CN105177031A - Chimeric antigen receptor-modified T cells and uses thereof - Google Patents
Chimeric antigen receptor-modified T cells and uses thereof Download PDFInfo
- Publication number
- CN105177031A CN105177031A CN201510324558.XA CN201510324558A CN105177031A CN 105177031 A CN105177031 A CN 105177031A CN 201510324558 A CN201510324558 A CN 201510324558A CN 105177031 A CN105177031 A CN 105177031A
- Authority
- CN
- China
- Prior art keywords
- cell
- seqidno
- car
- nucleotide sequence
- antigen receptor
- 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.)
- Granted
Links
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 55
- 239000000427 antigen Substances 0.000 title abstract description 18
- 102000036639 antigens Human genes 0.000 title abstract description 18
- 108091007433 antigens Proteins 0.000 title abstract description 18
- 239000002773 nucleotide Substances 0.000 claims abstract description 43
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 43
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims abstract description 27
- 230000003834 intracellular effect Effects 0.000 claims abstract description 17
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 12
- 241000700605 Viruses Species 0.000 claims description 33
- 108020004707 nucleic acids Proteins 0.000 claims description 19
- 102000039446 nucleic acids Human genes 0.000 claims description 19
- 150000007523 nucleic acids Chemical class 0.000 claims description 19
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 11
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 8
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 7
- 206010028980 Neoplasm Diseases 0.000 claims description 7
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 5
- 102100022132 High affinity immunoglobulin epsilon receptor subunit gamma Human genes 0.000 claims description 3
- 108091010847 High affinity immunoglobulin epsilon receptor subunit gamma Proteins 0.000 claims description 3
- 101000679851 Homo sapiens Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 claims description 3
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 claims description 3
- 239000013600 plasmid vector Substances 0.000 claims description 3
- 230000001177 retroviral effect Effects 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 79
- 238000000034 method Methods 0.000 description 20
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 12
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 11
- 230000009466 transformation Effects 0.000 description 11
- 210000004698 lymphocyte Anatomy 0.000 description 9
- 230000006907 apoptotic process Effects 0.000 description 8
- 210000001185 bone marrow Anatomy 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000013467 fragmentation Methods 0.000 description 7
- 238000006062 fragmentation reaction Methods 0.000 description 7
- 238000012239 gene modification Methods 0.000 description 7
- 230000005017 genetic modification Effects 0.000 description 7
- 235000013617 genetically modified food Nutrition 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 238000005336 cracking Methods 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 108090000695 Cytokines Proteins 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 238000004806 packaging method and process Methods 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 230000011664 signaling Effects 0.000 description 4
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 3
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 3
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 description 3
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 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 3
- -1 carbonyl cyanine Chemical compound 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000000139 costimulatory effect Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 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 3
- 239000012634 fragment Substances 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 230000001665 lethal effect Effects 0.000 description 3
- 229960002378 oftasceine Drugs 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241001430294 unidentified retrovirus Species 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 2
- 240000007019 Oxalis corniculata Species 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- BQRGNLJZBFXNCZ-UHFFFAOYSA-N calcein am Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=C(OC(C)=O)C=C1OC1=C2C=C(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(=O)C)C(OC(C)=O)=C1 BQRGNLJZBFXNCZ-UHFFFAOYSA-N 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000022534 cell killing Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000007421 fluorometric assay Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 231100000652 hormesis Toxicity 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000000527 lymphocytic effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 108010082808 4-1BB Ligand Proteins 0.000 description 1
- 108010083359 Antigen Receptors Proteins 0.000 description 1
- 102000006306 Antigen Receptors Human genes 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- 208000011691 Burkitt lymphomas Diseases 0.000 description 1
- 206010050685 Cytokine storm Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 101100508818 Mus musculus Inpp5k gene Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 101100366438 Rattus norvegicus Sphkap gene Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- 108010083312 T-Cell Antigen Receptor-CD3 Complex Proteins 0.000 description 1
- 102100032101 Tumor necrosis factor ligand superfamily member 9 Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000000448 cultured tumor cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 206010052015 cytokine release syndrome Diseases 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000009126 molecular therapy Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 108700001624 vesicular stomatitis virus G Proteins 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- 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]
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- 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
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/48—Blood cells, e.g. leukemia or lymphoma
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides chimeric antigen receptor-modified T cells and uses thereof, wherein the chimeric antigen receptor contains a signal peptide, an extracellular binding region, an optional hinge region, a transmembrane region and an intracellular signal region, the signal peptide, the extracellular binding region, the optional hinge region, the transmembrane region and the intracellular signal region are sequentially connected, and the nucleotide sequence of the extracellular binding region is represented by SEQ ID No:4 or SEQ ID No:5.
Description
Technical field
The present invention relates to immunology and biology field, relate more specifically to T cell of Chimeric antigen receptor modification and uses thereof.
Background technology
Major part has the patient of B cell malignant tumour-comprise chronic lymphocytic leukemia (ChronicLymphocyticLeukemia, CLL), use the curative ratio of chemotherapy, targeted therapy and prognosis all very poor.A method for the treatment of these patients be genetic modification T cell with the expression by Chimeric antigen receptor (ChimericAntigenReceptor, CAR), the antigen that target is expressed on tumour cell.CAR is designed to the antigen receptor with the mode identification cell-surface antigens of human leucocyte antigen (HLA)-dependent/non-dependent.The trial utilizing the T cell of genetic modification expressing CAR to treat these types patient success (MolecularTherapy, 2010,18:4,666-668 to a certain extent; Blood, 2008,112:2261-2271).
Along with the development of Chimeric antigen receptor T cell (ChimericAntigenReceptor-Tcell, CAR-T) technology, current CAR-T mainly can be divided into three generations.
First-generation CAR-T cell is by born of the same parents outer land---single-chain antibody (single-chainfragmentvariable, scF
v), cross-film district (transmembraneregion, and intracellular signal district---immunoreceptor tyrosine-based activation motif (immunoreceptortyrosine-basedactivationmotif TM), ITAM) form, wherein Chimeric antigen receptor each several part connects by following form: scFv-TM-CD3 ζ.This kind of CAR-T cell can excite antitumoral cytotoxic effect, but cytokine secretion is fewer, and lasting anti-tumour effect [ZhangT.et.al.ChimericNKG2D-modifiedTcellsinhibitsystemic T-celllymphomagrowthinamannerinvolvingmultiplecytokinesa ndcytotoxicpathways can not be excited in vivo, CancerRes2007,67 (22): 11029-11036.].
