CN116589598A - 共表达fosb的嵌合抗原受体及其应用 - Google Patents
共表达fosb的嵌合抗原受体及其应用 Download PDFInfo
- Publication number
- CN116589598A CN116589598A CN202310555126.4A CN202310555126A CN116589598A CN 116589598 A CN116589598 A CN 116589598A CN 202310555126 A CN202310555126 A CN 202310555126A CN 116589598 A CN116589598 A CN 116589598A
- Authority
- CN
- China
- Prior art keywords
- cells
- seq
- antigen receptor
- fosb
- amino acid
- 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
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 title claims abstract description 37
- 101000931462 Homo sapiens Protein FosB Proteins 0.000 title claims abstract description 23
- 102100020847 Protein FosB Human genes 0.000 title claims abstract description 23
- 210000004027 cell Anatomy 0.000 claims abstract description 68
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims abstract description 18
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims abstract description 18
- 210000002865 immune cell Anatomy 0.000 claims abstract description 15
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims abstract description 13
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 13
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims abstract description 13
- 230000003834 intracellular effect Effects 0.000 claims abstract description 13
- 101800001494 Protease 2A Proteins 0.000 claims abstract description 7
- 101800001066 Protein 2A Proteins 0.000 claims abstract description 7
- 210000004899 c-terminal region Anatomy 0.000 claims abstract description 4
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 28
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 25
- 239000002773 nucleotide Chemical group 0.000 claims description 23
- 125000003729 nucleotide group Chemical group 0.000 claims description 23
- 206010028980 Neoplasm Diseases 0.000 claims description 20
- 239000013598 vector Substances 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 9
- 238000011282 treatment Methods 0.000 claims description 7
- 102000001398 Granzyme Human genes 0.000 claims description 6
- 108060005986 Granzyme Proteins 0.000 claims description 6
- 241000713666 Lentivirus Species 0.000 claims description 4
- 239000000427 antigen Substances 0.000 claims description 4
- 102000036639 antigens Human genes 0.000 claims description 4
- 108091007433 antigens Proteins 0.000 claims description 4
- 210000005259 peripheral blood Anatomy 0.000 claims description 3
- 239000011886 peripheral blood Substances 0.000 claims description 3
- 230000008685 targeting Effects 0.000 claims description 3
