6)重复步骤5两次。
(7)用0.2ml含1~4%人血白蛋白的D-PBS(-)溶液重悬细胞,并向离心管中加入0.2ul PE-SA,混匀,37℃避光孵育15min。
(8)向离心管中加入1ml含1~4%人血白蛋白的D-PBS(-)溶液重并混匀,350g离心5min,弃上清。
(9)用1ml D-PBS(-)溶液重悬细胞沉淀,350g离心5min,弃上清。
(10)重复步骤9两次。
(11)用0.4ml D-PBS(-)溶液重悬细胞沉淀,流式细胞仪进行检测。
(12)CAR基因转导效率及免疫分型检测检测结果显示(如图6所示),CAR-CIK细胞的转导效率明显高于CAR-T细胞,CAR-CIK细胞的纯度达到了92%以上,CAR-T细胞的纯度达到了97%以上。
(13)上述实验结果表明,本发明提供的CAR-CIK细胞和CAR-T细胞培养转导体系,能够高效的生产出适合临床应用的免疫细胞。
实施例4 CAR-CIK细胞和CAR-T细胞的功能检测。
一、细胞因子分泌及杀伤效果评估。
(1)分别培养CD19+K562细胞和效应细胞CAR-CIK细胞、CAR-T细胞;
(2)收集靶细胞(CD19+K562)4x105cells和效应细胞(CAR-CIK细胞和CAR-T细胞)2.8x106cells,800g,6min离心,弃上清;
(3)用1ml D-PBS(-)溶液分别重悬靶细胞和效应细胞,800g,6min离心,弃上清;
(4)重复步骤3一次;
(5)用700ul培养基(AIM-V培养基+1~10%FBS)重悬效应细胞,用2ml培养基(AIM-V培养基+1~10%FBS)重悬靶细胞;
(6)设置效靶比为1:1、5:1、10:1的实验孔,并设置对照组,每组3个复孔;
(7)250g,5min平板离心;
(8)37℃,5%CO2培养箱中培养4小时;
(9)250g,5min平板离心;
(10)取每个孔的50ul上清到新96孔板中,并且每孔加入50ul底物溶液(避光操作);
(11)避光孵育25min;
(12)每孔加入50ul终止液;
(13)酶标仪检测490nm吸光度;
(14)将3个复孔取平均值;将所有实验孔、靶细胞孔和效应细胞孔的吸光值减去培养基背景吸光值的均值;将靶细胞最大值的吸光值减去体积校正对照吸光值的均值。
(15)将步骤14中获得的经过校正的值带入下面公式,计算每个效靶比所产生的细胞毒性百分比。结果如图7所示,CAR-CIK细胞在不同效靶比条件下杀伤效率明显高于CAR-T细胞;
杀伤效率=(实验孔-效应细胞孔-靶细胞孔)/(靶细胞最大孔-靶细胞孔)X100%
(16)重复准备一份步骤6中的10:1的样品用于CBA检测细胞因子表达水平。CBA检测细胞因子含量的步骤为,细胞因子标准品管、样品管、阴性对照管中各加入50ul细胞因子捕获微球悬液,加样前混匀微球;各管分别加入50ul PE信号抗体;细胞因子标准品管加入细胞因子标准品稀释液;样品管、阴性对照管中分别加入稀释后样品和阴性对照液;室温避光孵育3h;各管加入1ml洗液,200g离心5min;弃上清;各管加入300ul洗液,重新悬浮微球;使用NxT流式细胞仪分析样本,当日上机,上机前充分混匀3-5s。结果如图8所示,CAR-CIK细胞的IFN-γ明显高于CAR-T细胞,并且抑制性细胞因子IL-10的表达水平明显低于CAR-T细胞;
(17)上述实验结果表明,CAR-CIK细胞的杀伤效率高,抑制因素少,因此CAR-CIK细胞治疗在肿瘤的细胞治疗中拥有广阔的应用前景。
二、根据文献报道(C.Hontscha,et al.Clinical trials on CIK cells:first report of the international registry on CIK cells(IRCC).J Cancer Res Clin Oncol.DOI10.1007/s00432-010-0887-7.),CIK细胞疗法针对肿瘤的免疫治疗有一定的效果。
本实施例统计了全球11项CIK细胞治疗的临床研究,共有426名患者参与临床研究,其中男性313人,女性113人。肿瘤类型包括hepatocellular carcinoma,gastric cancer,and Hodgkin or non-Hodgkin disease。这些临床实验共分为3组,对照组、3疗程CIK组和6疗程CIK组。结果如图9所示,3年和5年的无病生存期,3疗程CIK组和6疗程CIK组要明显优于对照组,显示出了CIK细胞治疗拥有一定的临床应用前景。
Claims (9)
- 一种CAR-CIK转基因细胞,其特征在于,其包含靶向CD19抗原的嵌合抗原受体CAR基因、CD56细胞以及CD3细胞,所述CAR基因表达阳性,所述CD56细胞表达阳性,所述CD3细胞表达阳性。
- 一种如权利要求1所述的CAR-CIK转基因细胞的制备方法,其特征在于,包括如下步骤:步骤1,分离外周血单个核细胞PBMC;步骤2,激活CIK细胞;步骤3,加入靶向CD19抗原的hCAR19慢病毒转基因载体转导及CAR-CIK细胞诱导培养;步骤4,CAR-CIK细胞体外扩增,得到CAR-CIK转基因细胞。