The s-generation CAR-T cell developed subsequently adds the intracellular signal district of CD28 or CD137 (having another name called 4-1BB), and wherein Chimeric antigen receptor each several part connects by following form: scFv-TM-CD28-ITAM or scFv-TM-CD137-ITAM.B7/CD28 or 4-1BBL/CD137 that intracellular signal district occurs altogether hormesis causes the continuous proliferation of T cell, and the level of the cytokines such as T cell secretion IL-2 and IFN-γ can be improved, improve CAR-T time to live in vivo and antitumous effect [DottiG.et.al.CD28costimulationimprovesexpansionandpersis tenceofchimericantigenreceptormodifiedTcellsinlymphomapa tients.JClinInvest simultaneously, 2011,121 (5): 1822-1826.].
The third generation CAR-T cell developed in recent years, wherein Chimeric antigen receptor each several part connects by following form: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further increase CAR-T time to live in vivo and its antitumous effect [CarpenitoC., etal.Controloflargeestablishedtumorxenograftswithgenetic allyretargetedhumanTcellscontainingCD28andCD137domains.P NAS, 2009,106 (9): 3360-3365.].
Although CAR-T cell has tempting prospect in immunotherapy of tumors, some potential risks also need to consider.Such as, due to some/kind of the healthy tissues low expression CAR specific antigens that can identify may cause CAR-T cell to the damage of the healthy tissues of expression corresponding antigens.In addition, costimulatory signal too much in CAR can reduce effector cell and activate required threshold value, make the T cell of genetic modification low-level antigen or do not have antigen to trigger condition under also may be activated, cause the release of a large amount of cytokine so that may cause so-called " cytokine storm ".This signal leak outside (signalleakage) can cytotoxicity of missing the target be caused, thus produce nonspecific tissue injury.
Composition and the method for the treatment of human cancer is disclosed at present in number of patent application 201180067173.X, this invention comprises the T cell relating to and use genetic modification, to express CAR, wherein CAR comprises antigen binding domain (that is, the outer land of born of the same parents), trans-membrane region, costimulatory signal conducting region and CD3 ζ intracellular signaling district.But the antigen binding domain scFv right and wrong of this CAR-T cell surface humanized (mouse source), this causes CAR-T cell itself to have immunogenicity.
In summary, this area also exists the tight demand to the CAR-T cell overcoming above-mentioned defect.
Summary of the invention
A first aspect of the present invention provides the nucleic acid molecule that coding is expressed in the Chimeric antigen receptor on T cell surface, described Chimeric antigen receptor comprise be linked in sequence signal peptide, the outer land of born of the same parents, optional hinge area (hingeregion), cross-film district and intracellular signal district (signalregion), the nucleotide sequence of the outer land of wherein said born of the same parents is as shown in SEQIDNo:4 or SEQIDNo:5.
A second aspect of the present invention provides the carrier being expressed in the nucleic acid molecule of the Chimeric antigen receptor on T cell surface comprising first aspect present invention.
A third aspect of the present invention provides the virus of the carrier comprising second aspect present invention, the virus with infection ability after it comprises packaging, also comprise be packaged as the infectious virus institute of tool must the virus to be packaged of composition.
A fourth aspect of the present invention provides the T cell of genetic modification, and it comprises the virus of the nucleotide sequence of first aspect present invention or the carrier of second aspect present invention or third aspect present invention.
A fifth aspect of the present invention provides the medicine of the T cell comprising fourth aspect present invention.
A sixth aspect of the present invention provides the purposes of T cell for the preparation of the medicine for the treatment of tumour of fourth aspect present invention.
The antigen binding domain scFv of CAR-T cell surface of the present invention is through humanization modified, and therefore its immunogenicity reduces greatly.
Accompanying drawing explanation
According to the detailed description of carrying out referring to accompanying drawing, above and other aspects, features and advantages of the present invention can become clearly.
Fig. 1 shows the detected result expressing the T cell that Chimeric antigen receptor is modified.
Fig. 2 shows the result marked CAR-T19 cell with carbonyl cyanine dye Did.
Fig. 3 shows the result marked Daudi cell with carbonyl cyanine dye Did.
Fig. 4 shows by the result of Flow cytometry CAR-T19 cell to Daudi killing functions of immunocytes.
Fig. 5 shows T cell and CAR-T19 cell to the comparative result of the fragmentation effect of target cell Daudi.
Fig. 6 shows CAR-T19 containing different scFv region to the comparative result of the fragmentation effect of target cell Daudi.
Fig. 7 shows the result of the different CAR-T19 of vitro detection to the lethal effect of bone-marrow-derived lymphocyte leukaemic cell.
Embodiment
As mentioned above, a first aspect of the present invention provides the nucleic acid molecule that coding is expressed in the Chimeric antigen receptor on T cell surface, described Chimeric antigen receptor comprise be linked in sequence signal peptide, the outer land of born of the same parents, optional hinge area, cross-film district and intracellular signal district, the nucleotide sequence of the outer land of wherein said born of the same parents is as shown in SEQIDNo:4 or SEQIDNo:5.
Term of the present invention " nucleic acid " can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-strand.DNA can be coding strand or noncoding strand.The invention still further relates to the varient of described nucleic acid, its coding has the polypeptide of identical aminoacid sequence or fragment, the sum analogous to general Dedekind sum of polypeptide with the present invention.The varient of described nucleic acid can be the allelic variant of natural generation or the varient of non-natural generation.These nucleic acid variants comprise and replace varient, Deletion variants and insertion varient.As would be known to one of ordinary skill in the art, allelic variant is a kind of replacement form of polynucleotide, and it may be the replacement of one or more Nucleotide, disappearance or insertion, but can not from the function of polypeptide changing in fact its coding.
In one embodiment, described signal peptide is GMCSF signal peptide, and its nucleotide sequence is as shown in SEQIDNo:1
In one embodiment, described signal peptide is CD8 signal peptide, and its nucleotide sequence is as shown in SEQIDNo:2.
In one embodiment, described hinge area can be selected from the hinge area of the albumen such as CD8 or CD28.Described CD8 or CD28 is the native marker thing on T cell surface.
In a specific embodiment, described hinge area is CD8 hinge area (CD8-hinge), and its nucleotide sequence is as SEQIDNo:6.
In a specific embodiment, described hinge area is CD28 hinge area (CD28-hinge), and its nucleotide sequence is as shown in SEQIDNo:7.
In one embodiment, described cross-film district can be selected from the cross-film district of the albumen such as CD8 or CD28.
In a specific embodiment, described cross-film district is CD8 cross-film district (CD8-TM), and its nucleotide sequence is as shown in SEQIDNo:8.