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 claims description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 claims description 2
- 208000031671 Large B-Cell Diffuse Lymphoma Diseases 0.000 claims description 2
- 206010012818 diffuse large B-cell lymphoma Diseases 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 210000004700 fetal blood Anatomy 0.000 claims description 2
- 210000000581 natural killer T-cell Anatomy 0.000 claims description 2
- 210000000822 natural killer cell Anatomy 0.000 claims description 2
- 230000003248 secreting effect Effects 0.000 claims description 2
- 241001430294 unidentified retrovirus Species 0.000 claims description 2
- 108010083359 Antigen Receptors Proteins 0.000 claims 3
- 102000006306 Antigen Receptors Human genes 0.000 claims 3
- 210000004881 tumor cell Anatomy 0.000 abstract description 10
- 239000013612 plasmid Substances 0.000 description 21
- 238000000338 in vitro Methods 0.000 description 14
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 238000011534 incubation Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 108010036781 Fumarate Hydratase Proteins 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000003556 assay Methods 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 230000005909 tumor killing Effects 0.000 description 6
- 102100036160 Fumarate hydratase, mitochondrial Human genes 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 108010074328 Interferon-gamma Proteins 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000010361 transduction Methods 0.000 description 4
- 230000026683 transduction Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 102100037850 Interferon gamma Human genes 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 208000008839 Kidney Neoplasms Diseases 0.000 description 3
- 206010038389 Renal cancer Diseases 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 3
- 239000007640 basal medium Substances 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000009169 immunotherapy Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 201000010982 kidney cancer Diseases 0.000 description 3
- 230000002147 killing effect Effects 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 208000011691 Burkitt lymphomas Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000222418 Lentinus Species 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000006285 cell suspension Substances 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
