CN107337736A - The double targeting Chimeric antigen receptors of OCTS CAR, encoding gene, recombinant expression carrier and its structure and application - Google Patents
The double targeting Chimeric antigen receptors of OCTS CAR, encoding gene, recombinant expression carrier and its structure and application Download PDFInfo
- Publication number
- CN107337736A CN107337736A CN201710418260.4A CN201710418260A CN107337736A CN 107337736 A CN107337736 A CN 107337736A CN 201710418260 A CN201710418260 A CN 201710418260A CN 107337736 A CN107337736 A CN 107337736A
- Authority
- CN
- China
- Prior art keywords
- octs
- car
- seq
- gene
- cell
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 66
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 title claims abstract description 43
- 230000008685 targeting Effects 0.000 title claims abstract description 29
- 238000003259 recombinant expression Methods 0.000 title claims abstract description 17
- 102000005962 receptors Human genes 0.000 claims abstract description 54
- 108020003175 receptors Proteins 0.000 claims abstract description 54
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 claims abstract description 43
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 claims abstract description 43
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 claims abstract description 41
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 claims abstract description 41
- 239000000427 antigen Substances 0.000 claims abstract description 34
- 108091007433 antigens Proteins 0.000 claims abstract description 34
- 102000036639 antigens Human genes 0.000 claims abstract description 34
- 101000998120 Homo sapiens Interleukin-3 receptor subunit alpha Proteins 0.000 claims abstract description 30
- 101000633784 Homo sapiens SLAM family member 7 Proteins 0.000 claims abstract description 30
- 102100033493 Interleukin-3 receptor subunit alpha Human genes 0.000 claims abstract description 30
- 102100029198 SLAM family member 7 Human genes 0.000 claims abstract description 30
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 claims abstract description 29
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 claims abstract description 29
- 238000010276 construction Methods 0.000 claims abstract description 17
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims abstract description 14
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims abstract description 14
- 239000000969 carrier Substances 0.000 claims abstract description 14
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 claims abstract description 11
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 10
- 230000006044 T cell activation Effects 0.000 claims abstract description 10
- 102000006240 membrane receptors Human genes 0.000 claims abstract description 8
- 108020004084 membrane receptors Proteins 0.000 claims abstract description 8
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 claims abstract 4
- 210000004027 cell Anatomy 0.000 claims description 194
- 241000700605 Viruses Species 0.000 claims description 72
- 230000029087 digestion Effects 0.000 claims description 47
- 239000013612 plasmid Substances 0.000 claims description 44
- 206010028980 Neoplasm Diseases 0.000 claims description 31
- 239000006228 supernatant Substances 0.000 claims description 24
- 230000014509 gene expression Effects 0.000 claims description 21
- 241000713666 Lentivirus Species 0.000 claims description 16
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 16
- 239000013598 vector Substances 0.000 claims description 16
- 201000011510 cancer Diseases 0.000 claims description 14
- 239000013604 expression vector Substances 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 10
- 238000004806 packaging method and process Methods 0.000 claims description 9
- 230000003612 virological effect Effects 0.000 claims description 8
- 230000003211 malignant effect Effects 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 7
- 241000555745 Sciuridae Species 0.000 claims description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 5
- 229960000723 ampicillin Drugs 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 208000002672 hepatitis B Diseases 0.000 claims description 5
- 230000001124 posttranscriptional effect Effects 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- 208000031261 Acute myeloid leukaemia Diseases 0.000 claims description 4
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 4
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 4
- 208000032612 Glial tumor Diseases 0.000 claims description 3
- 206010018338 Glioma Diseases 0.000 claims description 3
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 claims description 3
- 208000007452 Plasmacytoma Diseases 0.000 claims description 3
- 208000006593 Urologic Neoplasms Diseases 0.000 claims description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 3
- 201000001441 melanoma Diseases 0.000 claims description 3
- 208000010626 plasma cell neoplasm Diseases 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 2
- 238000012986 modification Methods 0.000 claims description 2
- 230000006798 recombination Effects 0.000 claims description 2
- 238000005215 recombination Methods 0.000 claims description 2
- 208000008771 Lymphadenopathy Diseases 0.000 claims 1
- 208000013228 adenopathy Diseases 0.000 claims 1
- 239000013613 expression plasmid Substances 0.000 claims 1
- 210000005075 mammary gland Anatomy 0.000 claims 1
- 210000004994 reproductive system Anatomy 0.000 claims 1
- 210000003705 ribosome Anatomy 0.000 claims 1
- 241000701161 unidentified adenovirus Species 0.000 claims 1
- 241001430294 unidentified retrovirus Species 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 10
- SAZUGELZHZOXHB-UHFFFAOYSA-N acecarbromal Chemical compound CCC(Br)(CC)C(=O)NC(=O)NC(C)=O SAZUGELZHZOXHB-UHFFFAOYSA-N 0.000 abstract description 5
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 95
- 108020004414 DNA Proteins 0.000 description 77
- 239000012634 fragment Substances 0.000 description 28
- 239000000243 solution Substances 0.000 description 28
- 239000000047 product Substances 0.000 description 24
- 238000000246 agarose gel electrophoresis Methods 0.000 description 23
- 239000003550 marker Substances 0.000 description 18
- 239000000523 sample Substances 0.000 description 18
- 238000001962 electrophoresis Methods 0.000 description 17
- 239000007788 liquid Substances 0.000 description 17
- 230000026683 transduction Effects 0.000 description 17
- 238000010361 transduction Methods 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 230000000694 effects Effects 0.000 description 16
- 239000000499 gel Substances 0.000 description 16
- 238000001514 detection method Methods 0.000 description 15
- 239000000203 mixture Substances 0.000 description 15
- 238000005119 centrifugation Methods 0.000 description 14
- 238000011084 recovery Methods 0.000 description 13
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000002158 endotoxin Substances 0.000 description 12
- 238000010079 rubber tapping Methods 0.000 description 12
- 210000004881 tumor cell Anatomy 0.000 description 12
- 229910001868 water Inorganic materials 0.000 description 12
- 238000013394 immunophenotyping Methods 0.000 description 11
- 108091008146 restriction endonucleases Proteins 0.000 description 10
- 108700019146 Transgenes Proteins 0.000 description 9
- 238000001976 enzyme digestion Methods 0.000 description 9
- 238000009169 immunotherapy Methods 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 238000010586 diagram Methods 0.000 description 8
- 238000001556 precipitation Methods 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 238000002560 therapeutic procedure Methods 0.000 description 8
- 102000008100 Human Serum Albumin Human genes 0.000 description 7
- 108091006905 Human Serum Albumin Proteins 0.000 description 7
- 206010035226 Plasma cell myeloma Diseases 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 239000012636 effector Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 241000204031 Mycoplasma Species 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 239000001110 calcium chloride Substances 0.000 description 5
- 229910001628 calcium chloride Inorganic materials 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 108010074708 B7-H1 Antigen Proteins 0.000 description 4
- 102000008096 B7-H1 Antigen Human genes 0.000 description 4
- 238000011357 CAR T-cell therapy Methods 0.000 description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 4
- 229920001917 Ficoll Polymers 0.000 description 4
- 208000034578 Multiple myelomas Diseases 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 230000002147 killing effect Effects 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 210000002751 lymph Anatomy 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 102100037850 Interferon gamma Human genes 0.000 description 3
- 108010074328 Interferon-gamma Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 235000011148 calcium chloride Nutrition 0.000 description 3
- 230000009514 concussion Effects 0.000 description 3
- 230000001351 cycling effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000005923 long-lasting effect Effects 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 210000004180 plasmocyte Anatomy 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- OOIBFPKQHULHSQ-UHFFFAOYSA-N (3-hydroxy-1-adamantyl) 2-methylprop-2-enoate Chemical compound C1C(C2)CC3CC2(O)CC1(OC(=O)C(=C)C)C3 OOIBFPKQHULHSQ-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 108010083359 Antigen Receptors Proteins 0.000 description 2
- 102000006306 Antigen Receptors Human genes 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000019838 Blood disease Diseases 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 2
- 101001136592 Homo sapiens Prostate stem cell antigen Proteins 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 108010004729 Phycoerythrin Proteins 0.000 description 2
- 102100036735 Prostate stem cell antigen Human genes 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000001994 activation Methods 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 208000014951 hematologic disease Diseases 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108010056030 retronectin Proteins 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 2
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 102000000074 ADP-ribosyl Cyclase Human genes 0.000 description 1
- 108010080394 ADP-ribosyl Cyclase Proteins 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 1
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 1
- 101001005269 Arabidopsis thaliana Ceramide synthase 1 LOH3 Proteins 0.000 description 1
- 101001005312 Arabidopsis thaliana Ceramide synthase LOH1 Proteins 0.000 description 1
- 208000004736 B-Cell Leukemia Diseases 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 102100025221 CD70 antigen Human genes 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 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
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 description 1
- 101001076418 Homo sapiens Interleukin-1 receptor type 1 Proteins 0.000 description 1
- 101001076430 Homo sapiens Interleukin-13 Proteins 0.000 description 1
- 101000795167 Homo sapiens Tumor necrosis factor receptor superfamily member 13B Proteins 0.000 description 1
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 1
- 101000762805 Homo sapiens Tumor necrosis factor receptor superfamily member 19L Proteins 0.000 description 1
- 101000679907 Homo sapiens Tumor necrosis factor receptor superfamily member 27 Proteins 0.000 description 1
- 102100026016 Interleukin-1 receptor type 1 Human genes 0.000 description 1
- 241000239218 Limulus Species 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 206010050551 Lupus-like syndrome Diseases 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 101150053046 MYD88 gene Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102100024134 Myeloid differentiation primary response protein MyD88 Human genes 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 240000005373 Panax quinquefolius Species 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 101000668858 Spinacia oleracea 30S ribosomal protein S1, chloroplastic Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 101000898746 Streptomyces clavuligerus Clavaminate synthase 1 Proteins 0.000 description 1
- 230000033540 T cell apoptotic process Effects 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 102100029675 Tumor necrosis factor receptor superfamily member 13B Human genes 0.000 description 1
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 description 1
- 102100026716 Tumor necrosis factor receptor superfamily member 19L Human genes 0.000 description 1
- 102100022202 Tumor necrosis factor receptor superfamily member 27 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 108091093126 WHP Posttrascriptional Response Element Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 108091006004 biotinylated proteins Proteins 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 101150058049 car gene Proteins 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 239000008004 cell lysis buffer Substances 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000004940 costimulation Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 229960002204 daratumumab Drugs 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 230000003694 hair properties Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 208000018706 hematopoietic system disease Diseases 0.000 description 1
- 102000019207 human interleukin-13 Human genes 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000022983 regulation of cell cycle Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4631—Chimeric Antigen Receptors [CAR]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
-
- 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
-
- 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
- A61K39/464416—Receptors for cytokines
- A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
-
- 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
- A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
-
- 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
- A61K39/464454—Enzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
-
- C—CHEMISTRY; METALLURGY
- 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
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
- A61K2239/29—Multispecific CARs
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
The invention provides a kind of double targeting Chimeric antigen receptors of OCTS CAR based on OCTS technologies, encoding gene, OCTS CAR T recombinant expression carriers and its construction method and application.The double targeting Chimeric antigen receptors of the OCTS CAR include the CD8 leader membrane receptor signal peptides being sequentially connected in series,Double antigen binding domains,CD8 Hinge Chimerical receptor hinges,CD8 Transmembrane Chimerical receptor transmembrane regions,CD28 Chimerical receptor costimulating factors,CD134 Chimerical receptors costimulating factor and TCR Chimerical receptor t cell activations domain,Wherein,Double antigen binding domains include the heavy chain VH and light chain VL of two single-chain antibodies connected in a certain way,Hinge Inter Linker between antibody inner hinge Inner Linker and single-chain antibody,Two single-chain antibodies refer to BCMA single-chain antibodies,CD319 single-chain antibodies,CD38 single-chain antibodies,PDL1 single-chain antibodies,Any combination of two between CD123 single-chain antibodies.In addition, additionally provide the gene of the double targeting Chimeric antigen receptors of the coding OCTS CAR, recombinant expression carrier and its construction method and application.