- 如权利要求2所述的方法,其特征在于,步骤1中,所述分离外周血单个核细胞PBMC的具体步骤包括:抽取健康供者新鲜外周血,加入淋巴细胞分离液及血细胞稀释液,离心,吸取离心后的白膜层细胞,加入人血白蛋白重悬细胞沉淀。
- 如权利要求2所述的方法,其特征在于,步骤2中,所述激活CIK细胞的具体步骤包括:(1)使用50-500ug/L CD3单克隆抗体加入24孔板,封口膜封口,4℃过夜包被;(2)将分离得到的外周血单个核细胞PBMC按1×109/L密度,置于37℃、5%CO2培养箱中培养,RPMI 1640培养基中含有1×106~1×107U/L的rIFN-γ;(3)24h后,将细胞加入包被CD3单克隆抗体的24孔板中,置于37℃、5%CO2培养箱中培养。
- 如权利要求2所述的方法,其特征在于,步骤3中,所述加入靶向CD19抗原的hCAR19慢病毒转基因载体转导及CAR-CIK细胞培养的具体步骤包括:(1)提前一天包被1×103ug/L~1×104ug/L RetroNectin于24孔板内,封口膜封口,4℃过夜包被;(2)往24孔板中,根据每孔5×105细胞量,按MOI=5~20的量,加入hCAR19慢病毒转基因载体,同时添加3×105~3×106U/L rIL-2,500-2000ug/L rIL-1a,37℃、5%CO2继续培养。
- 如权利要求2或5所述的方法,其特征在于,步骤3中,所述靶向CD19抗原的hCAR19慢病毒转基因载体包括:用于质粒复制的原核复制子pUC Ori序列,如SEQ ID NO.2所示;用于目的菌株大量扩增的含氨苄青霉素抗性基因AmpR序列,如SEQ ID NO.1所示;用于增强真核细胞内的复制的病毒复制子SV40 Ori序列,如SEQ ID NO.3所示;用于真核细胞表达绿色荧光的ZsGreen1绿色荧光蛋白,如SEQ ID NO.11所示;用于共同转录表达蛋白质的IRES核糖体结合序列,如SEQ ID NO.12所示;用于嵌合抗原受体基因的真核转录的人EF1α启动子,如SEQ ID NO.14所示;用于增强转基因的表达效率的eWPRE增强型土拨鼠乙肝病毒转录后调控元件,如SEQ ID NO.13所示;用于组成集识别、传递、启动于一体的三代CAR的嵌合抗原受体包括:如SEQ ID NO.15所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.17所示的CD19单链抗体轻链VL、如SEQ ID NO.19所示的Optimal Linker C、如SEQ ID NO.16所示的CD19单链抗体重链VH、如SEQ ID NO.21所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.22所示的CD8 Transmembrane嵌合受体跨膜区、CD28、如SEQ ID NO.23所示的CD137嵌合受体共刺激因子、如SEQ ID NO.24所示的TCR嵌合受体T细胞激活域、以及如SEQ ID NO.20所示的CD28嵌合受体共刺激因子;用于慢病毒包装的慢病毒包装顺式元件,采用第三代慢病毒载体包括:如SEQ ID NO.5所示的慢病毒5 terminal LTR、如SEQ ID NO.6所示的慢病毒3 terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、 如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件所述慢病毒包装顺式元件,以及如SEQ ID NO.4所示的RSV启动子;所述靶向CD19抗原的hCAR19慢病毒转基因载体的核苷酸序列如SEQ ID NO.18所示。
- 如权利要求2所述的方法,其特征在于,步骤4中,所述CAR-CIK细胞体外扩增的具体为:每2天等量补加含1×105~1×106U/L rIL-2RPMI 1640培养基,使PH值维持在6.5~7.5之间,细胞密度维持在5×105~2×106/ml之间,37℃、5%CO2继续培养14-21天。
- 如权利要求2所述的方法,其特征在于,在步骤4之后增加如下步骤:(1)用含1×105~1×106U/L rIL-2RPMI 1640培养基调整细胞密度至2×106~4×106/mL之间;(2)按照细胞的转导效率、回输剂量和患者体重,用下列公式计算需要回输的细胞总量:total cell=(回输剂量X患者体重)/转导效率;(3)离心,弃上清,加生理盐水吹打混匀;重复该步骤;并过筛网,计数并计算细胞活率;(4)按照计数结果,取适量细胞悬液到离心管内,补加生理盐水,离心后弃上清,细胞沉淀用生理盐水重悬,控制细胞密度在1×107~4×107/mL之间,吹打混匀之后,加入10%人血白蛋白,混匀;(5)将细胞悬液注入回输袋中,封口,贴好标签后,-80℃保存。