- 238000005516 engineering process Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 101150064107 fosB gene Proteins 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229960002378 oftasceine Drugs 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000002476 tumorcidal effect Effects 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 102100023990 60S ribosomal protein L17 Human genes 0.000 description 1
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 108091033409 CRISPR Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 1
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 1
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 1
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 229940126547 T-cell immunoglobulin mucin-3 Drugs 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 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 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 210000003162 effector t lymphocyte Anatomy 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
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing 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
- 230000035772 mutation Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001111—Immunoglobulin superfamily
- A61K39/001112—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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
-
- 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
- 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
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
-
- 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
- C07K2319/01—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
- 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
- C12N2740/10041—Use of virus, viral particle or viral elements as a vector
- C12N2740/10043—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
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- General Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Toxicology (AREA)
- Virology (AREA)
- Marine Sciences & Fisheries (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明公开了一种共表达FOSB的嵌合抗原受体及其应用，所述嵌合型抗原受体从N端到C端顺次拼接信号肽、靶向CD19的单链抗体ScFv、strepII、CD8hinge、CD28跨膜区、CD28胞内结构域、胞内共刺激域4‑1BB和CD3ζ链；在CD3ζ链的C端进一步拼接有P2A肽和FOSB。以CD19抗体为例，共表达FOSB的抗CD19嵌合抗原受体用于修饰免疫细胞，修饰后的免疫细胞对肿瘤细胞的杀伤优于普通的Anti‑CD19CAR‑T细胞。
Description
优先权信息
本申请要求享有申请号为2023101691805、发明名称为“共表达FOSB的嵌合抗原受体及其应用”的中国发明专利申请的优先权。
技术领域
本发明涉及医药生物领域，具体是指一种以共表达FOSB的嵌合抗原受体(CAR)及其在肿瘤治疗中的应用。
背景技术
CAR-T介绍
肿瘤免疫治疗是指应用免疫学的原理和方法，提高肿瘤的免疫原性，利用自身的免疫系统攻击肿瘤细胞，从而抑制或杀伤肿瘤细胞。按其治疗策略分为两大类：免疫检查点抑制剂(如CTLA-4、PD-1、PD-L1等)和细胞免疫治疗(如CAR-T等)。其中CAR-T细胞全称为嵌合抗原受体T细胞(Chimeric Antigen ReceptorT-Cell)，原理为通过基因工程的方法，将能识别某种肿瘤抗原的抗体单链可变区(Scfv)与CD3-ζ链的胞内区在体外偶联为一个嵌合蛋白，通过基因转导的方法转染体外培养的患者T细胞，使其表达嵌合抗体受体(CAR)。患者的T细胞被“重编程”后，生成大量具有杀伤性的CAR-T细胞，能够特异性的靶向肿瘤细胞。CAR-T与传统的免疫疗法相比，具有治疗更精确、靶向更精准、杀瘤更广泛、效果更持久等显著优势。