Description
Technical field
The invention belongs to immunotherapy of tumors technical field, and in particular to a kind of for malignant tumour immunization therapy, base
In the CAR of OCTS technologies double targeting Chimeric antigen receptor, encoding gene, OCTS-CAR recombinant expression carriers and its construction methods
And application.
Background technology
The theoretical foundation of immunotherapy of tumors is that immune system has identification tumor associated antigen, regulation and control body attack tumour
The ability of cell (e.g., the cell dissolving of high degree of specificity).Generation nineteen fifty, Burnet and Thomas are proposed " immunosurveillance "
It is theoretical, it is believed that the tumour cell of the mutation often occurred in body can be identified and removed by immune system, be tumour immunity
Theoretical foundation [Burnet FM.Immunological aspects of malignant have been established in treatment
disease.Lancet,1967;1: 1171-4].Then, various tumour immunotherapies include cytokine therapy, monoclonal resists
The sequential uses such as autogenic therapy, adoptive immunotherapy, vaccine therapy are in clinic.
A kind of more advanced tumour immunotherapy in 2013 --- CAR-T therapies are used successfully to clinic, and are demonstrated by preceding institute not
Some clinical efficacies.CAR-T, full name are Chimeric Antigen Receptor T-Cell Immunotherapy, are fitted together to
Antigen receptor T cell immunotherapy.The therapy is the means by transgenosis, by promoter, antigen recognizing district, costimulation because
The chimeric molecule that son, effect area etc. collectively constitute, import in T cell genome, so that identification of the T cell to target cell, letter
Number transduction, killing etc. function combine together, realize specific killing [the Eleanor J.Cheadle, et to target cell
al.CAR T cells:driving the road from the laboratory to the
clinic.Immunological Reviews 2014.Vol.257: 91–106].CAR-T therapies are clinically most leading
There is the CLT019 of Novartis, refractory Patients With Acute Lymphoblastic Leukemia is recurred using CLT019 treatments, the tumour of six months is without entering
Exhibition survival rate reaches 67%, wherein most long response time reached more than 2 years.General headquarters are located at the excellent Ka Disheng in Shanghai of Chinese Shanghai
Thing Pharmaceutical Technology Co., Ltd cooperates with hospital, and by the end of 2 months 2017, the refractory white blood of acute lymphoblastic was recurred in treatment altogether
Patient 36, wherein complete 24, alleviation ratio reaches 66.6%.Therefore, CAR-T cell therapies are the tops of anticancer research
The property covered breaks through, it may be possible to one of most possible means for curing cancer, and by《Science》It is big that magazine was chosen as 2013 years ten
First of technological breakthrough.
CAR-T at present evident in efficacy in terms of the neoplastic hematologic disorder of the treatment several types such as B- lymphocytic leukemias, but
A kind of antigenic targets can only be identified by being that there is also some limitations, such as the previous Chimeric antigen receptor of mesh, but tumour cell is
Individual complicated colony, after the tumour cell containing corresponding antigens is eliminated, the tumour cell without corresponding antigens can increase rapidly
Grow, tumor recurrence is caused after a period of time.
CAR-T identifications is identified two kinds of antigens simultaneously, there are two schemes optional:First, by two groups of Chimeric antigen receptors
Structure enters a slow virus transgene carrier, and two groups of Chimeric antigen receptor transductions disposably are entered into primary T lymphocytes;
Second, being transduceed at twice with two slow virus transgene carriers, two groups of Chimeric antigen receptors are transduceed into primary T lymphs respectively
Cell.
The shortcomings that scheme one, is the valuable capacity for taking slow virus transgene carrier, is unfavorable for loading other Functional Units
Part;Transgene carrier packaging efficiency is low;Gene transduction efficiency is very low, it is difficult to transduce into primary T lymphocytes.
The shortcomings that scheme two, is needs by transduceing twice, and the overall efficiency transduceed twice is relatively low, transduces cycle time
It is long, the easy aging of primary cell, cause multiplication capacity to fail, killing ability declines, and influences tumor clearance curative effect.
As the primary study object of immunotherapy of tumors, the tumour of Huppert's disease (Multiple myeloma) is thin
Born of the same parents originate from the thick liquid cell in marrow, and thick liquid cell is cell of the bone-marrow-derived lymphocyte development to final function phases, therefore are one
Kind is using the malignant plasma cell tumour that existing treatment method is difficult healing.The life cycle of usual patient is 3 years, it is short even only
Have 6-12 months.
But research is found, Cucumber exists in B cell or malignant cell surface-stable or height is expressed, and can be used as thin
Born of the same parents' label or target.Such as:
BCMA (B-cell maturation antigen) is (including more in B cell as a kind of mark selective expression
Hair property myeloma cell) surface, can be as the target guiding CAR-T cell killing tumour cells (B-cell of CAR-T cells
Maturation Antigen is a Promising Target for Adoptive T cell Therapy of
Multiple Myeloma. Robert O.Carpenter,et al.Clin Cancer Res.2013April 15;19
(8):2048–2060.)。
CD319 is also known as CS1, SLAMF7, is a kind of cell surface marker of stabilization, in normal plasma cells and pernicious
Thick liquid cell expression has expression.CD319 is a kind of and its stable antigen, even isolated by coarse means
Malignant plasma cell system, even after freezing, what still can be stablized detects expression [Frigyesi I, Adolfsson
J, Ali M,Christophersen MK,Johnsson E,Turesson I,Gullberg U,Hansson M,Nilsson
B(Feb 2014)."Robust isolation of malignant plasma cells in multiple myeloma"
.Blood.123(9): 1336–40.]。
CD38 is also referred to as cyclic ADP ribose hydrolase, is detected in many immunocyte surface energies, including
B cell and NK cells.CD38 is a kind of mark of cell activation, is joined with HIV, leukaemia, myeloma, solid tumor etc.
It is tied.Clinically have at present using targeting CD38 antibody to treat Huppert's disease [Lokhorst, Henk M.;
Plesner,Torben;Laubach,Jacob P.;Nahi,Hareth;Gimsing,Peter;Hansson,Markus;
Minnema, Monique C.;Lassen,Ulrik;Krejcik,Jakub(2015-09-24)."Targeting
CD38with Daratumumab Monotherapy in Multiple Myeloma".The New England Journal
of Medicine.373(13): 1207–1219.]。
CD123 is the alpha chains of human interleukin-13 acceptor, in most acute myeloid leukemia (AML) cell and a lot
There is expression on hematopoietic cell surface, and CD123 is an extraordinary immunotherapeutic targets, because even seldom in CD123 expression
Cell in, its expression can increase gradually enhancing with the time, and acute myeloid leukemia is likely due to be in white blood
The candidate stem cell of sick early stage passes through clone evolution, using CD123 as target spot, then can play a part of clear marrow (Saar
Gill,Carl H.June et al.Preclinical targeting of human acute myeloid leukemia
and myeloablation using chimeric antigen receptor-modified T
cells.Blood.2014;123(15):2343- 2354.).
PD-L1 overexpressions in most cancerous tissues, including NSCLC, melanoma, breast cancer, glioma, lymph
[the Intlekofer AM, Thompson such as knurl, leukaemia and various urological cancers, tumor in digestive tract, system genitale tumour
CB.At the bench:preclinical rationale for CTLA-4and PD-1blockade as cancer
immunotherapy[J].J Leukoc Biol,2013,94(1):25-39.].Parsa in the tumour cell of mouse and people,
It was found that the IFN-γ of T cell abnormal secretion, IFN-γ can with induced tumor cell the high expression of PD-L1 [Ding H, Wu X,
Wu J,et al.Delivering PD-1 inhibitory signal concomitant with blocking ICOS
co-stimulation suppresses lupus-like syndrome in autoimmune BXSB mice[J].Clin
Immunol,2006,118(2/3):258-267.].The high expression of PD-L1, can be led to by suppressing RAS and PI3K/AKT signals
Road, and then cell cycle regulation checkpoint albumen and cell multiplication related protein expression, ultimately result in the suppression of T cell propagation
[11].The experiment in vitro such as Dong and mouse model also found that the activation of PD-1/PD-L1 signal paths can be with inducing specific
CTL adjust dies, CTL cell toxicant lethal effect sensitiveness is declined, promote tumour cell generation immunologic escape [Dong H,
Strome SE,Salomao DR,et al.Tumor-associated B7-H1promotes T-cell apoptosis:a
potential mechanism of immune evasion[J].Nat Med,2002,8(8):793-800.]。
The content of the invention
The present invention be based on above-mentioned discovery and carry out, and it is an object of the present invention to provide a kind of be used for malignant tumour, it is especially multiple
Property the double targeting Chimeric antigen receptors of the CAR based on OCTS technologies of myeloma immunization therapy, encoding gene, OCTS-CAR restructuring
Expression vector and its construction method and application.
OCTS full name is One CAR with Two ScFvs, passes through the OCTS that connects (Series OCTS) or corner
OCTS (Turn OCTS) connected mode, by two sections of scFv and it is integrated into a chimeric molecule (shown in Fig. 1), assigns T lymphs
Mode non-dependent cell HLA identifies the ability of two kinds of tumour antigens, can be identified relative to traditional CAR-T cells wider
General target, further expand the removing scope of tumour cell.
OCTS basic engineering includes two tumor associated antigens (tumor-associated antigen, TAA) knot
Close area (the scFv sections for being typically derived from monoclonal antibody antigen calmodulin binding domain CaM), an extracellular hinge area, a transmembrane region, two
Individual intracellular signal transduction area and a response element area.Specificity, validity and genetic modification of the scFv regions for OCTS
It is crucial determinant for the security of T cell itself.
OCTS-CAR-T technologies of the present invention, it is on the basis of current CAR-T cell therapies, by embedding
Close the Optimizing Reconstruction of antigen receptor (CAR) structure so that Chimeric antigen receptor can identify two kinds of antigens, greatly expand
The identification range of CAR-T cells, the removing for cancer colonies is more thorough, and curative effect is more longlasting.
The first aspect of the present invention, there is provided a kind of double targeting Chimeric antigen receptors of CAR based on OCTS technologies, including
CD8leader membrane receptors signal peptide, double antigen binding domains, CD8Hinge Chimerical receptors hinge, the CD8 being sequentially connected in series
Transmembrane Chimerical receptors transmembrane region, CD28 Chimerical receptors costimulating factor, CD134 Chimerical receptors costimulating factor with
And TCR Chimerical receptor t cell activations domain.Wherein, double antigen binding domains include two single-chain antibodies connected in a certain way
Hinge Inter-Linker between heavy chain VH and light chain VL, antibody inner hinge Inner-Linker and single-chain antibody;Two lists
Chain antibody refers to that BCMA single-chain antibodies, CD319 single-chain antibodies, CD38 single-chain antibodies, PDL1 single-chain antibodies, CD123 are single-stranded anti-
Any combination of two between body.
Further, the double targeting Chimeric antigen receptors of OCTS-CAR of the invention, also with following technical characteristic:Two single-stranded
Antibody with connect or the connected mode of corner connect.