- 一种如权利要求1所述的CAR-CIK转基因细胞在制备肿瘤的细胞治疗药物中的应用。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710187804.0A CN108660111B (zh) | 2017-03-27 | 2017-03-27 | Car-cik转基因细胞及其制备方法和应用 |
CN201710187804.0 | 2017-03-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2018176844A1 true WO2018176844A1 (zh) | 2018-10-04 |
Family
ID=63674097
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2017/110659 WO2018176844A1 (zh) | 2017-03-27 | 2017-11-13 | Car-cik转基因细胞及其制备方法和应用 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN108660111B (zh) |
WO (1) | WO2018176844A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112120012A (zh) * | 2020-09-30 | 2020-12-25 | 广东康盾生物工程技术有限公司 | 一种car-t细胞冻存方法 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114807044B (zh) * | 2021-04-30 | 2023-09-08 | 四川大学华西医院 | 一种具有高nkt细胞比例的car-cik细胞制备方法及应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104829726A (zh) * | 2015-01-21 | 2015-08-12 | 武汉友芝友生物制药有限公司 | 一种双特异性抗体cd19xcd3的构建及应用 |
CN105602992A (zh) * | 2016-03-17 | 2016-05-25 | 上海优卡迪生物医药科技有限公司 | 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 |
-
2017
- 2017-03-27 CN CN201710187804.0A patent/CN108660111B/zh active Active
- 2017-11-13 WO PCT/CN2017/110659 patent/WO2018176844A1/zh active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104829726A (zh) * | 2015-01-21 | 2015-08-12 | 武汉友芝友生物制药有限公司 | 一种双特异性抗体cd19xcd3的构建及应用 |
CN105602992A (zh) * | 2016-03-17 | 2016-05-25 | 上海优卡迪生物医药科技有限公司 | 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 |
Non-Patent Citations (2)
Title |
---|
VIRNA MARIN: "Characterization of in vitro migratory properties of an- ti- CD 19 chimeric receptor-redirected CIK cells for their potential use in B- ALL immunotherapy", EXPERIMENTAL HEMATOLOGY, vol. 34, no. 9, 30 September 2006 (2006-09-30), pages 1218 - 1228, XP027879630, DOI: 10.1016/j.exphem.2006.05.004 * |
YAO, WEIQI: "Progesses of Anti-Tumor research or Chimeric Antigen Receptor Modified CIK cells", CHINESE JOURNAL OF CANCER BIOTHERAPY, vol. 23, no. 5, 31 October 2016 (2016-10-31), pages 720 - 724, ISSN: 1007-385X * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112120012A (zh) * | 2020-09-30 | 2020-12-25 | 广东康盾生物工程技术有限公司 | 一种car-t细胞冻存方法 |