尽管目前在CAR-T治疗肿瘤领域捷报不断，但是CAR-T细胞治疗肿瘤过程中仍然面临着诸多问题。其中，CAR-T细胞需要浸润到肿瘤中，发挥功能是导致CAR-T疗法仅在小部分病人中有效果的主要原因，并直接导致了CAR-T疗法在治疗过程中的局限性。因此，促进CAR-T细胞浸润和激活、发挥功能成为CAR-T治疗有效性至关重要的一步。
延胡索酸水解酶FH(Fumarate hydratase)编码三羧酸循环中的线粒体蛋白，其基因突变或低表达在多种肿瘤中高发，尤其对肾癌的发展起重要作用。临床上发现，在肾癌病人中，FH低表达伴随更差的生存率。同时，FH低表达的肾癌病人伴随低的功能性CD8+T细胞浸润。在小鼠肿瘤细胞中敲低FH或抑制FH酶活，均可有效地抑制功能性CD8+T细胞的浸润，促进肿瘤生长。进一步的，在抗CD19 CAR-T细胞中抑制FH酶活，亦显著阻断CAR-T细胞对CD19肿瘤的杀伤和抑制。然而，过表达FH是否促进CAR-T细胞治疗肿瘤的效果，尚未发现。
发明内容
针对现有技术的不足，本发明提供一种共表达FOSB蛋白的嵌合抗原受体(CAR)，携带靶向CD19的ScFv序列的CAR-T细胞增加了CAR T细胞的功能，减少了终端分化和提高了抗肿瘤能力。CAR T细胞过度表达FOSB使CAR T具有更高的肿瘤杀伤能力，解决了这类新兴治疗药物进展的主要障碍。
本发明一方面涉及一种嵌合型抗原受体，其特征在于，从N端到C端顺次拼接信号肽、靶向CD19的单链抗体ScFv、strepII、CD8 hinge、CD28跨膜区、CD28胞内结构域、胞内共刺激域4-1BB、和CD3ζ链，在CD3ζ链的C端进一步拼接有P2A核苷酸序列和能够表达FOSB蛋白的核苷酸序列。
优选地，所述单链抗体ScFv的氨基酸序列如SEQ ID NO.1所示，所述单链抗体ScFv的核苷酸序列如SEQ ID NO.2所示，所述单链抗体ScFv特异性识别肿瘤细胞表面的CD19抗原蛋白。
本发明另一方面提供的嵌合型抗原受体CAR，从N端到C端顺次拼接信号肽、靶向CD19的单链抗体ScFv、strepII、CD8hinge、CD28跨膜区、CD28胞内结构域、胞内共刺激域4-1BB和CD3ζ链；在CD3ζ链的C端进一步拼接有P2A肽和FOSB。
优选地，所述能够识别肿瘤细胞表面CD19抗原的单链抗体ScFv的氨基酸序列如SEQ ID NO.1，所示ScFv的核苷酸序列如SEQ ID NO.2所示。
优选地，所述信号肽的氨基酸序列如SEQ ID NO.3，核苷酸序列如SEQ ID NO.4所示；CD8hinge的氨基酸序列如SEQ ID NO.5，核苷酸序列如SEQ ID NO.6所示；CD28跨膜区、CD28胞内结构域的氨基酸序列分别如SEQ ID NO.7、SEQ ID NO.9，核苷酸序列分别如SEQID NO.8、SEQ ID NO.10所示；胞内共刺激域4-1BB的氨基酸序列如SEQ ID NO.11，核苷酸序列如SEQ ID NO.12所示；CD3ζ的氨基酸序列如SEQ ID NO.13所示，核苷酸序列如SEQ IDNO.14所示；优选地，所述P2A蛋白的氨基酸序列如SEQ ID NO.15所示，所述P2A蛋白的核苷酸序列如SEQ ID NO.16所示；优选地，所述FOSB蛋白的氨基酸序列如SEQ ID NO.17所示，所述FOSB蛋白的核苷酸序列如SEQ ID NO.18所示。
本发明另一方面还涉及一种重组嵌合型抗原受体的基因载体，其特征在于，以PTK881-EF1α载体为骨架，插入上述的嵌合型抗原受体编码核苷酸序列的慢病毒、逆转录病毒或转座子载体；优选地，以PTK881-EF1α载体为骨架，插入如上述嵌合型抗原受体编码核苷酸序列的慢病毒载体；优选地，CRISPR、RNA干扰技术联合FOSB和嵌合型抗原受体原件的表达。
本发明另一方面还涉及一种表达嵌合型抗原受体的免疫细胞，其特征在于，由上述嵌合型抗原受体的编码核苷酸序列或上述重组嵌合型抗原受体基因载体转染免疫细胞得到，所述免疫细胞选自脐带血、外周血、肿瘤组织或iPSC来源的T细胞、NK细胞、NKT细胞、αβT细胞、γδT细胞、CD4+T细胞，CD8+T细胞，优选为外周血来源的T细胞；所述嵌合型抗原受体的单链抗体ScFv结合CD19受体。
本发明另一方面涉及上述免疫细胞的用途，所述免疫细胞用于制备治疗细胞膜外表达CD19抗原的肿瘤的药物中的应用。
在本发明的一个优选实施方式中，所述肿瘤包括但不限于急性淋巴细胞白血病(r/rALL)，弥漫性大B细胞淋巴瘤(r/rDLBCL)。本发明另一方面涉及上述免疫细胞的用途，所述免疫细胞用于过表达FOSB和分泌颗粒酶B。
在一些实施例中，表达嵌合型抗原受体的免疫细胞进行体外功能检测时，所选择为细胞膜外表达CD19抗原的细胞系。
本发明的有益效果在于：
1、本发明提供的CD19为靶点的嵌合抗原受体包括特定的单链抗体ScFv，其用于修饰免疫细胞，修饰后的免疫细胞能用于表面CD19阳性的肿瘤的治疗。
2、本发明提供的嵌合抗原受体过表达FOSB，使CART细胞强激活，分泌更多的颗粒酶B，提高其杀瘤效果。
附图说明
图1为实施例中Anti-CD19 CAR的DNA片段的示意图；
图2为实施例中Anti-CD19 CAR-FOSB的DNA片段的示意图；
图3为实施例中PTK881-EF1α-anti-CD19 CAR质粒图谱；
图4为实施例中PTK881-EF1α-anti-CD19 CAR-FOSB质粒图谱；
图5为Anti-CD19 CAR T和Anti-CD19 CAR-FOSB T细胞的CAR表达效率
图6为CAR-T细胞对Burkitt＇s淋巴瘤细胞系Raji体外杀伤结果示意图；
图7为CAR-T细胞与Burkitt＇s淋巴瘤细胞系Raji体外共孵育前后细胞因子IFN-γ、TNF-α和GranzymeB释放的结果示意图；