The second aspect of the invention, there is provided encode the gene of the double targeting Chimeric antigen receptors of above-mentioned CAR, coding is above-mentioned
The sequence number control of the gene of each several part is as follows:CD8leader membrane receptors signal peptide (SEQ ID NO.15), BCMA are single-stranded
Antibody light chain VL (SEQ ID NO.16), BCMA single-chain antibody heavy chains VH (SEQ ID NO.17), CD319 single-chain antibody light chains
VL (SEQ ID NO.18), CD319 single-chain antibody heavy chains VH (SEQ ID NO.19), CD38 single-chain antibody light chain VL (SEQ ID
NO.20), CD38 single-chain antibodies heavy chain VH (SEQ ID NO.21), PDL1 single-chain antibody light chains VL (SEQ ID NO.22),
PDL1 single-chain antibody heavy chains VH (SEQ ID NO.23), CD123 single-chain antibody light chains VL (SEQ ID NO.24), CD123 are single-stranded
Hinge between heavy chain of antibody VH (SEQ ID NO.25), antibody inner hinge Inner-Linker (SEQ ID NO.26), single-chain antibody
Inter-Linker (SEQ ID NO.27), CD8Hinge Chimerical receptors hinge (SEQ ID NO.28),
CD8Transmembrane Chimerical receptors transmembrane region (SEQ ID NO.29), CD28 Chimerical receptors costimulating factor (SEQ ID
NO.30), CD134 Chimerical receptors costimulating factor (SEQ ID NO.31), TCR Chimerical receptor t cell activations domain (SEQ ID
NO.32)。
The third aspect of the present invention, there is provided a kind of OCTS-CAR recombinant expression carriers, there is such technical characteristic:Bag
Include the gene of expression vector, people's EF1 α promoters and the double targeting Chimeric antigen receptors of coding CAR-T.Wherein, people EF1 α are opened
The gene order of mover is as shown in SEQ ID NO.14, and the double genes for targetting each several part in Chimeric antigen receptors of coding CAR-T are such as
It is upper described.Wherein, the gene order of antigen binding domain includes any two kinds in BCMA, CD319, CD38, PDL1, CD123 of list
The light chain VL and heavy chain VH of chain antibody gene order.
Further, present invention also offers another OCTS-CAR recombinant expression carriers, relative to above-mentioned recombination expression
Carrier, the recombinant expression carrier also include the gene of coding PDL1 single-chain antibodies, its gene order such as SEQ ID NO.33 institutes
Show.
Expression vector skeleton of the present invention is third generation slow virus carrier (shown in Fig. 2A), and 3 ' SIN LTR are removed
U3 regions, eliminate the possibility of slow virus carrier self-replacation, substantially increase security;Add cPPT and WPRE
Element, improve transduction efficiency and the expression efficiency of transgenosis;Core when ensure that slow virus carrier packaging using RSV promoters
Heart RNA lasting efficient transcription;Using the EF1 α promoters of people itself, enable CAR genes in human body long lasting for table
Reach.
The Lentiviral includes the sequences of AmpR containing ampicillin resistance gene, prokaryotic replions pUC Ori sequences
Row, Viral Replicon SV40Ori sequences, RSV promoters, slow virus 5terminal LTR, slow virus 3terminal Self-
Inactivating LTR, Gag cis elements, RRE cis elements, env cis elements, cPPT cis elements, ZsGreen1 are green
The enhanced marmot hepatitis B posttranscriptional regulatory element of color fluorescin, IRES ribosome binding sequences, eWPRE.
The present invention be by people EF1 α promoters, CD8leader Chimerical receptor signal peptides, BCMA, CD319, CD38, PDL1,
Any two kinds of single-chain antibody light chain VL and heavy chain VH in CD123, antibody inner hinge Inner-Linker, cut with scissors between single-chain antibody
Chain Inter-Linker, CD8Hinge Chimerical receptor hinge, CD8Transmembrane Chimerical receptor transmembrane regions, CD28 are fitted together to
Acceptor costimulating factor, CD134 Chimerical receptor costimulating factors, TCR Chimerical receptor t cell activations domain, and PDL1 are single-stranded anti-
The expressing gene structure of body enters recombined lentivirus vector, starts whole OCTS expression of structural gene by people EF1 α promoters.
CD8leader Chimerical receptors signal peptide is located at the N-terminal of OCTS albumen, for guiding OCTS albumen to be positioned at cell
Film;The heavy chain and light chain of any two groups of single-chain antibodies are mutually combined to form double antigen recognizing districts, for identifying corresponding target antigen;
CD8Hinge Chimerical receptors hinge is used to scFv being anchored on the outside of cell membrane;CD8Transmembrane Chimerical receptor cross-films
Area is used to whole Chimerical receptor being fixed on cell membrane;CD28 Chimerical receptors costimulating factor is used to stimulate T lymphocyte bodies
Outer activation and interior tumor cell lethal effect;CD134 Chimerical receptors costimulating factor be used for promote T lymphopoiesis and
Cytokine secretion, strengthen tumour immunity, be advantageous to the long-term surviving of memory T cell;TCR Chimerical receptor t cell activations domain is used to swash
The expression of downstream signaling pathway living;PDL1 single-chain antibody can effectively close PDL1, blocking immunity negative regulator signal path, face
On bed can be used for suppress tumour immune escape, improve CAR-T cellular immunotherapies the effect of.
When antigen recognition region is combined with target antigen, signal is transferred into the cell by Chimerical receptor, thin so as to produce T
One systems such as born of the same parents' propagation, cytokine secretion increase, Anti-apoptotic proteins secretion increase, cell death delay, cracking target cell
Row biological effect.
In the present invention, BCMA single-chain antibody light chain VL, BCMA single-chain antibody heavy chain VH, CD319 single-chain antibody light chains VL,
CD319 single-chain antibody heavy chain VH, CD38 single-chain antibody light chain VL, CD38 single-chain antibody heavy chain VH, PDL1 single-chain antibody light chains
VL, PDL1 single-chain antibody heavy chain VH, CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH, PDL1 single-chain antibody,
With with shown nucleotide sequence>=80% homology is (preferably,>=90% homology;Deng preferably,>=95% is homologous
Property;Most preferably,>=97% homology;) nucleotide sequence.
In the present invention, BCMA single-chain antibody light chain VL, BCMA single-chain antibody heavy chain VH, CD319 single-chain antibody light chains VL,
CD319 single-chain antibody heavy chain VH, CD38 single-chain antibody light chain VL, CD38 single-chain antibody heavy chain VH, PDL1 single-chain antibody light chains
VL, PDL1 single-chain antibody heavy chain VH, CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH, PDL1 single-chain antibody,
With the amino acid sequence corresponding with shown nucleotide sequence>=80% homology is (preferably,>=90% homology;Deng
Preferably,>=95% homology;Most preferably,>=97% homology) amino acid sequence.
In the present invention, BCMA single-chain antibody light chain VL, BCMA single-chain antibody heavy chain VH, CD319 single-chain antibody light chains VL,
CD319 single-chain antibody heavy chain VH, CD38 single-chain antibody light chain VL, CD38 single-chain antibody heavy chain VH, PDL1 single-chain antibody light chains
VL, PDL1 single-chain antibody heavy chain VH, CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH, PDL1 single-chain antibody are equal
By humanization modified, the production that the anti-mouse of internal people resists (Human anti-mouse antibodies, HAMA) can be effectively reduced
It is raw, extend scFv half-life period and action effect.
The transgene carrier that the present invention uses is the replication defect type slow virus carrier after restructuring, can integrate exogenous sequences
Enter host gene, it is disposable, it can not replicate and breed, securely and reliably.
The costimulating factor region in OCTS Chimerical receptors in the present invention can be 4-1BB, ICOS, CD27, OX40,
CD28, MYD88, IL1R1, CD70, TNFRSF19L, TNFRSF27, TNFRSF1OD, the tumour such as TNFRSF13B, TNFRSF18
One kind in necrosis factor superfamily (tumor necrosis factor receptor superfamily, TNFRSF), two
Kind, three kinds, several, tens kinds of combination.
The replication defect type slow virus transgene carrier used in the present invention can be two generations or the slow virus of three generations
Transgene carrier.
The fourth aspect of the invention, there is provided the construction method of OCTS-CAR recombinant expression carriers, comprise the following steps:
A. ampicillin resistance gene AmpR sequences (SEQ ID NO.1), prokaryotic replions pUC Ori sequences will be contained
(SEQ ID NO.2), Viral Replicon SV40Ori sequences (SEQ ID NO.3), RSV promoters (SEQ ID NO.4), slow disease
Malicious 5terminal LTR (SEQ ID NO.5), slow virus 3terminal Self-Inactivating LTR (SEQ ID
NO.6), Gag cis elements (SEQ ID NO.7), RRE cis elements (SEQ ID NO.8), env cis elements (SEQ ID
NO.9), cPPT cis elements (SEQ ID NO.10), ZsGreen1 green fluorescent proteins (SEQ ID NO.11), IRES ribose
Body binding sequence (SEQ ID NO.12), enhanced marmot hepatitis B posttranscriptional regulatory element (the SEQ ID of eWPRE
NO.13) it is stored on third generation slow virus skeleton plasmid pLenti-3G basic, this method is developed by our company and built,
And it is disclosed in《It is a kind of based on the CAR-T transgene carriers and its construction method of replication defective recombinant slow virus and application》
201610008360.5 in patent;
B. will coding CD8leader membrane receptors signal peptide, double antigen binding domains, CD8Hinge Chimerical receptors hinge, CD8
Transmembrane Chimerical receptors transmembrane region, CD28 Chimerical receptors costimulating factor, CD134 Chimerical receptors costimulating factor with
And the gene in TCR Chimerical receptor t cell activations domain is cloned into slow virus skeleton plasmid by digestion, connection, recombining reaction
In pLenti-3G basic, obtain the recombinant slow virus plasmid that the third generation is designed based on OCTS, as pOCTS123BCMAs,
pOCTS319BCMAs、pOCTS38BCMAs、pOCTS-PDL1BCMAs、 pOCTS123BCMAt、pOCTS319BCMAt、
POCTS38BCMAt, pOCTS-PDL1BCMAt etc., wherein, the last letter " s " represents two sections of scFv and is connected in series, letter
" t " represents two sections of scFv corners connections.
C. recombinant slow virus plasmid step B obtained respectively with slow virus packaging plasmid pPac-GP, pPac-R with
And memebrane protein plasmid pEnv-G transfects HEK293T/17 cells jointly, and gene transcript expression is carried out in HEK293T/17 cells
Afterwards, packing successfully recombined lentivirus vector can be discharged into cells and supernatant, collect the upper of the recombined lentivirus vector that includes
Clear liquid;
D. obtained recombinant slow virus supernatant is purified using the post way of purification for filtering, adsorbing, eluting, respectively
To CAR-T recombinant slow virus expression vectors, (be named as lvOCTS123BCMAs, lvOCTS319BCMAs,
lvOCTS38BCMAs、lvOCTS-PDL1BCMAs、lvOCTS123BCMAt、lvOCTS319BCMAt、 lvOCTS38BCMAt、
lvOCTS-PDL1BCMAt)。
In addition, in stepb, will can also encode the genes of PDL1 single-chain antibodies together with other genes by digestion,
Connection, recombining reaction are cloned into slow virus skeleton plasmid pLenti-3G basic, obtain what the third generation was designed based on OCTS
Recombinant slow virus plasmid.
The fifth aspect of the present invention, there is provided a kind of OCTS-CAR-T cells, the OCTS-CAR-T cells are in genome
The T lymphs for being imported with OCTS-CAR recombinant expression carriers or being modified through the double targeting Chimeric antigen receptors of the CAR based on OCTS are thin
Born of the same parents.
After Workshop Production of the OCTS-CAR-T cells that the present invention uses by GMP ranks, available for human clinical trial.
The sixth aspect of the present invention, there is provided application of the OCTS-CAR-T cells in treating malignant tumor medicine is prepared.
Further, the treating malignant tumor medicine is that treatment malignant tumour is that malignant plasma cell tumour, acute myeloid are white
Blood disease, melanoma, breast cancer, glioma, lymthoma, urological cancer, the medicine of tumor in digestive tract or system genitale tumour.