In a specific embodiment, described cross-film district is CD28 cross-film district (CD28-TM), and its nucleotide sequence is as shown in SEQIDNo:9.
Described " the outer land of born of the same parents " comprises the scFv (anti-CD19scFv) of specific recognition people CD19 epi-position.
Term used herein " scFv " refers to such antibody fragment---it comprises the variable region of heavy chain (variableregionofheavychain connected by joint (linker), and variable region of light chain (variableregionoflightchain VH), VL) recombinant protein, joint makes these two structural domains be associated, finally to form antigen binding site.The size of scFv is generally 1/6 of a complete antibody.The aminoacid sequence that scFv is preferably encoded by a nucleotide chain.The scFv that the present invention uses is by being used alone or in combination routine techniques known in the art, and such as aminoacid deletion, insertion, replacement, increase and/or restructuring and/or other modifying method are done to modify further.A kind of method introducing this modification according to aminoacid sequence of antibody in its DNA sequence dna is known (see such as a person skilled in the art, Sambrook molecular cloning: laboratory manual, ColdSpringHarborLaboratory (1989) N.Y.).Described modification is preferably carried out in nucleic acid level.Above-mentioned scFv can also comprise its derivative.
Term used herein " specific recognition " mean bi-specific antibody of the present invention not with or substantially not with target antigen beyond any polypeptide cross reaction.Its specific degree can be judged by immunological technique, includes but not limited to immunoblotting, immunoaffinity chromatography, flow cytometry etc.
In one embodiment, described intracellular signal district can be selected from the intracellular signal district of CD3 ζ, Fc ε RI γ, CD28, CD137, CD134 albumen, and combination.CD3 molecule is made up of five subunits, and wherein containing 3 ITAM motifs, this motif is signal zone of transformation important in TCR-CD3 complex body in CD3 ζ subunit (also known as CD3zeta, being called for short Z).CD3 δ Z is the CD3 ζ sequence without ITAM motif of sudden change, the general structure component as negative control in an embodiment of the present invention.Fc ε RI γ is mainly distributed in mastocyte and basophil cellular surface, and it contains an ITAM motif, in structure, distribution and functionally similar with CD3 ζ.In addition as previously mentioned, CD28, CD137, CD134 are costimulatory signal molecules, with respective ligand binding after the common hormesis that produces of its intracellular signal section cause the continuous proliferation of T cell, and the level of the cytokines such as T cell secretion IL-2 and IFN-γ can be improved, improve CAR-T cell time to live in vivo and antitumous effect simultaneously.
The present invention use intracellular signal district have multiple combination, it comprise be selected from following signaling zone or its combination:
CD28 signaling zone (CD28-signal), its nucleotide sequence is as shown in SEQIDNo:10;
CD137 signaling zone (CD137-signal), its nucleotide sequence is as shown in SEQIDNo:11; With
CD3 ζ signaling zone (CD3 ζ-signal), its nucleotide sequence is as shown in SEQIDNo:12.
In a specific embodiment, the Chimeric antigen receptor albumen being expressed in T cell surface of the present invention be selected from following comprise be linked in sequence signal peptide, the outer land of born of the same parents, optional hinge area, cross-film district and intracellular signal district Chimeric antigen receptor albumen:
GMCSF – scFv-S1 – CD8-hinge – CD8-TM – CD137-signal – CD3 ζ-signal, its nucleotide sequence is as shown in SEQIDNo:13;
GMCSF – scFv-S2 – CD8-hinge – CD8-TM – CD137-signal – CD3 ζ-signal, its nucleotide sequence is as shown in SEQIDNo:14;
GMCSF – scFv-S1 – CD28-TM – CD28-signal – CD3 ζ-signal, its nucleotide sequence is as shown in SEQIDNo:15;
GMCSF – scFv-S2 – CD28-TM – CD28-signal – CD3 ζ-signal, its nucleotide sequence is as shown in SEQIDNo:16;
GMCSF – scFv-S1 – CD8-hinge – CD8-TM – CD28-signal – CD137-signal – CD3 ζ-signal, its nucleotide sequence is as shown in SEQIDNo:17;
GMCSF – scFv-S2 – CD8-hinge – CD8-TM – CD28-signal – CD137-signal – CD3 ζ-signal, its nucleotide sequence is as shown in SEQIDNo:18;
As mentioned above, a second aspect of the present invention provides the carrier being expressed in the nucleic acid molecule of the Chimeric antigen receptor on T cell surface comprising first aspect present invention.
In a specific embodiment, the carrier that the present invention uses is a kind of slow virus plasmid vector pLenti-CMV-eGFP.This plasmid belongs to the third generation from deactivation slow virus carrier system, and this system has the packaging plasmid psPAX2 of three plasmids and proteins encoded Gag/Pol, coding Rev albumen; The envelope plasmid pVSVG of coding vesicular stomatitis virus-G protein; And empty carrier pLenti-CMV-eGFP, it may be used for restructuring and introduces object nucleotide sequence, the nucleotide sequence of the CAR that namely encodes.By elongation factor-1 α (elongationfactor-1 α in empty carrier pLenti-CMV-eGFP (itself being the mock in follow-up test), EF-1 α) expression of promoter regulation enhanced green fluorescence protein (enhancedgreenfluorescentprotein, eGFP).And the recombinant expression vector pLenti-CMV-eGFP comprising the object nucleotide sequence of coding CAR is by realizing the coexpression of eGFP and CAR by the ribosomal skip sequence (ribosomalskippingsequence2A) (being called for short F2A) from foot and mouth disease virus (foodandmouthviresdisease, FMDV).
In another embodiment, the carrier that the present invention uses is a kind of retroviral vector pMSCV-Ubc-GFP, and this plasmid is used for process LAN object nucleotide sequence, the nucleotide sequence of the CAR that namely encodes.
As mentioned above, a third aspect of the present invention provides the virus of the carrier comprising second aspect present invention, the virus with infection ability after it comprises packaging, also comprise be packaged as the infectious virus institute of tool must the virus to be packaged of composition.
In one embodiment, described virus is the slow virus of the pLenti-CMV-eGFP-F2A-CAR recombinant vectors comprising second aspect present invention.In another embodiment, described virus is the retrovirus of the pMSCV-Ubc-GFP-CAR recombinant vectors comprising second aspect present invention.Should be understood that the virus of other transfecting T cells as known in the art and the plasmid vector of correspondence thereof also can be used for the present invention.
As mentioned above, a fourth aspect of the present invention provides the T cell of genetic modification, wherein transforms the virus having the nucleotide sequence of first aspect present invention or the carrier of second aspect present invention or third aspect present invention.