图8为Anti-CD19 CAR T和Anti-CD19 CAR-FOSB T细胞细胞分型的变化。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：PTK881-EF1α-CD19、PTK881-EF1α-CD19-FOSB、质粒的构建
1、分别人工合成片段CD19、FOSB，人工合成SP、strepII-CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ片段。
2、采用OverlapPCR扩增分别以SP、CD19和CD8hinge-CD28TM、ICD-4-1BB-CD3ζ，或以SP、CD19和CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ+P2A肽+FOSB为模版，获得带有酶切位点EcoRI和BamHI的CD19-CAR和CD19-CAR-FOSB片段的结构示意图分别如图1、图2所示。其中，信号肽(SP)的氨基酸序列如SEQ ID NO.3所示，CD8 hinge的氨基酸序列如SEQ ID NO.5所示，CD28TM的氨基酸序列如SEQ ID NO.7，CD28ICD的氨基酸序列如SEQ ID NO.9所示，4-1BB的氨基酸序列如SEQ ID NO.11所示，CD3ζ的氨基酸序列如SEQ ID NO.13所示,所述P2A肽的氨基酸序列如SEQ ID NO.15所示，FOSB的氨基酸序列如SEQ ID NO.17所示，更优选地，信号肽(SP)的核苷酸序列如SEQ ID NO.4所示，CD8 hinge的核苷酸序列如SEQ ID NO.6所示，CD28TM的核苷酸序列如SEQ ID NO.8，CD28ICD的核苷酸序列如SEQ ID NO.10所示，4-1BB的核苷酸序列如SEQ ID NO.12所示，CD3ζ的核苷酸序列如SEQ ID NO.14所示，所述F2A肽的核苷酸序列如SEQ ID NO.16所示，FOSB的核苷酸序列如SEQ ID NO.18所示。
3、将质粒PTK881-EF1α-Kan使用EcoRI和BamHI限制性内切酶进行双酶切，产物经过0.8％的琼脂糖凝胶电泳，并割胶回收置于Eppendorf管内，用Axygen公司的琼脂糖凝胶回收试剂盒回收相应的片段，并测定产物的纯度和浓度。
4、将片段以1:2摩尔比加入Eppendorf管加入ExnaseⅡ连接酶(Vazyme)与同源重组酶5×CEⅡbuffer，37℃反应0.5小时；将连接液取出10μL加入100μL DH5α感受态细胞冰浴30min后42℃热激90s，完成后加入500μL SOC培养基37℃、220rpm培养2小时；2小时后将Eppendorf管4000g离心1min移除400μL多余液体。将剩余液体涂布在LB平板37℃培养12小时；在平板上挑取单菌落，接种到5mLLB液体培养基中37℃、220rpm培养12小时。
5、用Axygen小提试剂盒提取质粒，获得质粒PTK881-EF1α-CD19、PTK881-EF1α-CD19-FOSB；送生工生物工程(上海)股份有限公司科技公司一代测序验证无误后，进行含质粒PTK881-EF1α-CD19、PTK881-EF1α-CD19-FOSB的完整图谱示意图分别如图3、图4所示。
实施例2、质粒的制备与测序
1、质粒的制备
将含质粒PTK881-EF1α-CD19、PTK881-EF1α-CD19-FOSB的大肠杆菌DH5α菌种分别接种至250mL含100μg/mL氨苄霉素的LB培养液中，37℃、220rpm培养过夜。培养液在4℃于6000g离心20min，弃上清。
取出EndoFree plasmid mega kit(Qiagen)中的Buffers P1，向离心得到的大肠杆菌沉淀中加120mL提前预冷的Buffers P1，盖上离心瓶盖，剧烈振荡离心瓶使大肠杆菌沉淀在BuffersP1中完全分散。
向离心瓶中加120mL Buffers P2，盖上瓶盖放置在滚轴混匀仪上，慢慢提速至50rpm，彻底混匀后室温放置5min。
向离心瓶中加120mL Buffers P3，盖上瓶盖放置在滚轴混匀仪上，慢慢提速至滚轴混匀仪最大转速70rpm，彻底混匀直至呈白色不粘稠蓬松的混合液。在4℃于9000g离心15min。
向QIAfilter Cartridge倒入50mL Buffer FW，将离心所得上清液倒入QIAfilterCartridge中，轻轻地搅拌混匀。将混合液抽滤入已标记好对应的玻璃瓶中。
向每个玻璃瓶中加入20mL Buffer ER，上下颠倒混匀6次，在-20℃孵育30min。
将标记好的mega柱放入对应的架子上，向每个mega柱内加入35mL Buffers QBT平衡，重力作用使之流尽。
将玻璃瓶中的液体分批全部倒入对应标记的mega柱中，待柱中液体流尽后，向每个mega柱分批加入200mL Buffer QC进行清洗。待柱中液体流尽后，将废液收集盘中的废液倒入50mL洁净离心管内。
再向每个mega柱内加入40mL Buffer QN，使用50mL洁净离心管收集流出液，上下颠倒6次混匀，分装20mL至另一洁净已标记的50mL离心管内。
向每个50mL离心管加入14mL异丙醇(常温)，上下颠倒6次混匀。在4℃于15000g离心50min。
超净工作台内吸尽上清，每管加入3.5mL Endotoxin-free water漂洗，不要将底部沉淀冲散。在4℃于15000g离心30min。将EndoFree plasmid mega kit中的Buffer TE放入烘箱内预热。
在超净工作台内吸尽离心后的上清，于超净工作台内吹干(挥发残留的无水乙醇，时间在10min左右)。
在烘箱内拿出Buffer TE，在超净工作台内向每管加入1mL Buffer TE，用枪吹打10次后放入65℃烘箱，期间不间断地敲击管壁促使沉淀完全溶解。在4℃于4000g离心1min将管壁上的液体甩到管底后吹打混匀。
在超净工作台内将液体全部转移至无内毒素无热源无核酸酶对应标记的EP管中。吸出2μL，用微量分光光度计测质粒浓度，并标记在对应的EP管上，获得质粒PTK881-EF1α-CD19、PTK881-EF1α-CD19-FOSB。