The effect of invention and effect
The present invention uses OCTS technologies on the basis of current traditional CAR-T cell therapies, by Chimeric antigen receptor
(CAR) Optimizing Reconstruction of structure so that Chimeric antigen receptor can identify two kinds of antigens, and it is thin on the one hand to have expanded CAR-T significantly
The identification range of born of the same parents, the removing for cancer colonies is more thorough, and curative effect is more longlasting;On the other hand batch culture CAR-T is avoided
Cell, cost is greatlyd save, so as to avoid patient from repeatedly feeding back different targeting CAR-T cells, saved the economic branch of patient
Go out, reduce the probability of recurrence, improve the life quality of patient indirectly.
By the OCTS that connects (Series OCTS) or corner OCTS (Turn OCTS) connected mode, by two sections
ScFv is integrated into a chimeric molecule, not only assigns the energy that the non-dependent modes of T lymphocytes HLA identify two kinds of tumour antigens
Power, and wider target can be identified relative to traditional CAR-T cells, further expand the removing of tumour cell
Scope, as OCTS-CAR-T will enter clinical investigation phase, indicate that CAR-T cell therapies will enter for 2.0 epoch.
In addition, BCMA single-chain antibody light chain VL, BCMA single-chain antibody heavy chain VH, CD319 single-chain antibody light chains of the present invention
VL, CD319 single-chain antibody heavy chain VH, CD38 single-chain antibody light chain VL, CD38 single-chain antibody heavy chain VH, PDL1 single-chain antibody are light
Chain VL, PDL1 single-chain antibody heavy chain VH, CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH, PDL1 single-chain antibody
By humanization modified, it can effectively reduce the anti-mouse of internal people and resist (Human anti-mouse antibodies, HAMA)
Generation, extend scFv half-life period and action effect, increase the existence time of OCTS-CAR-T cells.
In addition, one kind of the costimulating factor used in the present invention or several combination, by increasing capacitance it is possible to increase cell after transduction
The characteristics such as multiplication rate, time-to-live, killing-efficiency, immunological memory.
Therefore, OCTS-CAR-T cells of the present invention will treat to tumour cell and provide reliable guarantee.
Brief description of the drawings
Fig. 1 is the schematic diagram of the double targeting Chimeric antigen receptors (OCTS) of CAR of the present invention, wherein (A) is series connection OCTS
The schematic diagram of (Series OCTS), (B) are corner OCTS (Turn OCTS) schematic diagram.
Fig. 2 is the slow virus carrier structural representation of the present invention;Wherein (A) figure is the third generation slow virus that the present invention uses
Carrier structure schematic diagram, (B) figure are the second generation and third generation slow virus carrier structure comparison schematic diagram.
Fig. 3 is the structure flow chart of the recombined lentivirus vector of the present invention;Wherein, (A) figure is slow virus skeleton plasmid
PLenti-3G basic structural representation;(B) figure is the schematic diagram of 8 OCTS plasmids;(C) figure is slow virus packaging plasmid
The structural representation of pPac-GP plasmids;(D) figure is the structural representation of slow virus packaging plasmid pPac-R plasmids;(E) figure is
Memebrane protein plasmid pEnv-G structural representation.
Fig. 4 is the element orders schematic diagram of the double targeting Chimeric antigen receptors (OCTS) of CAR, wherein, (A) figure is series connection
OCTS (Series OCTS) structural representation, (B) figure are corner OCTS (Turn OCTS) structural representations.
Fig. 5 is recombinant slow virus plasmid pOCTS123BCMAs (A), pOCTS319BCMAs (B), pOCTS38BCMAs
(C)、pOCTS-PDL1BCMAs(D)、pOCTS123BCMAt(E)、pOCTS319BCMAt (F)、pOCTS38BCMAt(G)、
POCTS-PDL1BCMAt (H) digestion prognostic chart and digestion agarose gel electrophoresis figure.Wherein, in A~H is schemed, lane1 is
1kb DNA ladder Marker digestion prognostic chart;Lane2 is the digestion prognostic chart of above-mentioned each recombinant slow virus plasmid;
Lane3 is 1kb DNA ladder Marker digestion agarose gel electrophoresis figures;Lane4 is above-mentioned each recombinant slow virus plasmid
Digestion agarose gel electrophoresis figure.
Fig. 6 is the titre testing result of recombined lentivirus vector.
Fig. 7 is the schematic flow sheet of the OCTS-CAR-T cell constructions of the present invention, comprising be separately cultured, activate, gene turns
Lead, the stage such as OCTS-CAR-T cellular identifications.
Fig. 8 is the detection of mycoplasma result of OCTS-CAR-T cells, lane1 DL2000marker, from top to bottom band
Band is followed successively by from top to bottom:2kb、1kb、750bp、500bp、250bp、100bp;Lane2 is positive control;Lane3 is the moon
Property control;Lane4 is PBS;Lane5 is lysate;Lane6 is OCTS123BCMAs-CAR-T cells;Lane7 is
OCTS319BCMAs-CAR-T cells;Lane8 is OCTS38BCMAs-CAR-T cells;Lane9 is OCTS-PDL1BCMAs-
CAR-T cells;Lane10 is OCTS123BCMAt-CAR-T cells;Lane11 is OCTS319BCMAt-CAR-T cells;
Lane12 is OCTS38BCMAt-CAR-T cells;Lane13 is OCTS-PDL1BCMAt-CAR-T cells.
Fig. 9 is the transduction efficiency and immunophenotyping result of flow cytometer detection OCTS-CAR-T cells.Scheme A to represent
The transduction efficiency result of OCTS123BCMAs-CAR-T cells;Scheme the immunophenotyping that B represents OCTS123BCMAs-CAR-T cells
As a result;Scheme the transduction efficiency result that C represents OCTS319BCMAs-CAR-T cells;Scheme D and represent that OCTS319BCMAs-CAR-T is thin
The immunophenotyping result of born of the same parents;Scheme the transduction efficiency result that E represents OCTS38BCMAs-CAR-T cells;Scheme F to represent
The immunophenotyping result of OCTS38BCMAs-CAR-T cells;Scheme the transduction effect that G represents OCTS-PDL1BCMAs-CAR-T cells
Rate result;Scheme the immunophenotyping result that H represents OCTS-PDL1BCMAs-CAR-T cells;Scheme I and represent OCTS123BCMAt-
The transduction efficiency result of CAR-T cells;Scheme the immunophenotyping result that J represents OCTS123BCMAt-CAR-T cells;Scheme K to represent
The transduction efficiency result of OCTS319BCMAt-CAR-T cells;Scheme the immunophenotyping that L represents OCTS319BCMAt-CAR-T cells
As a result;Scheme the transduction efficiency result that M represents OCTS38BCMAt-CAR-T cells;Scheme N and represent OCTS38BCMAt-CAR-T cells
Immunophenotyping result;Scheme the transduction efficiency result that O represents OCTS-PDL1BCMAt-CAR-T cells;Scheme P and represent OCTS-
The immunophenotyping result of PDL1BCMAt-CAR-T cells.
Under the conditions of Figure 10 is different effect target, OCTS-CAR-T cells are to target cell killing-efficiency block diagram:Scheme A to represent
The Mortaility results of OCTS123BCMAs-CAR-T cells and OCTS123BCMAt-CAR-T cells to different target cells;Scheme B to represent
The Mortaility results of OCTS319BCMAs-CAR-T cells and OCTS319BCMAt-CAR-T cells to different target cells;Scheme C to represent
The Mortaility results of OCTS38BCMAs-CAR-T cells and OCTS38BCMAt-CAR-T cells to different target cells;Scheme D to represent
The Mortaility results of OCTS-PDL1BCMAs-CAR-T cells and OCTS-PDL1BCMAt-CAR-T cell to different target cells.
Embodiment
Following examples are merely to illustrate the present invention, and without with limiting the scope of the present invention.Unreceipted tool in embodiment
The experimental method of concrete conditions in the establishment of a specific crime, generally according to normal condition, or according to the condition proposed by manufacturer.
Embodiment is combined into dual anti-original with CD319, CD38, PDL1, CD123 single-chain antibody respectively with BCMA single-chain antibodies
Exemplified by cog region, to structure, OCTS-CAR-T cell constructions and the function test method of the double targeting Chimeric antigen receptors of CAR
Illustrate.In every kind of combination, two sections of scFv are connected in a manner of being connected in series and being connected with corner respectively, form eight altogether
The double antigen recognizing districts of kind.
The light chain and heavy chain of the single-chain antibody of other four kinds of materials can also be combined into double antigen recognizing districts, the double targetings of its CAR
Structure, OCTS-CAR-T cell constructions and the function test method of Chimeric antigen receptor and the side described in the present embodiment
Method is identical.
Experiment material used in embodiment is as follows:
(1) slow virus skeleton plasmid pLenti-3G basic, slow virus packaging plasmid pPac-GP, pPac-R and film egg
White matter grain pEnv-G, HEK293T/17 cell, homologous recombination enzyme, Oligo Annealing Buffer, mycoplasma test reagent
Box, endotoxin detection kit, PSCA+K562, PDL1+K562, PDL1+PSCA+K562, K562 cell take wing purchased from generation (on
Sea) biological medicine Science and Technology Ltd.;;
(2) people's fresh peripheral blood is provided by health donors;
(3)OCTS123BCMAs、OCTS319BCMAs、OCTS38BCMAs、OCTS-PDL1BCMAs、
OCTS123BCMAt, OCTS319BCMAt, OCTS38BCMAt, OCTS-PDL1BCMAt DNA sequence dna combination designed, designed (ginseng
It is shown in Table 1), gives Shanghai Jierui Biology Engineering Co., Ltd's synthesis, and preserve with oligonucleotides dry powder or plasmid form;
The slow virus recombinant plasmid of table 1 designs
(4) toolenzyme Cla I, Pst I, BsrG I, Nde I, EcoR I, BamH I, ApaL I, Spe I, T4DNA connect
Connect enzyme and be purchased from NEB companies;
(5) 0.22 μm of -0.8 μm of PES filters are purchased from millipore companies;D-PBS (-), 0.4% trypan blue, screen cloth,
All types of Tissue Culture Dish, culture bag, culture plate are purchased from corning companies;
(6)Opti-MEM、Pen-Srep、Hepes、FBS、AIM-V、RPMI 1640、DMEM、lipofectamine
3000 are purchased from invitrogen companies;Biotinylated protein L are purchased from GeneScript companies;LDH detection kits
Purchased from promega companies;Ficoll lymphocyte separation mediums are purchased from GE companies;20% human serum albumin injection is purchased from Ztel
Belling company;CryoPremium frozen stock solutions, sorting buffer solution voluntarily configure;RIL-2, rIL-7, rIL-15, rIL-21 purchase
From peprotech companies;CD3 monoclonal antibodies, CD28 monoclonal antibodies, CD3/CD28 magnetic bead CD4/CD8 magnetic beads are purchased from Germany
Miltenyi companies;
(7) refrigerated centrifuge is purchased from ThermoScientific companies of the U.S.;FACS flow cytometers are public purchased from Thermo
Department;Fluorescence inverted microscope is purchased from Olympus companies.
(8) CD4-FITC, CD8-APC are purchased from BioLegend companies;0.9% physiological saline is purchased from Jin Mai companies;
ProteinL Magnetic Beads are purchased from BioVision companies;PrimeSTAR, RetroNectin are purchased from Takara companies;
Phycoerythrin (PE)-conjugated streptavidin are purchased from BD Bioscience companies;Plasmid extraction reagent
Box, Ago-Gel QIAquick Gel Extraction Kit are purchased from MN companies;Competent cell TOP10 is purchased from tiangen companies;NaCl、KCl、
Na2HPO4.12H2O、KH2PO4、Trypsin、EDTA、CaCl2, NaOH, PEG6000 be purchased from Shanghai life work.