The nuclear transformation method of this area routine---comprise non-viral and viral method for transformation and may be used to the present invention.Electroporation and transposon method is comprised based on non-viral method for transformation.Foreign gene transfered cell core directly can be obtained the Efficient Conversion of goal gene by the nucleofector consideration convey dye instrument of recent Amaxa company research and development.In addition, the more common electroporation of transformation efficiency based on Transposon Systems such as sleeping beauty transposon stand (SleepingBeautysystem) or PiggyBac transposons improves a lot, nucleofector transfection instrument and the system combined application of SB sleeping beauty transposon stand are had been reported [DaviesJK., etal.CombiningCD19redirectionandalloanergizationtogenera tetumor-specifichumanTcellsforallogeneiccelltherapyofB-c ellmalignancies.CancerRes, 2010, 70 (10): OF1-10.], the method can either have the site-directed integration that higher transformation efficiency can realize again goal gene.
In one embodiment of the invention, the lymphocytic method for transformation of T realizing Chimeric antigen receptor genetic modification is based on the method for transformation of virus as retrovirus or slow virus.It is high that the method has transformation efficiency, and foreign gene can stably express, and can shorten vitro culture T lymphocyte and arrive the advantages such as the time of clinical grade quantity.At this transgenosis T lymphocytic cell surface, the nucleic acid of conversion by transcribing, accurate translation thereon.Prove the cell in vitro poison experiment that various different cultured tumor cells carries out, the transgenosis T lymphocyte of surface expression Chimeric antigen receptor of the present invention has the tumor cytotoxicity effect (also known as cytotoxicity) of high degree of specificity.Therefore the nucleic acid molecule of encoding chimeric antigen acceptor of the present invention, the plasmid comprising this nucleic acid, the virus comprising this plasmid and conversion have the transgenosis T lymphocyte of above-mentioned nucleic acid, plasmid or virus can effectively for the immunotherapy of tumour.
As mentioned above, a fifth aspect of the present invention provides the medicine of the T cell comprising fourth aspect present invention.
In one embodiment, described medicine also comprises pharmaceutically useful thinner, vehicle or carrier etc.
As mentioned above, a sixth aspect of the present invention provides the purposes of T cell for the preparation of the medicine for the treatment of tumour of fourth aspect present invention.
Below by way of specific embodiment, content of the present invention is described.Should be understood that described specific embodiment only for the purpose of illustration, and do not mean that content of the present invention is only limitted to specific embodiment.
The preparation of the nucleotide sequence of embodiment 1:CAR molecule
CAR molecule of the present invention comprises signal peptide, the outer land of born of the same parents, optional hinge area, cross-film district and intracellular signal district.The nucleotide sequence of the outer land of described born of the same parents is that (at this called after anti-CD19scFv-S0, referred to as scFv-S0, it derives from mouse, see JImmunother.2009September for the antigen binding domain of the Chimeric antigen receptor at anti-CD19; 32 (7): 689 – 702.) nucleotide sequence (SEQIDNo:3) basis on carry out humanization modified and obtain.Should be understood that the principle that antibody humanization transforms is while guarantee affinity of antibody, skeleton district (frameworkregion, FM) is changed for people's source sequence to greatest extent, to reduce the immunogenicity of antibody.In this embodiment, antigen recognition district in SEQIDNo:3 is remained unchanged, remaining sequence is changed accordingly, carry out the humanization design more than 40 kinds, obtain these sequences by the method synthesis of gene chemical synthesis again, other parts of CAR molecule are all utilize round pcr, clone respectively and obtain from human cDNA library, then carry out bridging to connect, finally prepare the nucleotide sequence of CAR molecule.These CAR molecules are proceeded to T cell, compare comprising containing the T cell kill capability to target cell of these T cell through the CAR molecule of humanization modified sequence with the CAR molecule comprised containing scFv-S0, final screening obtains two kinds through humanization modified born of the same parents outer land sequence---and the kill rate comprising the CAR-T19 of these two kinds of humanization modified sequences omits good than the kill rate of the CAR-T19 comprising scFv-S0 or does not have with it difference (Fig. 6 see in embodiment 4), it is called anti-CD19scFv-S1 (referred to as scFv-S1, its nucleotide sequence is as shown in SEQIDNo:4) and anti-CD19scFv-S2 (referred to as scFv-S2, its nucleotide sequence is as shown in SEQIDNo:5).Below for SEQIDNo:15 and SEQIDNo:16, illustrate the preparation process of the nucleotide sequence of CAR molecule.
First carry out design of primers, the primer sequence used in the present embodiment is as follows:
1-1：5’-ATGCTTCTCCTGGTGACAAG-3’
1-2：5’-TGGGATCAGGAGGAATGCTG-3’
2-1：5’-TACATCTGGGCGCCCTTGGCCGG-3’
2-2：5’-GGAGCGATAGGCTGCGAAGTCGCG-3’
3-1：5’-AGAGTGAAGTTCAGCAGGAGCG-3’
3-2：5’-TTAGCGAGGGGGCAGGGCCT-3’
4-1：5’-ATGCTTCTCCTGGTGACAAGCC-3’
4-2：5’-TGAGGAGACGGTGACTGAGGTTCCTTGG-3’
5-1：5’-GCGGCCGCAATTGAAGTTATGTA-3’
5-2：5’-TTAGCGAGGGGGCAGGGCCTGC-3’
6-1：5’-CTAGACTAGTATGCTTCTCCTGGTGACAAGCC-3’
6-2：5’-CGACGCGTTTAGCGAGGGGGCAGGGCCTGC-3’
With the cDNA library of people for template, respectively with 1-1 and 1-2,2-1 and 2-2,3-1 and 3-2 for primer, clone corresponding CAR molecular moiety by PCR, be respectively GMCSF, CD28-TM+CD28-signal (these two portions are connected) and CD3 ζ-signal.GMCSF+scFv fragment is obtained again by bridging primer 4-1 and 4-2, CD28-TM+CD28-signal+CD3 ζ-signal fragment is obtained by bridging primer 5-1 and 5-2, obtained the nucleotide sequence of complete CAR molecule subsequently by bridging primer 6-1 and 6-2, restriction enzyme site is SpeI and MluI.
Embodiment 2: the structure comprising the virus vector of the nucleotide sequence of CAR molecule
By the nucleotide sequence of the CAR molecule of preparation in embodiment 1 through SpeI (Fermentas) and MluI (Fermentas) double digestion, the SpeI-MluI site being connected insertion slow virus pLenti-CMV-eGFP carrier through T4 ligase enzyme (Fermentas), be transformed into competence E.coli (DH5 α), through checking order correctly, the plasmid purification kit of Qrigene company is used to extract and plasmid purification, for follow-up slow virus Packaging experimentation.