2、目的基因测序
分别取20μL(500ng)质粒DNA，外送测序，根据原始种子序列，检查质粒生产所得产品的目的基因有无发生改变，稳定的工艺下，工作种子在进行发酵培养放大过程中，目的基因不会发生改变，可用于下一环节的生产和正确表达蛋白。
实施例3、PTK881-EF1α-CD19、PTK881-EF1α-CD19-FOSB慢病毒载体的制备与活滴检测
1、慢病毒载体的制备
在多层细胞培养瓶(Hyperflask)接入130.0～140.0×106数目的293T细胞(Takara)，共560mL DMEM完全培养基(50mL胎牛血清、5mL Antibiotic-Antimycotic(100×))，在37℃含5％CO2培养箱中培养24小时。分别将混有320μg质粒(PTK881-EF1α-CD19、PTK881-EF1α-CD19-FOSB：BZ1质粒：BZ2质粒：BZ3质粒＝12：10：5：6)的DMEM完全培养基加入960μgPEI管中，漩涡震荡,室温平衡10min。分别将上述35mLPEI与质粒的混合液与525mLDMEM完全培养基混匀，换入上述多层细胞培养瓶中。将多层细胞培养瓶置于37℃含5％CO2培养箱培养3天后，收集细胞培养上清液。
分别将上清液4000rpm(或3000g)离心30min后，向离心后上清中加入cryonase酶(Takara)置于4℃。6个小时后，使用0.22μm的滤膜对慢病毒上清液进行抽滤，4℃于30000g离心2.5h。去除上清，加入1mLT细胞培养基重悬沉淀。重悬后，留20μL做病毒活性滴度检测，剩余慢病毒浓缩液分装，标记为Lenti3-CD19-CAR、Lenti3-CD19-CAR-FOSB并置于-80℃保存备用。
2、慢病毒载体活性滴度检测
原理：商业化的靶向FCM63的抗体用作细胞表面CAR表达的验证，通过流式细胞仪检测到的荧光信号间接反应了CAR在293T细胞中的表达情况。
方法：在6孔板中接入5.0×105个/孔293T细胞，慢病毒浓缩液每孔分别加入0.1μL、0.5μL、1μL，并设1个阴性对照。置于37℃含5％CO2培养箱内培养。三日后，用Versene溶液(Gibco)收集293T细胞送流式细胞学检测CAR阳性293T细胞比例，并换算得到PTK881-EF1α-CD19、PTK881-EF1α-CD19-FOSB慢病毒浓缩液活性滴度。
目前的慢病毒浓缩液活性滴度在1×108～10×108(TU/mL)范围内，检测分析结果见表1。表明各个慢病毒载体均能获得较高的活性滴度，可用于后续的嵌合抗原受体免疫细胞的制备。
表1慢病毒活性滴度检测分析结果
| 样品编号 | 活性滴度(TU/mL) |
| Lenti3-CD19-CAR | 1.8×108 |
| Lenti3-CD19-CAR-FOSB | 1.4×108 |
实施例4、Anti-CD19 CAR-T、Anti-CD19-FOSB CAR-T细胞的制备
1、CAR-T细胞制剂制备：
采集健康供者外周血100mL，采用Ficoll淋巴细胞分离液分离单个核细胞。计数后，使用适量CD3 MicroBeads,human(美天旎)分选CD3阳性细胞，并以1.0～2.0×106个/mL密度在T细胞完全培养液(OpTmizerTMCTSTMT-Cell Expansion Basal Medium，OpTmizerTMCTS T-Cell Expansion Supplement(Invitrogen)，500IU/mL的IL-2(双鹭药业))中培养，同时按每106个细胞加入25ul Dynabeads Human T-Activator CD3/CD28(Invitrogen)活化T细胞。
48小时(Day2)后，按MOI为3分别加入Lenti3-CD19-CAR、Lenti3-CD19-CAR-FOSB慢病毒载体进行转导，混匀后置于CO2培养箱孵育，4小时后补加适量的T细胞完全培养基进行培养。
慢病毒转导24小时后将转导后的细胞换入新鲜T细胞完全培养液，并调整活细胞密度为1.0-2.0×106/mL，继续培养扩增10～20天，每天进行观察和计数，并根据计得的细胞数量进行补液扩大培养，始终保持细胞培养密度为1.0-2.0×106/mL。
2、Anti-CD19 CAR-T、Anti-CD19-FOSB CAR-T细胞转导效率检测
取1.0×106个转导后T细胞，与1ug/mLFITC-Protein-L室温孵育30分钟，生理盐水清洗两次后，通过流式细胞仪检测FITC荧光信号，测量FITC阳性细胞比率，反映了CAR-T细胞在总细胞中的比率。Anti-CD19 CAR-T、Anti-CD19-FOSB CAR-T细胞转导效率检测结果如图5所示。图5表明成功制备了CAR-T细胞，而且Anti-CD19 CAR-T、Anti-CD19-FOSB CAR-T的CAR表达效率分别有28.7％、39.5％。
实施例5、Anti-CD19 CAR-T、Anti-CD19-FOSB CAR-T细胞体外功能检测
1.体外杀瘤检测：
采用钙黄绿素检测法分别对Anti-CD19 CAR-T、Anti-CD19 CAR-FOSB-T细胞进行体外杀瘤功能检测，靶细胞为CD19阳性的B急淋肿瘤细胞系Raji。
取适量的靶细胞，在1×106/mL的细胞悬液(PBS，5％胎牛血清)加入钙黄绿素-乙酰羟甲基酯(Calcein-AM)至终浓度25μM，培养箱中孵育30min。常温，洗两遍后将细胞重悬至0.5×105/mL，96孔板中每孔加入0.5×105/mL个细胞，按25:1的效靶比分别加入Anti-CD19 CAR-T、Anti-CD19 CAR-FOSB-T，37℃孵育2～3小时。孵育完成后取上清，测量其中钙黄绿素的荧光强度，并根据自发释放对照和最大释放对照，计算靶细胞裂解百分数。
Anti-CD19 CAR-T、Anti-CD19 CAR-FOSB-T细胞对CD19高表达的肿瘤细胞系Raji体外杀伤裂解结果如图6所示，
由以上体外杀瘤结果可知，Anti-CD19 CAR-T、Anti-CD19 CAR-FOSB-T中，Anti-CD19 CAR-FOSB-T的杀伤显著高于普通的Anti-CD19 CAR-T，所以更优选Anti-CD19 CAR-FOSB-T用于治疗肿瘤。