(9) DNeasy kits are purchased from Shanghai JaRa company;SA-HRP is purchased from Shanghai Yi Sheng companies;
(10) primer:Primer according to needed for design of primers principle designs amplification of DNA fragments and target site, the primer is by upper
Marine growth company synthesizes, and is specially:
EF1α-F:5’-attcaaaattttatcgatgctccggtgcccgtcagt-3’(SEQ ID NO.34)
EF1α-R:5’-TCACGACACCTGAAATGGAAGA-3’(SEQ ID NO.35)
OCTS-F:CATTTCAGGTGTCGTGAGGATCCGCCACCATGGCGCTGCCGGTGAC(SEQ ID NO.36)
OCTS-R:GGGGAGGGAGAGGGGCTTAGCGCGGCGGCAGCG(SEQ ID NO.37)
IRES-F:GCCCCTCTCCCTCCCCC(SEQ ID NO.38)
IRES-R:ATTATCATCGTGTTTTTCAAAGGAA(SEQ ID NO.39)
PDL1scab-F:AAAACACGATGATAATGCCACCATGAACTCCTTCTCCACAAGCG(SEQ ID NO.40)
PDL1scab-R:AATCCAGAGGTTGATTGTCGACGAATTCTCATTTGCCCGGGCTCAG(SEQ ID
NO.41)
WPRE-QPCR-F:5’-CCTTTCCGGGACTTTCGCTTT-3’(SEQ ID NO.42)
WPRE-QPCR-R:5’-GCAGAATCCAGGTGGCAACA-3’(SEQ ID NO.43)
Actin-QPCR-F:5’-CATGTACGTTGCTATCCAGGC-3’(SEQ ID NO.44)
Actin-QPCR-R:5’-CTCCTTAATGTCACGCACGAT-3’(SEQ ID NO.45).
The OCTS-CAR-T cell constructions of embodiment one
First, recombined lentivirus vector lvOCTS123BCMAs, lvOCTS319BCMAs, lvOCTS38BCMAs, lvOCTS-
PDL1BCMAs, lvOCTS123BCMAt, lvOCTS319BCMAt, lvOCTS38BCMAt, lvOCTS-PDL1BCMAt structure
Build, purify, detect
As shown in figure 3, the construction method of recombined lentivirus vector of the present invention is as follows:
1st, by people EF1 α promoters, the double targeting Chimeric antigen receptors of the CAR based on OCTS (OCTS123BCMAs,
OCTS319BCMAs、OCTS38BCMAs、OCTS-PDL1BCMAs、OCTS123BCMAt、 OCTS319BCMAt、
OCTS38BCMAt, OCTS-PDL1BCMAt), PDL1 single-chain antibodies be cloned into slow virus skeleton plasmid pLenti-3G basic,
Respectively obtain recombinant slow virus plasmid pOCTS123BCMAs, pOCTS319BCMAs, pOCTS38BCMAs, pOCTS-
PDL1BCMAs、pOCTS123BCMAt、 pOCTS319BCMAt、pOCTS38BCMAt、pOCTS-PDL1BCMAt。
(1) slow virus skeleton plasmid pLenti-3G basic are carried out using Cla I and EcoR I restriction enzymes double
Digestion, product pass through 1.5% agarose gel electrophoresis, confirm 5823bp fragment V1, and recovery of tapping rubber is placed in
In Eppendorf pipes, corresponding fragment (being shown in Table 2) is reclaimed with the Ago-Gel QIAquick Gel Extraction Kit of MN companies, and determine product
Purity and concentration;
The Ago-Gel recycling step of table 2
Step | Concrete operations |
Colloidal sol | Sol solutionses are added in 200 μ l NTI/100mg gel ratios, 5-10 minutes are placed in 50 DEG C of water-baths. |
With reference to DNA | 11000g is centrifuged 30 seconds, discards filtrate. |
Wash film | 700 μ l NT3,11000g centrifugation 30 seconds is added, discards filtrate. |
Wash film | Repeat the 3rd step once |
Dry | 11000g is centrifuged 1 minute, the collecting pipe renewed, and room temperature is placed 1 minute. |
Eluted dna | 15-30 μ l NE are added, room temperature is placed 1 minute, and 11000g is centrifuged 1 minute, collects filtrate. |
(2) with primer EF1 α-F and EF1 α-R using the SEQ ID NO.14 synthesized as template, using the system in table 3,
PCR cycle condition is:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 2min) * 35cycle, 72 DEG C of 10min.Product
By 1.5% agarose gel electrophoresis, 1208bp fragment a is confirmed, and recovery of tapping rubber is placed in Eppendorf pipes, uses MN
The Ago-Gel QIAquick Gel Extraction Kit of company reclaims corresponding fragment (being shown in Table 2), and determines the purity and concentration of product.
The μ L PCR reaction systems of table 3 50
Reagent | Volume (μ L) |
H2O | 32.5 |
5×Buffer(with Mg2+) | 10 |
DNTP (each 2.5mM) | 4 |
Primer1(+)(10μM) | 1 |
Primer2 (-) (10 μM) | 1 |
Template | 1 |
PrimeSTAR | 0.5 |
(3) with primer OCTS-F and OCTS-R using the OCTS123BCMAs synthesized as template, using the system in table 3,
PCR cycle condition is:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product
By 1.5% agarose gel electrophoresis, 2357bp fragment b is confirmed, and recovery of tapping rubber is placed in Eppendorf pipes, uses MN
The Ago-Gel QIAquick Gel Extraction Kit of company reclaims corresponding fragment (being shown in Table 2), and determines the purity and concentration of product;
(4) with primer OCTS-F and OCTS-R using the OCTS319BCMAs synthesized as template, using the system in table 3,
PCR cycle condition is:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product
By 1.5% agarose gel electrophoresis, 2375bp fragment c is confirmed, and recovery of tapping rubber is placed in Eppendorf pipes, uses MN
The Ago-Gel QIAquick Gel Extraction Kit of company reclaims corresponding fragment (being shown in Table 2), and determines the purity and concentration of product;
(5) system in table 3, PCR are used using the OCTS38BCMAs synthesized as template with primer OCTS-F and OCTS-R
Cycling condition is:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis is crossed, confirms 2378bp fragment d, and recovery of tapping rubber is placed in Eppendorf pipes, it is public with MN
The Ago-Gel QIAquick Gel Extraction Kit of department reclaims corresponding fragment (being shown in Table 2), and determines the purity and concentration of product;
(6) with primer OCTS-F and OCTS-R using the OCTS-PDL1BCMAs synthesized as template, using the system in table 3,
PCR cycle condition is:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product
By 1.5% agarose gel electrophoresis, 2372bp fragment e is confirmed, and recovery of tapping rubber is placed in Eppendorf pipes, uses MN
The Ago-Gel QIAquick Gel Extraction Kit of company reclaims corresponding fragment (being shown in Table 2), and determines the purity and concentration of product;
(7) with primer OCTS-F and OCTS-R using the OCTS123BCMAt synthesized as template, using the system in table 3,
PCR cycle condition is:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product
By 1.5% agarose gel electrophoresis, 2387bp fragment f is confirmed, and recovery of tapping rubber is placed in Eppendorf pipes, uses MN
The Ago-Gel QIAquick Gel Extraction Kit of company reclaims corresponding fragment (being shown in Table 2), and determines the purity and concentration of product;
(8) with primer OCTS-F and OCTS-R using the OCTS319BCMAt synthesized as template, using the system in table 3,
PCR cycle condition is:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product
By 1.5% agarose gel electrophoresis, 2403bp fragment g is confirmed, and recovery of tapping rubber is placed in Eppendorf pipes, uses MN
The Ago-Gel QIAquick Gel Extraction Kit of company reclaims corresponding fragment (being shown in Table 2), and determines the purity and concentration of product;
(9) system in table 3, PCR are used using the OCTS38BCMAt synthesized as template with primer OCTS-F and OCTS-R
Cycling condition is:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis is crossed, confirms 2406bp fragment h, and recovery of tapping rubber is placed in Eppendorf pipes, it is public with MN
The Ago-Gel QIAquick Gel Extraction Kit of department reclaims corresponding fragment (being shown in Table 2), and determines the purity and concentration of product;
(10) body in table 3 is used using the OCTS-PDL1BCMAt synthesized as template with primer OCTS-F and OCTS-R
System, PCR cycle condition are:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.
Product passes through 1.5% agarose gel electrophoresis, confirms 2433bp fragment i, and recovery of tapping rubber is placed in Eppendorf pipes,
Corresponding fragment (being shown in Table 2) is reclaimed with the Ago-Gel QIAquick Gel Extraction Kit of MN companies, and determines the purity and concentration of product;
(11) with primer I RES-F and IRES-R using the SEQ ID NO.12 synthesized as template, using the system in table 3,
PCR cycle condition is:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product
By 1.5% agarose gel electrophoresis, 575bp fragment j is confirmed, and recovery of tapping rubber is placed in Eppendorf pipes, uses MN
The Ago-Gel QIAquick Gel Extraction Kit of company reclaims corresponding fragment (being shown in Table 2), and determines the purity and concentration of product;
(12) with primer PDL1scab-F and PDL1scab-R using the SEQ ID NO.33 synthesized as template, using in table 3
System, PCR cycle condition is:98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C
5min.Product passes through 1.5% agarose gel electrophoresis, confirms 1557bp fragment k, and recovery of tapping rubber is placed in Eppendorf
In pipe, corresponding fragment (being shown in Table 2) is reclaimed with the Ago-Gel QIAquick Gel Extraction Kits of MN companies, and determine product purity and
Concentration;
(16) by recombinant slow virus DNA fragment combination (being shown in Table 4) with 5 μ l cumulative volumes and mol ratio 1:1:1:1 ratio
Example is added in Eppendorf pipes, is added the μ l of homologous recombination enzyme reaction solution 15, is incubated 30 minutes at 42 DEG C after mixing, is transferred to ice
Upper placement 2-3 minutes, reaction solution was added in 50 μ l TOP10, gently rotated to mix content, 30 points are placed in ice
Clock, pipe is put into pre-heating heat shock 90 seconds into 42 DEG C of thermostat water bath, quickly pipe is transferred in ice bath, makes cell cold
But 2-3 minutes, often pipe, is then transferred on 37 DEG C of shaking tables by pipe plus 900 μ l LB nutrient solutions, and incubating 1 hour makes bacteria resuscitation,
Take 100 μ l transformed bacteria solution to be coated on Amp LB agar plates, be inverted plate, 37 DEG C of cultures in constant incubator, 16 is small
When.
The recombinant slow virus DNA fragment combination of table 4
Recombinant slow virus plasmid | Fragment combination |
pOCTS123BCMAs | a、b、j、k |
pOCTS319BCMAs | a、c、j、k |
pOCTS38BCMAs | a、d、j、k |
pOCTS-PDL1BCMAs | a、e、j、k |
pOCTS123BCMAt | a、f、j、k |
pOCTS319BCMAt | a、g、j、k |
pOCTS38BCMAt | a、h、j、k |
pOCTS-PDL1BCMAt | a、i、j、k |
Picked clones carry out bacterium colony PCR identifications, identify that correctly clone is recombinant slow virus plasmid pOCTS123BCMA
s、pOCTS319BCMAs、pOCTS38BCMAs、pOCTS-PDL1BCMAs、 pOCTS123BCMAt、pOCTS319BCMAt、
POCTS38BCMAt, pOCTS-PDL1BCMAt, digestion identification (see Fig. 5) is carried out to correctly clone, and send sequencing review knot
Fruit.
As shown in figure 5, A figures represent pOCTS123BCMAs digestion prognostic chart and digestion agarose gel electrophoresis figure.
Lane1 is 1kb DNA ladder Marker digestion prognostic chart, and band is followed successively by from top to bottom:10kb、8kb、6kb、
5kb、 4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;Lane2 is pOCTS123BCMAs
BamH I digestions prediction, band is followed successively by from top to bottom:9712bp、1277bp、1023bp、511bp;Lane3 is 1kb
DNA ladder Marker electrophoresis result;Lane4 is pOCTS123BCMAs BamH I restriction enzyme digestion and electrophoresis results.