The structure of embodiment 3:CAR-T cell
In this embodiment, the recombinant viral vector of preparation is first made in embodiment 2 to be packaged into virus, then by T cell that this virus infection T cell is modified to realize Chimeric antigen receptor.Present invention employs based on the method for transformation of virus as retrovirus or slow virus.It is high that the method has transformation efficiency, and foreign gene can stably express, and can shorten culturing T cells in vitro and arrive the advantages such as the time of clinical grade quantity.Concrete steps are as follows:
Slow virus is packed: first day: inoculation 293T cell is in 10cm culture dish, and during guarantee transfection in second day, cell confluency degree is 80-90%; Second day: transfection, before transfection, 1h was replaced by 5mL serum-free DMEM high glucose medium the substratum of 293T; In EP pipe, add 500 μ LDMEM, add 4 μ gpR8.74,400ngpVSVG subsequently, 4 μ gpLenti-CMV-eGFP-F2A-CAR vortex oscillation 8s mix; Add the PEI (1 μ g/ μ L, totally 25.2 μ L) of plasmid (pR8.74+pLenti-CMV-eGFP-F2A-CAR) 3 times of volumes, vortex oscillation 20s mixes, and room temperature leaves standstill 15min; Mixed dropwise is added in 293T cell; After transfection 6h, substratum is replaced by the full substratum containing 10%FBS.After transfection 48h, collecting cell culture supernatant, 4 DEG C of centrifugal 15min of 3000rpm, get supernatant, with 0.45 μm of PVDF membrane filtration virus liquid, are sub-packed in EP pipe, are stored in-80 DEG C, to wait to infect T cell.
Recombinant slow virus infects T cell: the CD8 be separated from healthy human peripheral blood
+t lymphocyte, cultivate after activating and infect T cell with the above-mentioned recombinant slow virus of MOI=5, metainfective cell every other day adopts 2 × 10
5the density of/mL goes down to posterity.The CD8 infected
+t lymphocyte detects variant Chimeric antigen receptor and expresses when cultivation the 7th day, due to eGFP and CAR coexpression, detect the positive cell of eGFP be express Chimeric antigen receptor positive cell (result as shown in Figure 1, left figure is cell representative graph under light microscopic, right figure is the right figure of eGFP luciferase expression figure, left figure is the same visual field).
Embodiment 4: the structure of killing experiments in vitro model
Target cell selected by In vitro cell model is Daudi cell.Daudi clone derives from Burkitt lymphoma (Burkitt'slymphoma) patient, is a kind of typical B lymphoblast, is widely used in the research about mechanism such as leukemia generation and treatments in basis and preclinical test.Daudi cell high expression level B cell antigen CD19, can be killed and wounded by CAR-T19 (CAR-T of target CD19) cell recognition.Therefore, we utilize Daudi cell as target cell (T), CAR-T19 cell action effect cell (E), T cell cell (C) in contrast, construct the cell model of Cytotoxicity in vitro.This model simulates CAR-T19 cell or T cell in vivo to the leukemic therapeutic action of bone-marrow-derived lymphocyte in very high degree.
1) model of two kinds of monitoring Cell killing efficacy is set up
After T cell identification target cell, can make it that apoptosis occurs, and final cracking.Wherein late apoptic process and the cracking process of cell have and to overlap but not exclusively the same.Therefore we establish the model of two kinds of vitro detection Cell killing efficacy for apoptosis and cracking.
1-1) Flow cytometry target cell apoptosis
First, we utilize carbonyl cyanine dye Did to mark CAR-T19 cell, reach 99.17%, only have the CAR-T19 cell of 0.83% to be Did feminine gender (Fig. 2) by the cell of the Flow cytometry Did positive.
In addition, under same Testing index, the Daudi cell of 99.94% is Did feminine gender (Fig. 3).
Therefore, we first by Did in CAR-T19 cell marking, then hatch than with Daudi cytomixis according to certain ET, kill and wound; Then by the level of apoptosis of flow cytomery Did negative cells, acquired results can reflect that CAR-T19 cell kills and wounds level to Daudi cell.We utilize AnnexinV-FITC/PI to detect apoptosis, and what FITC/PI was double-negative is normal cell, and FITC is positive, PI is negative is viable apoptotic cell, and the two positive of FITC/PI is non-viable apoptotic cell.According to the ET ratio of 1:2, killing and wounding the apoptosis situation detecting Did negative cells after 4 hours, result is as shown in Figure 4: early apoptosis (LR=lowright) is 4.32%, and late apoptic (UR=upright) is 4.55%, and overall kill rate is 8.87%.
1-2) Fluorometric assay target cell lysis
Calcein-AM is that one can carry out fluorescently-labeled cell staining reagent to viable cell, because it strengthens hydrophobicity on the basis of Calcein, therefore, it is possible to penetrate membrane easily.After it enters into tenuigenin, can be that Calcein stays in cell by esterase hydrolyzed, thus send strong green fluorescence.We first by Calcein-AM on target cell Daudi mark, then utilize CAR-T19 cell to kill and wound it.After Daudi cell is killed and wounded also cracking by CAR-T19, Calcein can be discharged in supernatant liquor, detect the fluorescence intensity (TESTRELEASE) of supernatant, detect the fluorescence intensity (MAXIMUMRELEASE) of the maximum cracking of Daudi cell spontaneous apoptosis cracking (SPONTANEOUSRELEASE) and the Daudi cell that utilizes washing agent (TritonX-100 of 2%) to process simultaneously.Calculate the per-cent of lysis as follows: [(TESTRELEASE-SPONTANEOUSRELEASE)/(MAXIMUMRELEASE – SPONTANEOUSRELEASE)] * 100.
As shown in table 1, when ET is 1:2, killing and wounding the cleavage rate of DAUDI cell after 4 hours is 4.77%.The result of the basic late apoptic with being arrived by Flow cytometry of acquired results is similar.
Table 1 Fluorometric assay CAR-T19 cell is to the cleavage rate of DAUDI cell
2) the comparing of fragmentation effect of T cell and CAR-T19 cell
After establishing the model of cell in vitro fragmentation effect, we compare the CAR-T19 cell (comprising the CAR-T19 cell of GMCSF – scFv-S1 – CD28-TM – CD28-signal – CD3 ζ-signal or GMCSF – scFv-S2 – CD28-TM – CD28-signal – CD3 ζ-signal, respectively referred to as CAR-T19 (S1), CAR-T19 (S2)) of structure and do not have the T cell transformed to the fragmentation effect of target cell Daudi.Detecting step is with 1) described in, result as shown in Figure 5, under identical condition, the fragmentation effect of CAR-T19 cell improves at least 20 times (kill rate kill rate of T cell being set to 1, CAR-T19 (S1) and CAR-T19 (S2) is respectively 20.5,22).