Also Published As
Publication number | Publication date |
---|---|
CN108660111A (zh) | 2018-10-16 |
CN108660111B (zh) | 2020-01-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2018223600A1 (zh) | Octs-car双靶向嵌合抗原受体、编码基因、重组表达载体及其构建和应用 | |
WO2018223601A1 (zh) | 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 | |
WO2018218876A1 (zh) | 一种基于octs技术的淋系白血病car-t治疗载体及其构建方法和应用 | |
WO2018218877A1 (zh) | 一种基于octs技术的恶性胶质瘤car-t治疗载体及其构建方法和应用 | |
CN108409840B (zh) | 抗cd123单链抗体及其组合的嵌合抗原受体和应用 | |
CN105924533B (zh) | Ror1特异性嵌合抗原受体及其应用 | |
WO2018218879A1 (zh) | 一种基于octs技术的胰腺癌、恶性间皮瘤car-t治疗载体及其构建方法和应用 | |
EP3511347A1 (en) | Construction of recombined gene of chimeric antigen receptor for treatment of hiv infection, and application thereof | |
WO2016034094A1 (zh) | 制备dc-ctl的试剂盒及其应用 | |
WO2018188331A1 (zh) | 胆固醇转脂酶soat1被抑制的car-t细胞及其制备方法和应用 | |
WO2018058431A1 (zh) | 一种嵌合抗原受体分子及其应用 | |
CN108276497A (zh) | 靶向cd19的嵌合抗原受体t细胞及其应用 | |
CN109111525B (zh) | 一种hla-g嵌合抗原受体、编码序列和表达载体以及应用 | |
CN111234031B (zh) | 靶向肿瘤细胞表面muc1的新型嵌合抗原受体及muc1嵌合抗原受体t细胞的制备方法 | |
AU2020104307A4 (en) | Bispecific chimeric antigen receptor for treating hematological tumor complicated with HIV infection | |
CN110055269B (zh) | 人间皮素嵌合抗原受体、其t细胞及其制备方法和用途 | |
CN110317822B (zh) | Trop2嵌合抗原受体、其t细胞及其制备方法和用途 | |
WO2018176844A1 (zh) | Car-cik转基因细胞及其制备方法和应用 | |
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 | |
WO2021109977A1 (zh) | 一种能高效制备且应用安全的嵌合抗原受体t细胞及其制备方法与应用 | |
CN108699163B (zh) | 一种多基因重组嵌合抗原受体分子及其应用 | |
CN111560075B (zh) | 一种含双靶点嵌合抗原受体基因的载体、car-t细胞及其应用 | |
WO2018218875A1 (zh) | 一种基于octs技术的前列腺癌car-t治疗载体及其构建方法和应用 | |
WO2018218878A1 (zh) | 一种基于octs技术的髓系白血病car-t治疗载体及其构建方法和应用 | |
CN113122579B (zh) | 一种慢病毒转染免疫细胞的方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17904073 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
32PN | Ep: public notification in the ep bulletin as address of the adressee cannot be established |
Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 27/01/2020) |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 17904073 Country of ref document: EP Kind code of ref document: A1 |