B figures represent pOCTS319BCMAs digestion prognostic chart and digestion agarose gel electrophoresis figure.Lane1 is 1kb DNA
Ladder Marker digestion prognostic chart, band are followed successively by from top to bottom:10kb、8kb、6kb、5kb、4kb、3.5kb、 3kb、
2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;Lane2 is pOCTS319BCMAs ApaL I digestions prediction:
Band is followed successively by from top to bottom:6543bp、1961bp、1723bp、1234bp、488bp;Lane3 is 1kb DNA ladder
Marker electrophoresis result;Lane4 is pOCTS319BCMAs ApaL I restriction enzyme digestion and electrophoresis results.
C figures represent pOCTS38BCMAs digestion prognostic chart and digestion agarose gel electrophoresis figure.Lane1 is 1kb DNA
Ladder Marker digestion prognostic chart, band are followed successively by from top to bottom:10kb、8kb、6kb、5kb、4kb、3.5kb、 3kb、
2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;Lane2 be pOCTS38BCMAs BsrG I digestions prediction, bar
Band is followed successively by from top to bottom:10928bp、1087bp;Lane3 is 1kb DNA ladder Marker electrophoresis result;lane4
It is pOCTS38BCMAs BsrG I restriction enzyme digestion and electrophoresis results.
D figures represent pOCTS-PDL1BCMAs digestion prognostic chart and digestion agarose gel electrophoresis figure.Lane1 is 1kb
DNA ladder Marker digestion prognostic chart, band are followed successively by from top to bottom:10kb、8kb、6kb、5kb、4kb、 3.5kb、
3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;Lane2 is pOCTS-PDL1BCMAs Cla I digestions
Prediction, band are followed successively by from top to bottom:8876bp、3139bp;Lane3 is 1kb DNA ladder Marker electrophoresis knot
Fruit;Lane4 is pOCTS-PDL1BCMAs Cla I restriction enzyme digestion and electrophoresis results.
E figures represent pOCTS123BCMAt digestion prognostic chart and digestion agarose gel electrophoresis figure.Lane1 is 1kb DNA
Ladder Marker digestion prognostic chart, band are followed successively by from top to bottom:10kb、8kb、6kb、5kb、4kb、3.5kb、 3kb、
2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;Lane2 is pOCTS123BCMAt BamH I digestions prediction:Bar
Band is followed successively by from top to bottom:9545bp、1467bp、977bp;Lane3 is 1kb DNA ladder Marker electrophoresis result;
Lane4 is pOCTS123BCMAt BamH I restriction enzyme digestion and electrophoresis results.
F figures represent pOCTS319BCMAt digestion prognostic chart and digestion agarose gel electrophoresis figure.Lane1 is 1kb DNA
Ladder Marker digestion prognostic chart, band are followed successively by from top to bottom:10kb、8kb、6kb、5kb、4kb、3.5kb、 3kb、
2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;Lane2 be pOCTS319BCMAt Nde I digestions prediction, bar
Band is followed successively by from top to bottom:9686bp、2342bp;Lane3 is 1kb DNA ladder Marker electrophoresis result;lane4
It is pOCTS319BCMAt Nde I restriction enzyme digestion and electrophoresis results.
G figures represent pOCTS38BCMAt digestion prognostic chart and digestion agarose gel electrophoresis figure.Lane1 is 1kb DNA
Ladder Marker digestion prognostic chart, band are followed successively by from top to bottom:10kb、8kb、6kb、5kb、4kb、3.5kb、 3kb、
2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;Lane2 is pOCTS38BCMAt Spe I digestions prediction:Band
It is followed successively by from top to bottom:9976bp、1926bp;Lane3 is 1kb DNA ladder Marker electrophoresis result;Lane4 is
POCTS38BCMAt Spe I restriction enzyme digestion and electrophoresis results.
H figures represent pOCTS-PDL1BCMAt digestion prognostic chart and digestion agarose gel electrophoresis figure.Lane1 is 1kb
DNA ladder Marker digestion prognostic chart, band are followed successively by from top to bottom:10kb、8kb、6kb、5kb、4kb、3.5kb、
3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;Lane2 is pOCTS-PDL1BCMAt Pst I digestions
Prediction, band are followed successively by from top to bottom:10821bp、1340bp、411bp;Lane3 is 1kb DNA ladder Marker electricity
Swimming result;Lane4 is pOCTS-PDL1BCMAt Pst I restriction enzyme digestion and electrophoresis results.
From the electrophoresis result shown by Fig. 5, the restriction enzyme digestion and electrophoresis result of each recombinant slow virus plasmid to be measured and digestion are pre-
Survey result is basically identical, and the construction method of above-mentioned recombinant slow virus plasmid is effective.
2nd, recombined lentivirus vector lvOCTS123BCMAs, lvOCTS319BCMAs, lvOCTS38BCMAs, lvOCTS-
PDL1BCMAs, lvOCTS123BCMAt, lvOCTS319BCMAt, lvOCTS38BCMAt, lvOCTS-PDL1BCMAt packaging
2.1 solution allocation
(1) complete medium:Preheated fresh culture is taken out, adds 10%FBS+5ml Pen-Srep, up and down top
Mix;
(2) 1XPBS solution:Weigh NaCl 8g, KCl 0.2, Na2HPO4.12H2O 3.58g, KH2PO4 0.24g are placed in
In 1000ml beakers, the dissolving of 900ml Milli-Q grade ultra-pure waters is added, after the completion of dissolving, uses 1000ml graduated cylinder constant volumes
To 1000ml, 121 DEG C of high-temperature heat sterilization 20min;
(3) 0.25%Trypsin solution:Trypsin 2.5g are weighed, EDTA0.19729g is placed in 1000ml beakers, added
Enter 900ml 1XPBS dissolvings, after the completion of dissolving, 1000ml is settled to using 1000ml graduated cylinders, 0.22 μM of filtration sterilization, for a long time
Using can preserve to -20 DEG C of refrigerators;
(4) 0.5M CaCl2 solution:Weigh 36.75g CaCl2Dissolved with 400ml Milli-Q grade ultra-pure waters;With
Cumulative volume is settled to 500ml by Milli-Q grade ultra-pure waters, is mixed;0.22 μm of filtration sterilization, packing are saved in 50ml centrifugations
Guan Zhong, often pipe 45ml or so, 4 DEG C of preservations.
(5) 2XHBS solution:Weigh 4.09g NaCl, 0.269g Na2HPO4,5.96g Hepes, with 400ml Milli-
Q grade ultra-pure waters dissolve;After calibrating pH instrument, the pH of HBS solution is transferred to 7.05 with 2M NaOH solutions.Adjust every bottle of HBS
PH consumption 2M NaOH be 3ml or so.
2.2HEK293T/17 cell culture
(1) the HEK293T/17 cells frozen are taken out from liquid nitrogen container, are quickly transferred in 37 DEG C of water-baths, after 1~2min
It is transferred in super-clean bench, the liquid in cryopreservation tube is fully transferred to 10cm by sterile working2In culture dish, supply containing 10%FBS
DMEM to 8mL/10cm2Micro- sem observation cell after dish, 24h, the degree of cell confluency are passed on more than 80%;
(2) select that cell state is good, free of contamination HEK293T/17 cells, be one group per 2-6 culture dish, will be carefully
After the digestion of born of the same parents' pancreatin, 4-12ml complete mediums are drawn with electric pipettor, 2ml is added into each postdigestive culture dish, is kept away
Exempt from culture dish exsiccation;All cells are blown and beaten into single cell suspension using 1ml pipettors, are transferred in medium bottle;
(3) remaining cell in above-mentioned 2-6 culture dish is transferred in medium bottle, and rinsed again with culture medium once
Culture dish;
(4) culture medium bottle cap is covered tightly, turns upside down 10 times or so and fully mixes cell suspension, cell is passed to 8-24
10cm2In culture dish, the cell density per ware should about 4 × 106Individual/10ml complete mediums or so.If cell density and pre-
The difference of phase is larger, then needs to count cell, then according to 4 × 106The amount inoculation of individual/ware;
(5) every 6 culture dishes arrange piles up for one, pays attention to keeping the cooperation between ware up and down.By culture dish or so, front and rear rolling
Move for several times, cell is fully spread out, be then placed in 5%CO2Incubator.Remaining cell does same processing;
(6) institute's passage cell is checked, cell confluency degree should be 70-80%, and profile is full, adherent good, is trained in cell
Support and be uniformly distributed in ware;
(7) liquid is changed for cell, culture medium is replaced with into fresh complete medium, per ware 9ml, and by the CO of incubator2It is dense
Degree setting value brings up to 8%;
2.3 cell transfecting
(1) DNA/CaCl is matched somebody with somebody according to N+0.52Solution.Per ware HEK293T/17 cell transfecting plasmid amounts according to following ratio
Use:Recombinant slow virus plasmid (20 μ g), pPac-GP (15 μ g), pPac-R (10 μ g), pEnv-G (7.5 μ g).Take one it is new
5ml centrifuge tubes, add 0.5M CaCl2:0.25ml, the μ g of recombinant slow virus plasmid 20:pPac-GP 15μg:pPac-R 10μg:
The μ g of pEnv-G 7.5, supplement ultra-pure water to 0.5ml close the lid, and fully mix;
(2) it is another to take a 5ml centrifuge tube, add 0.5ml DNA/CaCl2 solution.Turbula shaker is opened, a hand is taken
The firmly upper end of 5ml centrifuge tubes, ttom of pipe is contacted oscillating end, liquid is scattered on tube wall flowing, another hand is by one 1mL
Liquid-transfering gun, 0.5mL 2 × HBS solution is drawn, is slowly added dropwise into centrifuge tube, coutroi velocity, being dripped off with half a minute is advisable. 2×
After HBS is added, continue vibration 5 seconds, stop oscillation, can be directly added into the cell for needing to transfect;
(3) take a ware cell, the 1mL calcium in centrifuge tube is turned into drop adds, make as far as possible calcium turn reagent be distributed to it is whole
In individual culture dish;
(4) after calcium turns liquid addition, covered in ware and carry out mark, culture dish is released to another 5%CO2In incubator.
Ensure that culture dish is horizontal positioned, often pile up culture dish and do not exceed 6.In 5%CO2(6-8h) is placed in incubator;
(5) by the CO of first incubator2Concentration set point adjusts back to 5%;
After (6) 24 hours, cell state is checked.Cell confluency degree should be 80-85% or so, in good condition.Will culture
Base siphons away, and changes the fresh DMEM complete mediums of 10ml;
After (7) 48 hours, transfection efficiency is observed.Most cells are still adherent.It is it can be seen that thin more than 95%
Born of the same parents can carry green fluorescence.Same virus is packed into supernatant collection to together, and continues addition 10mL into culture dish
Fresh culture;
After (8) 72 hours, same vial supernatant is collected into the virus together, collected twice again can be placed on one
Rise, abandon culture dish;Contained in the supernatant now collected recombined lentivirus vector lvOCTS123BCMAs,
lvOCTS319BCMAs、lvOCTS38BCMAs、lvOCTS-PDL1BCMAs、lvOCTS123BCMAt、 lvOCTS319BCMAt、
lvOCTS38BCMAt、lvOCTS-PDL1BCMAt。
3rd, ion exchange chromatography recombined lentivirus vector
(1) supernatant of collection is used into Thermo vavuum pumps, filtered through 0.22 μm -0.8 μm of PES filters, except impurity elimination
Matter;
(2) 1 is pressed:1~1:10 ratio is toward adding 1.5M NaCl 250mM Tris-HCl (pH 6-8) in supernatant;
(3) 2 ion exchange columns are placed in series, with 4ml 1M NaOH, 4ml 1M NaCl, 5ml 0.15M NaCl
25mM Tris-HCl (pH 6-8) solution crosses post successively;
(4) solution obtained in step 2 is given to ion exchange column loading by peristaltic pump with 1-10ml/min speed;
(5) after whole supernatants cross post, cleaned using 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution
One time;
(6) eluted according to applied sample amount using 1-5ml 1.5M NaCl 25mM Tris-HCl (pH 6-8), collection is washed
De- liquid;
(7) eluent is divided into 25 to 50 μ L mono- to manage, freezes -80 DEG C of refrigerators, preserved for a long time.