3) comparison of the fragmentation effect of the CAR-T19 containing different scFv region
In the present embodiment, the CAR-T19 (CAR-T19 as above (S1), CAR-T19 (S2)) two kinds of the present invention being contained the outer land scFv (scFv-s1 and scFv-s2) of humanization modified born of the same parents compares the kill capability of target cell with containing the CAR-T19 (comprising the CAR-T19 of GMCSF – scFv-S0 – CD28-TM – CD28-signal – CD3 ζ-signal (its nucleotide sequence is as shown in SEQIDNo:19), referred to as CAR-T19 (S0)) without the outer land scFv (scFv-s0) of humanization modified born of the same parents.Concrete detecting step is with 1) described in, as shown in Figure 6, carry out humanization modification to scFv does not affect the killing-efficiency of CAR-T19 to target cell to result.
4) CAR-T19 that vitro detection is different is to the lethal effect of bone-marrow-derived lymphocyte leukaemic cell
In this embodiment, first the cell of bone-marrow-derived lymphocyte leukaemic is isolated, mainly be divided into two to walk greatly: the first step, from Bone Marrow of Patients, isolate PBMC (peripheral blood lymphocytes) (this step well known to a person skilled in the art); Second step, isolates B cell from PBMC.The concrete steps of described second step are as follows: mixed with the sheep red blood cell (SRBC) (SRBC) processed with the bromine flower different sulfohydrate of diamino (being called for short AET) by lymphocyte, wherein all T lymphocyte all can adsorb AET-SRBC to form firm stable and huge E-garland, the per-cent of E-garland that the per-cent of this E-garland is formed higher than T lymphocyte and normal untreated SRBC, and it is formed fast, difficult drop-off, reproducible.Then use lymphocytes separating solution to be separated, now AET-E-garland is easily sunken at the bottom of pipe, and bone-marrow-derived lymphocyte directly can take from the interface of layering liquid.This embodiment have detected CAR-T19 different in the present invention in vitro to the lethal effect of bone-marrow-derived lymphocyte leukaemic cell.Result is as shown in Figure 7: compared to the T cell without transformation, different CAR-T19 of the present invention (dividing subrepresentation with different CAR in the figure) kill capability to bone-marrow-derived lymphocyte leukaemic cell has remarkable enhancing.
Claims (10)
1. coding is expressed in the nucleic acid molecule of the Chimeric antigen receptor on T cell surface, described Chimeric antigen receptor comprise be linked in sequence signal peptide, the outer land of born of the same parents, cross-film district and intracellular signal district, the nucleotide sequence of the outer land of wherein said born of the same parents is as shown in SEQIDNo:4 or SEQIDNo:5.
2. the nucleic acid molecule of claim 1, wherein said Chimeric antigen receptor also comprises hinge area between land and cross-film district outside its born of the same parents.
3. the nucleic acid molecule of claim 2, the nucleotide sequence of wherein said signal peptide is as shown in SEQIDNo:1 or SEQIDNo:2, the nucleotide sequence of described hinge area is as shown in SEQIDNo:6 or SEQIDNo:7, the nucleotide sequence in described cross-film district is as shown in SEQIDNo:8 or SEQIDNo:9, described intracellular signal district is selected from the intracellular signal district of CD3 ζ, Fc ε RI γ, CD28, CD137, CD134 albumen, and combination, the nucleotide sequence in preferred described intracellular signal district is made up of SEQIDNo:10 and/or SEQIDNo:11 and/or SEQIDNo:12.
4. the nucleic acid molecule any one of claim 1-3, its sequence is as shown in SEQIDNo:13, SEQIDNo:14, SEQIDNo:15, SEQIDNo:16, SEQIDNo:17 or SEQIDNo:18.
5. carrier, it comprises the nucleic acid molecule any one of claim 1-4.
6. the carrier of claim 5, it is slow virus plasmid vector pLenti-CMV-eGFP or retroviral vector pMSCV-Ubc-GFP.
7. virus, it comprises the carrier of claim 5 or 6.
8.T cell, wherein transforms the nucleic acid molecule of having the right any one of requirement 1-4 or the carrier of claim 5 or 6 or the virus of claim 7.
9. comprise the medicine of the T cell of claim 8, preferred described medicine also comprises pharmaceutically useful thinner, vehicle or carrier.
10. the T cell of claim 8 is for the preparation of the purposes of medicine for the treatment of tumour.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510324558.XA CN105177031B (en) | 2015-06-12 | 2015-06-12 | T cell of Chimeric antigen receptor modification and application thereof |
| PCT/CN2015/096775 WO2016197570A1 (en) | 2015-06-12 | 2015-12-09 | Chimeric antigen receptor modified t-cell and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510324558.XA CN105177031B (en) | 2015-06-12 | 2015-06-12 | T cell of Chimeric antigen receptor modification and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105177031A true CN105177031A (en) | 2015-12-23 |
| CN105177031B CN105177031B (en) | 2018-04-24 |
Family
ID=54899458
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510324558.XA Active CN105177031B (en) | 2015-06-12 | 2015-06-12 | T cell of Chimeric antigen receptor modification and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN105177031B (en) |
| WO (1) | WO2016197570A1 (en) |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567640A (en) * | 2016-01-27 | 2016-05-11 | 苏州佰通生物科技有限公司 | Chimeric antigen receptor adipose-derived stem cell and preparation method thereof |
| CN105647946A (en) * | 2016-03-18 | 2016-06-08 | 苏州康元生物科技有限公司 | Fc gamma RIII a-based chimeric gene and application thereof |
| CN105753991A (en) * | 2015-11-26 | 2016-07-13 | 广州中科蓝华生物科技有限公司 | Chimeric antigen receptor resistant to placenta chondroitin sulfate and application of chimeric antigen receptor |
| CN106544321A (en) * | 2016-10-13 | 2017-03-29 | 北京艺妙神州医疗科技有限公司 | Universal CAR T cells and its production and use |