2.体外共孵育前后CAR-T细胞分型检测：
采用流式细胞术分别对Anti-CD19 CAR-T、Anti-CD19 CAR-FOSB-T细胞进行细胞分型检测，靶细胞为CD19阳性的B急淋肿瘤细胞系Raji。
分别取2ⅹ106个Anti-CD19 CAR-T和Anti-CD19 CAR-FOSB-T细胞，分成两组，一组加入T细胞完全培养液(OpTmizerTMCTSTMT-Cell Expansion Basal Medium，OpTmizerTMCTST-Cell Expansion Supplement(Invitrogen)，500IU/mL的IL-2(双鹭药业))中培养，调整细胞密度为1.0～2.0×106个/mL；另一组按照5：1的效靶比加入靶细胞，并加入T细胞完全培养液(OpTmizerTMCTSTMT-Cell Expansion Basal Medium，OpTmizerTMCTS T-CellExpansion Supplement(Invitrogen)，500IU/mL的IL-2(双鹭药业))中培养，调整细胞密度为1.0～2.0×106个/mL。置于CO2培养箱孵育48h。
孵育完成后使用BV421-CD45RA/PE-CCR7/BV650-CD45RO标记细胞来评价细胞分化水平，使用PE-CD25/BV421-CD69/BV650-CD71来评价细胞激活水平，使用PE-PD-1/BV421-TIM-3/BV650-LAG-3评价细胞耗竭水平。结果如图8所示：孵育前后Anti-CD19 CAR-FOSB-T的TN、TSCM比例低于Anti-CD19 CAR-T细胞，表明FOSB基因可以使CAR-T细胞向效应T细胞方向分化；激活标志物(CD25/CD69/CD71)高于Anti-CD19 CAR-T细胞，表明FOSB基因使CAR-T细胞强激活，与体外杀瘤实验结果一致。
3.体外细胞因子检测：
取适量的靶细胞，在1×106/mL的细胞悬液(PBS，5％胎牛血清)常温，洗两遍后将细胞重悬至0.5×105/mL，96孔板中每孔加入0.05×105/mL个靶细胞，按25:1的效靶比加入T、Anti-CD19 CAR-T、Anti-CD19 CAR-FOSB-T细胞，200g离心30秒，37℃孵育18小时。孵育完成后取上清，分别检测上清中IFN-γ、TNF-α和Granzyme B的浓度。
Anti-CD19 CAR-T、Anti-CD19 CAR-FOSB-T细胞与CD19高表达细胞系Raji体外孵育前后IFN-γ、TNF-α和Granzyme B分泌结果分别如图7所示，孵育前后CD19 CAR-FOSB-T细胞分泌的IFN-γ和Granzyme B与CD19 CAR-T细胞相比有所提高，说明FOSB在CAR-T细胞中的过表达影响CART细胞的杀瘤能力。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (9)
1.一种嵌合型抗原受体，从N端到C端顺次拼接信号肽、靶向CD19的单链抗体ScFv、strepII、CD8hinge、CD28跨膜区、CD28胞内结构域、胞内共刺激域4-1BB和CD3ζ链；在CD3ζ链的C端进一步拼接有P2A核苷酸序列和能够表达FOSB蛋白的核苷酸序列。
2.根据权利要求1所述的抗原受体，所述单链抗体ScFv的氨基酸序列如SEQ ID NO.1所示。
3.根据权利要求1所述的抗原受体，所述信号肽的氨基酸序列如SEQ ID NO.3；CD8hinge的氨基酸序列如SEQ ID NO.5；CD28跨膜区、CD28胞内结构域的氨基酸序列分别如SEQ ID NO.7、SEQ ID NO.9所示；胞内共刺激域4-1BB的氨基酸序列如SEQ ID NO.11；CD3ζ的氨基酸序列如SEQ ID NO.13所示。
4.根据权利要求1所述的抗原受体，所述P2A蛋白的氨基酸序列如SEQ ID NO.15所示；所述FOSB蛋白的氨基酸序列如SEQ ID NO.17所示。
5.一种重组嵌合型抗原受体的基因载体，其特征在于，以PTK881-EF1α载体为骨架，插入权利要求1-4任意一项所述的嵌合型抗原受体编码核苷酸序列的慢病毒、逆转录病毒或转座子载体。
6.一种表达嵌合型抗原受体的免疫细胞，其特征在于，由权利要求1-4任意一项所述嵌合型抗原受体的编码核苷酸序列或权利要求5所述重组嵌合型抗原受体基因载体转染免疫细胞得到，所述免疫细胞选自脐带血、外周血、肿瘤组织或iPSC来源的T细胞、NK细胞、NKT细胞、αβT细胞、γδT细胞、CD4+T细胞，CD8+T细胞。
7.权利要求6所述的免疫细胞的用途，所述免疫细胞用于制备治疗表达肿瘤抗原的肿瘤治疗，更优选的，免疫细胞用于治疗表达CD19抗原的肿瘤治疗。
8.根据权利要求7所述的应用，所述肿瘤选自急性淋巴细胞白血病或弥漫性大B细胞淋巴瘤。
9.权利要求7所述的免疫细胞的用途，所述免疫细胞用于过表达FOSB和分泌颗粒酶B。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2023101691805 | 2023-02-27 | ||
| CN202310169180 | 2023-02-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116589598A true CN116589598A (zh) | 2023-08-15 |
Family
ID=87598620
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310555126.4A Pending CN116589598A (zh) | 2023-02-27 | 2023-05-17 | 共表达fosb的嵌合抗原受体及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116589598A (zh) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107312091A (zh) * | 2017-05-02 | 2017-11-03 | 重庆精准生物技术有限公司 | 靶向人cd19抗原的人源化单克隆抗体 |