4th, recombined lentivirus vector titer determination
(1) 24 orifice plates are taken to be inoculated with 293T cells.It is 5 × 10 per hole cell4Individual, added culture volume is 500ul, different
The vitro growth rates of species difference, cell confluency when carrying out virus infection is 40%-60%;
(2) prepare 3 sterile EP pipes, 90ul fresh complete medium (DMEM in high glucose+10% is added in each pipe
FBS) inoculating cell takes the cell in two holes to be counted with blood counting chamber after 24 hours, it is determined that infection when cell actual number
Mesh, it is designated as N;
(3) take virus stock solution used 10ul to be determined to be added in first pipe, after gently mixing, take 10ul to be added to second
In individual pipe, a to the last pipe is then operated successively;410ul complete medium (DMEM in high glucose+10% is added in every pipe
), FBS final volume 500ul;
(4) 20 hours after infection starts, culture supernatant is removed, is replaced by 500 μ l complete medium (DMEM in high glucose+10%
FBS), 5%CO2Continue culture 48 hours;
After (5) 72 hours, luciferase expression situation is observed, under normal circumstances, fluorecyte number increases and phase with extension rate
It should reduce, and take pictures;
(6) the pancreas enzyme -EDTA solution digestion cells of 0.2ml 0.25% are used, are placed 1 minute at 37 DEG C.Purged with culture medium whole
Individual cell face, is collected by centrifugation cell.Genomic DNA is extracted according to the explanation of DNeasy kits.Added in each sample cell
200 μ l eluents are washed lower DNA and quantified;
(7) preparing target DNA detection house steward qPCRmix I, (QPCR primer sequences are SEQ ID NO.42---SEQ ID
NO.43):
Component in table house steward 5qPCRmix I
2×TaqMan Master Mix | 25μl×n |
Forward primer(100pmol ml-1) | 0.1μl×n |
Reverse primer(100pmol ml-1) | 0.1μl×n |
Probe(100pmol ml-1) | 0.1μl×n |
H2O | 19.7μl×n |
N=number of reactions. are for example:Overall reaction number is 40, by 2 × TaqMan of 1ml Universal
PCR Master Mix, 4 μ l forward primer, 4 μ l reverse primer, 4 μ l probe and 788 μ l H2O are mixed
Be placed on ice after concussion.
(8) preparing internal reference DNA detection qPCRmix pipes II, (QPCR primer sequences are SEQ ID NO.44---SEQ ID
NO.45):
Component in house steward qPCRmix II of table 6
2×TaqMan Master Mix | 25μl×n |
10×RNaseP primer/probe mix | 2.5μl×n |
H2O | 17.5μl×n |
N=number of reactions. are for example:Overall reaction number is 40, by 2 × TaqMan of 1ml Universal
PCR Master Mix, 100 μ l 10 × RNaseP primer/probe mix and 700 μ l H2O is mixed, and ice is placed on after concussion
On.
(9) PCR system is completed in 96 hole PCR plates of precooling to establish.45 μ l are respectively taken to be added to each rows of A-D from house steward I
Hole in, respectively take 45 μ l to be added in the hole of each rows of E-G from house steward II.
(10) 5 μ l plasmid standards and testing sample genomic DNA is taken to be added in A-D rows respectively, each sample repeats 1
It is secondary.It is another to stay the water that 1 hole adds 5 μ l as no template control (no-template control).
(11) 5 μ l genomes standard items and testing sample genomic DNA is taken to be added in E-G rows respectively, each sample weight
It is multiple 1 time.It is another to stay the water that 1 hole adds 5 μ l as no template control (no-template control).
(12) it is the quantitative systems of ABI PRISM 7500 to use quantitative PCR apparatus.Cycling condition is set as:50 DEG C 2 minutes,
95 DEG C 10 minutes, followed by 95 DEG C 15 seconds, 60 DEG C of 40 of 1 minute circulations.
(13) data analysis:The slow virus carrier copy number integrated in the DNA sample measured is demarcated with genome number,
Obtain the viral copy number of every genome conformity.
Titre (integration units per ml, IU ml-1) calculation formula it is as follows:
IU ml-1=(C × N × D × 1000)/V
Wherein:Viral copy numbers of the C=averagely per genome conformity
The number (about 1 × 10 of cell when N=infects5)
The extension rate of D=viral vectors
The volume number for the dilution virus that V=is added
Recombined lentivirus vector lvOCTS123BCMAs, lvOCTS319BCMAs, lvOCTS38BCMAs, lvOCTS-
PDL1BCMAs, lvOCTS123BCMAt, lvOCTS319BCMAt, lvOCTS38BCMAt, lvOCTS-PDL1BCMAt titre
As a result it is as shown in Figure 6.
2nd, OCTS-CAR-T cell constructions
Fig. 7 shows OCTS-CAR-T cell construction flows, the construction method of the OCTS-CAR-T cells in the present embodiment
It is as follows:
1st, PBMC is separated.
(1) health donors fresh peripheral blood 50ml is extracted;
(2) blood taking bag spray is wiped into alcohol twice, and dried.
(3) haemocyte in bag is sucked out with 50ml syringes and moved in new 50ml pipes.
(4) 400g, 20 DEG C of centrifugation 10min.
(5) upper plasma is moved on in new 50ml centrifuge tubes, 56 DEG C, 30min inactivation blood plasma, recovered to room temperature,
2000g, 30min is centrifuged, takes supernatant stand-by into 50ml centrifuge tubes.
(6) mended with D-PBS (-) to 50ml, tighten lid, overturned and mix.
(7) 2 new 50ml centrifuge tubes are taken, often pipe adds 15ml Ficoll lymphocyte separation mediums.
(8) to being carefully added into haemocyte dilution 25ml on every pipe Ficoll.800g, 20 DEG C of centrifugation 20min.
(9) liquid is divided into four layers in centrifuge tube, is respectively from top to bottom:The plasma layer (recovery stand-by) of yellow, tunica albuginea layer,
The Ficoll layers of water white transparency, the cell mixing layer of reddish black.
(10) tunica albuginea layer is carefully drawn into new 50ml centrifuge tubes, is added D-PBS (-) to 50ml, is overturned 500g after mixing,
20 DEG C of centrifugation 10min.
(11) human serum albumins of 25ml 5% are added and cell is resuspended, 400g, 20 DEG C of centrifugation 10min.
(12) supernatant is abandoned, the human serum albumins of 25ml 5% is added and cell precipitation is resuspended, and crosses 70um screen clothes, is counted.
(13) 1 part is taken to contain 1.25x108Cells is used to activate;Remaining cell suspension 400g, 20 DEG C of centrifugation 10min, adds
CryoPremium simultaneously freezes.
2nd, CD4/CD8 positive T cells sort.
(1) PBMC of acquisition is counted, with 80ul/107Cells ratio adds sorting buffer solution, and cell precipitation is resuspended.
(2) again with 20ul/107Cells ratio adds CD4/CD8 magnetic beads, and piping and druming is put into 4 DEG C after mixing and is incubated
15min。
(3) magnetic bead-cell mixture is taken out, with 2ml/107Cells ratio adds sorting buffer solution, overturns after mixing,
250g, 4 DEG C of centrifugation 10min.
(4) with 500ul/108Cells ratio adds sorting buffer solution, and cell precipitation is resuspended.
(5) LS splitters are gripped to magnetic frame with tweezers.
(6) while prepare 2 15ml centrifuge tubes, mark respectively:CD4-/CD8- cell liquid (A pipes), CD4+/CD8+ cells
Liquid (B pipes).
(7) 3ml dissociating buffer rinse LS are used, and buffer solution is connect with A pipes.
(8) cell-magnetic bead mixed liquor is added, 3ml wash buffers pillar is added after dripping off (during each no liquid residual again
Add new liquid), altogether three times, collection obtains CD4/CD8- cells.
(9) LS splitters separate with magnetic frame, connect cell suspension with B pipes, add 5ml buffer solutions, will and be filled in in pillar
Slightly firmly rinse, be collected as CD4+/CD8+ cells, sampling counts.
(10) 1x10 is pressed6/ml-4x106/ ml cell density AIM-V culture mediums resuspension cell precipitation, and addition 2 ×
105~1 × 106The U/L IFN-γ factors.
3rd, t cell activation.
(1) the previous day is carried by 1 × 103Ug/L~1 × 104Ug/L CD3 monoclonal antibodies and 1 × 103Ug/L~1 ×
104Ug/L CD28 monoclonal antibodies add 24 orifice plates, and sealed membrane sealing, 4 DEG C are coated with overnight;
(2) coated T75 bottles are taken out, coating buffer is outwelled, washed once with D-PBS (-), and obtained cell will be sorted
Suspension is inoculated into T75 bottles, is shaken up, and is put into 37 DEG C, 5%CO2Cultivated in incubator.
4th, CAR gene transfers and OCTS-CAR-T cell induction cultures.
(1) the previous day coating 1 × 10 is put forward3Ug/L~1 × 104In in 24 orifice plates, sealed membrane seals ug/L RetroNectin
Mouthful, 4 DEG C are coated with overnight.
(2) toward in 24 orifice plates, according to every hole 5 × 105Cell concentration, by the amount of MOI=5~20, it is separately added into
lvOCTS123BCMAs、lvOCTS319BCMAs、lvOCTS38BCMAs、lvOCTS-PDL1BCMAs、 lvOCTS123BCMAt、
LvOCTS319BCMAt, lvOCTS38BCMAt, lvOCTS-PDL1BCMAt slow virus transgene carrier, while add containing 2 ×
105~5 × 105U/L rIL-2,5 × 103Ng/L~1 × 104Ng/L rIL-7,5 × 103Ng/L~1 × 104ng/L rIL-
15,5 × 103Ng/L~1 × 104Ng/L rIL-21 and 37 DEG C of AIM-V culture mediums, 5%CO containing 10% autoserum2Continue
Culture.
5th, OCTS-CAR-T cell expansion ex vivos.
(1) every 2 days equivalent is added containing 2 × 105~5 × 105U/L rIL-2,5 × 103Ng/L~1 × 104ng/L rIL-
7,5 × 103Ng/L~1 × 104Ng/L rIL-15,5 × 103Ng/L~1 × 104Ng/L rIL-21 and containing 10% autoserum
AIM-V culture mediums, make between pH value maintains 6.5~7.5, cell density maintains 5 × 105~2 × 106Between/ml, 37
DEG C, 5%CO2Continue culture 10-14 days.
(2) the 7th days or so, the OCTS-CAR-T cells for freezing culture were used for subsequent detection.
Embodiment 2OCTS-CAR-T cells Pathogen test and detection of expression
First, endotoxin detects;
(1) endotoxin working standard is 15EU/ branch;
(2) sensitivity of the limulus reagent λ=0.25EU/ml, 0.5ml/ are managed
(3) endotoxin standard dilutes:Endotoxin standard one is taken, is diluted to 4 λ and 2 λ in proportion with BET water respectively
Dissolving, sealed membrane sealing, concussion dissolving 15min;A step is often diluted during dilution all should mix 30s on eddy mixer;
(4) it is loaded:If taking the TAL Heavenly Stems and Earthly Branches, every adds BET water 0.5ml dissolvings, and as Heavenly Stems and Earthly Branches endotoxin-free is tried for packing
Guan Zhong, often pipe 0.1ml.Wherein 2 are negative control pipe, add BET water 0.1ml;2 are positive control pipe, and it is dense to add 2 λ
The endotoxin working standard solution 0.1ml of degree;2 are Sample Positive control tube, add 0.1ml and contain 2 λ endotoxin standards
Sample solution (the testing sample 1ml+4 λ endotoxin standard solution 1ml=2ml of 20 times of dilution contains 2 λ endotoxin standards
40 times of samples of dilution).