| CN107034193A (en) * | 2016-02-03 | 2017-08-11 | 北京马力喏生物科技有限公司 | Treat the therapeutic combination of B cell leukemia and B cell lymphoma |
| WO2017140256A1 (en) * | 2016-02-19 | 2017-08-24 | Huiru Wang | Antibodies against n-acetylglucosamine and n-acetyl-galactosamine |
| CN107226867A (en) * | 2017-07-25 | 2017-10-03 | 重庆精准生物技术有限公司 | The Chimeric antigen receptor of anti human CD 19 antigen and its application |
| CN107326014A (en) * | 2017-07-31 | 2017-11-07 | 时力生物科技（北京）有限公司 | A kind of T lymphocytes of bispecific chimeric antigen receptor modification and its preparation method and application |
| WO2018068257A1 (en) * | 2016-10-13 | 2018-04-19 | 北京艺妙神州医疗科技有限公司 | Universal car-t cell and preparation method and application therefor |
| CN109385402A (en) * | 2017-08-11 | 2019-02-26 | 上海恒润达生生物科技有限公司 | A kind of equal proportion mixes the preparation method and application of two kinds of target spot CART cells |
| CN109803983A (en) * | 2017-03-15 | 2019-05-24 | 南京凯地生物科技有限公司 | Target the specific chimeric antigen receptor T cell of NKG2DL, preparation method and application |
| WO2019129047A1 (en) * | 2017-12-28 | 2019-07-04 | 上海细胞治疗研究院 | Cd40 bidirectional-activated costimulatory molecule receptor, and use thereof |
| US10597461B2 (en) | 2014-08-22 | 2020-03-24 | B & H Biotechnologies, Llc | Saccharide-based biomarkers and therapeutics |
| CN114107214A (en) * | 2021-11-16 | 2022-03-01 | 东莞市麦亘生物科技有限公司 | Preparation method and application of universal allogenic CAR-T cells |
| WO2022121899A1 (en) | 2020-12-08 | 2022-06-16 | 北京艺妙神州医药科技有限公司 | Antibody specifically binding to strep-tag ii tag and use thereof |
| CN117247466A (en) * | 2023-11-20 | 2023-12-19 | 北京艺妙神州医药科技有限公司 | Chimeric antigen receptor against glypican 3 and uses thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111097043B (en) * | 2020-01-13 | 2023-07-04 | 广东昭泰体内生物医药科技有限公司 | Gastric cancer pharmaceutical composition and application thereof |
| CN113584083A (en) * | 2020-04-30 | 2021-11-02 | 深圳市深研生物科技有限公司 | Producer and packaging cells for retroviral vectors and methods for making the same |
| CN112626029B (en) * | 2020-12-22 | 2022-12-20 | 深圳市赛欧细胞技术有限公司 | Transgenic modified Daudi cell and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102775500A (en) * | 2012-08-03 | 2012-11-14 | 郑骏年 | Chimeric antigen receptor iRGD-scFv (G250)-CD8-CD28-CD137-CD3zeta and application thereof |
| CN103492406A (en) * | 2010-12-09 | 2014-01-01 | 宾夕法尼亚大学董事会 | Use of chimeric antigen receptor-modified t cells to treat cancer |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996030523A2 (en) * | 1995-03-31 | 1996-10-03 | Hans Wolf | Antigen presentation system based on retrovirus-like particles |
| WO2004027079A2 (en) * | 2002-09-19 | 2004-04-01 | Tanox, Inc. | Nuclear factor of activated t cells receptor |
-
2015
- 2015-06-12 CN CN201510324558.XA patent/CN105177031B/en active Active
- 2015-12-09 WO PCT/CN2015/096775 patent/WO2016197570A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103492406A (en) * | 2010-12-09 | 2014-01-01 | 宾夕法尼亚大学董事会 | Use of chimeric antigen receptor-modified t cells to treat cancer |
| CN102775500A (en) * | 2012-08-03 | 2012-11-14 | 郑骏年 | Chimeric antigen receptor iRGD-scFv (G250)-CD8-CD28-CD137-CD3zeta and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JAMES N. KOCHENDERFER等: "Construction and pre-clinical evaluation of an anti-CD19 Chimeric antigen receptor", 《J IMMUNOTHER》 * |
| 刘亚丹等: "CD19与B细胞恶性肿瘤的免疫治疗", 《中国肿瘤生物治疗杂志》 * |
Cited By (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11919968B2 (en) | 2014-08-22 | 2024-03-05 | B & H Biotechnologies, Llc | Methods of treating gastrointestinal diseases characterized by inflammatory cells |
| US10597461B2 (en) | 2014-08-22 | 2020-03-24 | B & H Biotechnologies, Llc | Saccharide-based biomarkers and therapeutics |
| US11421039B2 (en) | 2014-08-22 | 2022-08-23 | B & H Biotechnologies, Llc | Saccharide-based biomarkers and therapeutics |
| CN105753991A (en) * | 2015-11-26 | 2016-07-13 | 广州中科蓝华生物科技有限公司 | Chimeric antigen receptor resistant to placenta chondroitin sulfate and application of chimeric antigen receptor |
| CN105753991B (en) * | 2015-11-26 | 2019-12-27 | 广州中科蓝华生物科技有限公司 | Chimeric antigen receptor for resisting placenta-like chondroitin sulfate and application thereof |
| CN105567640A (en) * | 2016-01-27 | 2016-05-11 | 苏州佰通生物科技有限公司 | Chimeric antigen receptor adipose-derived stem cell and preparation method thereof |
| CN107034193A (en) * | 2016-02-03 | 2017-08-11 | 北京马力喏生物科技有限公司 | Treat the therapeutic combination of B cell leukemia and B cell lymphoma |
| CN107034193B (en) * | 2016-02-03 | 2020-06-05 | 北京马力喏生物科技有限公司 | Therapeutic compositions for the treatment of B-cell leukemia and B-cell lymphoma |
| WO2017140256A1 (en) * | 2016-02-19 | 2017-08-24 | Huiru Wang | Antibodies against n-acetylglucosamine and n-acetyl-galactosamine |
| US11446328B2 (en) | 2016-02-19 | 2022-09-20 | B & H Biotechnologies, Llc | Antibodies against N-acetylglucosamine and N-acetyl-galactosamine |
| CN105647946B (en) * | 2016-03-18 | 2019-09-20 | 江苏普瑞康生物医药科技有限公司 | A kind of mosaic gene and application thereof based on III a of Fc γ R |
| CN105647946A (en) * | 2016-03-18 | 2016-06-08 | 苏州康元生物科技有限公司 | Fc gamma RIII a-based chimeric gene and application thereof |
| WO2018068257A1 (en) * | 2016-10-13 | 2018-04-19 | 北京艺妙神州医疗科技有限公司 | Universal car-t cell and preparation method and application therefor |
| CN106544321A (en) * | 2016-10-13 | 2017-03-29 | 北京艺妙神州医疗科技有限公司 | Universal CAR T cells and its production and use |
| CN109803983B (en) * | 2017-03-15 | 2020-09-25 | 南京凯地生物科技有限公司 | Specific chimeric antigen receptor T cell targeting NKG2DL, preparation method and application thereof |
| CN109803983A (en) * | 2017-03-15 | 2019-05-24 | 南京凯地生物科技有限公司 | Target the specific chimeric antigen receptor T cell of NKG2DL, preparation method and application |