| US20200216534A1 (en) * | 2017-06-08 | 2020-07-09 | H. Lee Moffitt Cancer Center And Research Institute, Inc. | Chimeric antigen receptors with enhanced nfkb signaling |
| CN111773215A (zh) * | 2020-07-30 | 2020-10-16 | 曾辉 | 一种治疗aml的药物及应用 |
| CN114555788A (zh) * | 2019-09-06 | 2022-05-27 | 阿维塔斯有限公司 | 免疫细胞工程化用于体外细胞治疗 |
-
2023
- 2023-05-17 CN CN202310555126.4A patent/CN116589598A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107312091A (zh) * | 2017-05-02 | 2017-11-03 | 重庆精准生物技术有限公司 | 靶向人cd19抗原的人源化单克隆抗体 |
| US20200216534A1 (en) * | 2017-06-08 | 2020-07-09 | H. Lee Moffitt Cancer Center And Research Institute, Inc. | Chimeric antigen receptors with enhanced nfkb signaling |
| CN114555788A (zh) * | 2019-09-06 | 2022-05-27 | 阿维塔斯有限公司 | 免疫细胞工程化用于体外细胞治疗 |
| CN111773215A (zh) * | 2020-07-30 | 2020-10-16 | 曾辉 | 一种治疗aml的药物及应用 |
Non-Patent Citations (1)
| Title |
|---|
| DANIEL E. SPEISER等: "Regulatory circuits of T cell function in cancer", NATURE REVIEWS, vol. 16, 16 August 2016 (2016-08-16), pages 599 - 611 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112142854B (zh) | 免疫调节特异性嵌合抗原受体细胞及制备方法和应用 | |
| CN111909966B (zh) | 一种制备经修饰的免疫细胞的方法 | |
| CN110856751A (zh) | 包含核酸及tcr修饰的免疫细胞的治疗剂及其应用 | |
| CN110684120B (zh) | 一种靶向gpc3的嵌合抗原受体及其应用 | |
| JP2021536435A (ja) | 核酸とｃａｒ修飾免疫細胞とを含む治療薬およびその使用 | |
| CN109320615A (zh) | 靶向新型bcma的嵌合抗原受体及其用途 | |
| CN108395478A (zh) | 靶向bcma的嵌合抗原受体并对其双重修饰的方法及其用途 | |
| CN110590960B (zh) | 以cd99为靶点的嵌合抗原受体及其应用 | |
| CN111196858B (zh) | 一种治疗血液肿瘤合并hiv感染的双特异性嵌合抗原受体、基因、构建方法及其应用 | |
| CN113896801B (zh) | 靶向人Claudin18.2和NKG2DL的嵌合抗原受体细胞及其制备方法和应用 | |
| WO2016057986A1 (en) | Tandem epitope constructs for presentation of cd4 and cd8 epitopes and uses thereof | |
| CN113416260A (zh) | 靶向Claudin18.2的特异性嵌合抗原受体细胞及其制备方法和应用 | |
| CN116120451A (zh) | 抗cd70内化的抗体、抗体偶联物及其应用 | |
| CN109021114A (zh) | 联合两种单链抗体的双特异性嵌合抗原受体及表达载体 | |
| CN111378624B (zh) | 一种靶向性抗肿瘤t细胞及其制备方法和应用 | |
| CN109666074B (zh) | 一种趋化因子受体cxcr5的用途 | |
| 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 | |
| CN102532269B (zh) | γδT淋巴细胞中δ1链互补决定域3的优势序列及其TCR受体转染细胞与应用 | |
| CN111978412B (zh) | 武装靶向TGF-β的特异性嵌合抗原受体细胞及其制备方法和应用 | |
| CN110615847A (zh) | TCRγδ为靶点的嵌合抗原受体及其应用 | |
| CN116589598A (zh) | 共表达fosb的嵌合抗原受体及其应用 | |
| CN113736742B (zh) | Prtn3基因作为肿瘤免疫治疗中激活细胞毒性免疫细胞靶点的应用 | |
| CN114573713A (zh) | 以ifngr1为靶点的嵌合抗原受体及其应用 | |
| CN116514992B (zh) | 一种信号肽序列优化的靶向cd19的嵌合抗原受体及其应用 | |
| CN116813794A (zh) | 共表达spi1的嵌合抗原受体及其应用 |
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 |