| CN107226867A (en) * | 2017-07-25 | 2017-10-03 | 重庆精准生物技术有限公司 | The Chimeric antigen receptor of anti human CD 19 antigen and its application |
| CN107326014B (en) * | 2017-07-31 | 2019-09-24 | 时力生物科技（北京）有限公司 | A kind of T lymphocyte and its preparation method and application of bispecific chimeric antigen receptor modification |
| CN107326014A (en) * | 2017-07-31 | 2017-11-07 | 时力生物科技（北京）有限公司 | A kind of T lymphocytes of bispecific chimeric antigen receptor modification and its preparation method and application |
| CN109385402A (en) * | 2017-08-11 | 2019-02-26 | 上海恒润达生生物科技有限公司 | A kind of equal proportion mixes the preparation method and application of two kinds of target spot CART cells |
| CN109970867A (en) * | 2017-12-28 | 2019-07-05 | 上海细胞治疗研究院 | A kind of two-way activation costimulatory molecules receptor of CD40 and application thereof |
| WO2019129047A1 (en) * | 2017-12-28 | 2019-07-04 | 上海细胞治疗研究院 | Cd40 bidirectional-activated costimulatory molecule receptor, and use thereof |
| CN109970867B (en) * | 2017-12-28 | 2022-10-18 | 上海细胞治疗研究院 | CD40 bidirectional activation costimulatory molecule receptor and application thereof |
| WO2022121899A1 (en) | 2020-12-08 | 2022-06-16 | 北京艺妙神州医药科技有限公司 | Antibody specifically binding to strep-tag ii tag and use thereof |
| CN114107214A (en) * | 2021-11-16 | 2022-03-01 | 东莞市麦亘生物科技有限公司 | Preparation method and application of universal allogenic CAR-T cells |
| CN117247466A (en) * | 2023-11-20 | 2023-12-19 | 北京艺妙神州医药科技有限公司 | Chimeric antigen receptor against glypican 3 and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2016197570A1 (en) | 2016-12-15 |
| CN105177031B (en) | 2018-04-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105177031A (en) | Chimeric antigen receptor-modified T cells and uses thereof | |
| CN105949324B (en) | Target the Chimeric antigen receptor and application thereof of GPC3 | |
| CN108018299B (en) | Chimeric antigen receptor targeting BCMA and uses thereof | |
| CN104087607B (en) | The nucleic acid of encoding chimeric antigen receptor protein and the T lymphocytes of expression Chimeric antigen receptor albumen | |
| JP6682509B2 (en) | Nucleic acid encoding chimeric antigen receptor protein and T lymphocyte expressing chimeric antigen receptor protein | |
| CN104140974B (en) | Encode the nucleic acid of the Chimeric antigen receptor albumen of GPC 3 and express the T lymphocytes of the Chimeric antigen receptor albumen of GPC 3 | |
| CN108004259B (en) | Chimeric antigen receptor targeting B cell maturation antigen and uses thereof | |
| CN108504668A (en) | Target CD19 and CD22 Chimeric antigen receptors and application thereof | |
| CN110818802A (en) | Chimeric T cell receptor STAR and application thereof | |
| CN112979828A (en) | T lymphocyte targeting CLD18A2 and preparation method and application thereof | |
| CN109320615A (en) | Target the Chimeric antigen receptor and application thereof of novel B CMA | |
| CN108728459A (en) | Target the Chimeric antigen receptor of CD19 and the method and purposes of Combined expression IL-15 | |
| JP2021531008A (en) | GD2-based chimeric antigen receptor and its utilization | |
| CN108441505B (en) | Chimeric antigen receptor targeting ROR1 and application thereof | |
| CN113896801B (en) | Chimeric antigen receptor cell targeting human Claudin18.2 and NKG2DL, and preparation method and application thereof | |
| CN108330133A (en) | Target CD19 Chimeric antigen receptors and the method and application thereof to its dual modification | |
| CN109776671A (en) | Cell, code nucleic acid, expression vector, preparation method, pharmaceutical composition and the application of isolated T cell receptor, its modification | |
| CN108715616A (en) | The Chimeric antigen receptor method and purposes of targeting humanized mesothelin | |
| CN110423767A (en) | Express Chimeric antigen receptor CAR gene and the application of solubility PD-1 | |
| CN103965362A (en) | Chimeric chemokines receptors capable of making T cells tend to tumor locations | |
| CN108285489A (en) | Target the Chimeric antigen receptor and application thereof of BCMA-BBz-tEGFR | |
| CN108728460A (en) | Target the Chimeric antigen receptor and application thereof of GD2 | |
| WO2023236796A1 (en) | Cd79b humanized antibody-based chimeric antigen receptor and use thereof | |
| CN110526979A (en) | Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of FAP | |
| CN111499766A (en) | Immune effector cell aiming at chronic lymphocytic leukemia, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: No. 316, floor 3, building 1, block B, No. 80, xingshikou Road, Haidian District, Beijing 100093 Patentee after: Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd Address before: 102206 Beijing City, Changping District small town life Zhongguancun Life Science Park Road No. 8 Building 1, Beijing University Medical Industrial Park 218 Patentee before: BEIJING IMMUNO SHENZHOU MEDICAL TECHNOLOGY Co.,Ltd. |
|
| CP03 | Change of name, title or address | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200812 Address after: Room 402, 4th floor, No.16, Lane 118, Furonghua Road, Pudong New Area, Shanghai 201321 Patentee after: Shanghai xianbo Biotechnology Co., Ltd Address before: No. 316, floor 3, building 1, block B, No. 80, xingshikou Road, Haidian District, Beijing 100093 Patentee before: Beijing Yimiao Shenzhou Pharmaceutical Technology Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220105 Address after: 100195 No.316, 3rd floor, building 1, block B, 80 xingshikou Road, Haidian District, Beijing Patentee after: Beijing Yimiao Shenzhou Pharmaceutical Technology Co.,Ltd. Address before: 201321 room 402, 4th floor, No.16 Lane 118, Furonghua Road, Pudong New Area, Shanghai Patentee before: Shanghai xianbo Biotechnology Co., Ltd |
|
| TR01 | Transfer of patent right |