Add 0.1mL samples in sample cell, dilution ratio be shown in Table 7,37 ± 1 DEG C of water-baths (or incubator) insulation 60 ±
1min;
The endotoxin testing result of the endotoxin dilution ratio of table 7 and OCTS-CAR-T cells
As shown in table 7, the endotoxin content of all cells is respectively less than 2.5EU/ml, meets《Pharmacopoeia of People's Republic of China》
In be less than 10EU/ml standard.
2nd, detection of mycoplasma
(1) first three day is being tested, cell sample is cultivated with antibiotic-free culture medium;
(2) (cell number is more than 1 × 10 to collection 1mL cell suspending liquids5), it is placed in 1.5mL centrifuge tubes;
(3) 13000g centrifuges 1min, collects precipitation, discards culture medium;
(4) 500ul PBS pipette tips pressure-vaccum or vortex oscillation are added, precipitation is resuspended.13000g centrifuges 5min;
(5) step 4 is repeated once;
(6) 50 μ l Cell Lysis Buffer are added, with pipette tips pressure-vaccum, after fully mixing, are incubated in 55 DEG C of water-baths
20min;
(7) sample is placed in 95 DEG C and heats 5min;
(8) after 13000g centrifuges 5min, the 5 μ l supernatants are taken to be as template, 25 μ l PCR reaction systems:ddH20 6.5μl、
The μ l of Myco Mix 1,2x Taq Plus Mix Master (Dye Plus) 12.5 μ l, the μ l of template 5;PCR cycle condition is:95
DEG C 30sec, (95 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 30sec) * 30cycle, 72 DEG C of 5min.
Detection of mycoplasma result shown see Fig. 8, the judgement explanation of positive control, negative control and sample in Fig. 8
It is shown in Table 8.As shown in Fig. 8 results, mycoplasma is free of in OCTS-CAR-T cells.
The judgement explanation of the positive control of table 8, negative control and sample
3rd, the detection of OCTS gene transduction efficiencies and immunophenotyping detection;
(1) T cell after viral transduction is collected, cell is resuspended with D-PBS (-) solution containing 1~4% human serum albumin
And it is adjusted to 1 × 106/ml。
(2) D-PBS (-) solution 1ml containing 1~4% human serum albumin is added into centrifuge tube and is mixed, 350g centrifugations
5min, abandon supernatant.
(3) repeat step 2 is once.
(4) cell is resuspended with 0.2ml D-PBS (-) solution containing 1~4% human serum albumin, and is added into centrifuge tube
1ul 1mg/ul protein L, 5ul CD4-FITC, 5ul CD8-APC, mix, 4 DEG C of incubation 45min.
(5) D-PBS (-) solution of the 1ml containing 1~4% human serum albumin is added into centrifuge tube and is mixed, 350g centrifugations
5min, abandon supernatant.
(6) repeat step 5 is twice.
(7) cell is resuspended with D-PBS (-) solution of 0.2ml containing 1~4% human serum albumin, and is added into centrifuge tube
0.2ul PE-SA, mix, 37 DEG C of lucifuges are incubated 15min.
(8) add D-PBS (-) solution of 1ml containing 1~4% human serum albumin into centrifuge tube to weigh and mix, 350g centrifugations
5min, abandon supernatant.
(9) cell precipitation is resuspended with 1ml D-PBS (-) solution, 350g centrifugation 5min, abandons supernatant.
(10) repeat step 9 is twice.
(11) cell precipitation is resuspended with 0.4ml D-PBS (-) solution, flow cytometer is detected.
OCTS gene transduction efficiencies and immunophenotyping test result are as shown in figure 9, the OCTS-CAR-T cells prepared
Most of efficiency of infection between 37%~50%, the ratio of CD4 positive cells and CD8 positive cells is located at 1: 3~3: 1
Between, follow-up function detection can be carried out.
The Function detection of embodiment 3OCTS-CAR-T cells
First, target cell fragmentation effect is assessed.
(1) target cell [BCMA is cultivated respectively+K562、CD319+K562、CD38+K562、PDL1+K562、 CD123+K562、
BCMA+CD123+K562、BCMA+CD319+K562、BCMA+CD38+K562、 BCMA+PDL1+K562, K562 cell] and effect it is thin
Born of the same parents' [OCTS-CAR-T cells], the packet that effector cell is incubated altogether with single target cell and double target cells respectively are as shown in table 9.
The group list that the effector cell of table 9 is incubated altogether with single target cell and double target cells respectively
Effector cell | Target cell 1 | Target cell 2 | Target cell 3 |
OCTS123BCMAs-CAR-T | CD123+K562 | BCMA+K562 | BCMA+CD123+K562 |
OCTS319BCMAs-CAR-T | CD319+K562 | BCMA+K562 | BMCA+CD319+K562 |
OCTS38BCMAs-CAR-T | CD38+K562 | BCMA+K562 | BCMA+CD38+K562 |
OCTS-PDLlBCMAs-CAR-T | PDL1+K562 | BCMA+K562 | BCMA+PDL1+K562 |
OCTS123BCMAt-CAR-T | CD123+K562 | BCMA+K562 | BCMA+CD123+K562 |
OCTS319BCMAt-CAR-T | CD319+K562 | BCMA+K562 | BMCA+CD319+K562 |
OCTS38BCMAt-CAR-T | CD38+K562 | BCMA+K562 | BCMA+CD38+K562 |
OCTS-PDL1BCMAt-CAR-T | PDL1+K562 | BCMA+K562 | BCMA+PDL1+K562 |
(2) target cell 4x10 is collected5Cells and OCTS-CAR-T cells 2.8x106Cells, 800g, 6min are centrifuged, and are abandoned
Supernatant;
(3) target cell and effector cell are resuspended respectively with 1ml D-PBS (-) solution, 800g, 6min centrifugation, abandon supernatant;
(4) repeat step 3 is once;
(5) effector cell is resuspended with 700ul culture mediums (+1~10%FBS of AIM-V culture mediums), with 2ml culture mediums (AIM-
+ 1~10%FBS of V culture mediums) target cell is resuspended;
(6) it is 1 to set effect target ratio:1、5:1、10:1 experimental port, packet situation is as shown in table 9, and sets control group
(K562 cells), every group of 3 multiple holes;
(7) 250g, 5min flat board centrifuge;
(8) 37 DEG C, cultivated 4 hours in 5%CO2 incubators;
(9) 250g, 5min flat board centrifuge;
(10) the 50ul supernatants in each hole are taken into new 96 orifice plate, and 50ul substrate solutions (lucifuge behaviour is added per hole
Make);
(11) lucifuge is incubated 25min;
(12) 50ul terminate liquids are added per hole;
(13) ELIASA detection 490nm absorbances;
(14) 3 multiple holes are averaged;The light absorption value of all experimental ports, Target cell wells and effector cell hole is subtracted into training
Support the average of base background light absorption value;The light absorption value of target cell maximum is subtracted to the average of volume correction control light absorption value.
(15) bring the corrected value obtained in step 14 into formula below, calculate thin caused by each effect target ratio
Cellular toxicity percentage.
Killing-efficiency=(experimental port-effector cell hole-Target cell wells)/(target cell largest hole-Target cell wells) × 100%
As a result as shown in Figure 10, OCTS-CAR-T has preferably killing effect to respective single target cell and double target cells
Fruit, the CAR-T that the CAR-T cells of Turn OCTS structures are slightly above Series OCTS structures to the killing-efficiency of target cell are thin
Born of the same parents.
It is above-mentioned test result indicates that, by traditional CAR structures antigen recognizing district transformation formed OCTS structures,
OCTS-CAR-T cell recognitions can be significantly improved and kill the scope of target cell, therefore OCTS-CAR-T cells will be in future
The double positives of the BCMA positives/CD319 positives/CD38 positives/CD123 positives/PDL1 positives/BCMA, CD319/BCMA, CD38
In the cell therapies of malignant tumour such as the double positives of double positives/BCMA, CD123/double positive Huppert's diseases of BCMA, PDL1
Play huge effect.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, various changes and modifications of the present invention are possible without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and
Its equivalent defines.
SEQUENCE LISTING
<110>Shanghai You Kadi biological medicines Science and Technology Ltd.
<120>The double targeting Chimeric antigen receptors of OCTS-CAR, encoding gene, recombinant expression carrier and its structure and application
<130>Claims specification
<160> 45
<170> PatentIn version 3.5
<210> 1
<211> 837
<212> DNA
<213>Artificial sequence
<400> 1
atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240
cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300
gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360
tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540
cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctatcc 600
cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 660
gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 720
ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 780
acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcc 837
<210> 2
<211> 674
<212> DNA
<213>Artificial sequence
<400> 2
cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 60
ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 120
actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 180
gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 240
ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 300
gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 360
acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 420
tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 480
gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 540
cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 600
cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 660
ccttttgctc acat 674
<210> 3
<211> 147
<212> DNA
<213>Artificial sequence
<400> 3
atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 60
tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 120
ggcttttttg gaggcctaga cttttgc 147
<210> 4
<211> 228
<212> DNA
<213>Artificial sequence
<400> 4
gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 60
cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 120
tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc 180
gcattgcaga gatattgtat ttaagtgcct agctcgatac aataaacg 228
<210> 5
<211> 180
<212> DNA
<213>Artificial sequence
<400> 5
ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 60
tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 120
gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 180
<210> 6
<211> 234
<212> DNA
<213>Artificial sequence
<400> 6
tgctagagat tttccacact gactaaaagg gtctgaggga tctctagtta ccagagtcac 60
acaacagacg ggcacacact acttgaagca ctcaaggcaa gctttattga ggcttaagca 120
gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac cagagagacc 180
cagtacaagc aaaaagcaga tcttattttc gttgggagtg aattagccct tcca 234
<210> 7
<211> 353
<212> DNA
<213>Artificial sequence
<400> 7
atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc 60
ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg 120
agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa 180
tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata 240
atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag 300
ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caa 353
<210> 8
<211> 233
<212> DNA
<213>Artificial sequence
<400> 8
aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcctcaat 60
gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 120
gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 180
gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcc 233
<210> 9
<211> 489
<212> DNA
<213>Artificial sequence
<400> 9
tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta 60
gttggagtaa taaatctctg gaacagattg gaatcacacg acctggatgg agtgggacag 120
agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca 180
agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt 240
taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt 300
aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc 360
accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat 420
agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg 480
acggttaac 489
<210> 10
<211> 119
<212> DNA
<213>Artificial sequence
<400> 10
ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 60
agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaatttta 119
<210> 11
<211> 696
<212> DNA
<213>Artificial sequence
<400> 11
atggcccagt ccaagcacgg cctgaccaag gagatgacca tgaagtaccg catggagggc 60
tgcgtggacg gccacaagtt cgtgatcacc ggcgagggca tcggctaccc cttcaagggc 120
aagcaggcca tcaacctgtg cgtggtggag ggcggcccct tgcccttcgc cgaggacatc 180
ttgtccgccg ccttcatgta cggcaaccgc gtgttcaccg agtaccccca ggacatcgtc 240
gactacttca agaactcctg ccccgccggc tacacctggg accgctcctt cctgttcgag 300
gacggcgccg tgtgcatctg caacgccgac atcaccgtga gcgtggagga gaactgcatg 360
taccacgagt ccaagttcta cggcgtgaac ttccccgccg acggccccgt gatgaagaag 420
atgaccgaca actgggagcc ctcctgcgag aagatcatcc ccgtgcccaa gcagggcatc 480
ttgaagggcg acgtgagcat gtacctgctg ctgaaggacg gtggccgctt gcgctgccag 540
ttcgacaccg tgtacaaggc caagtccgtg ccccgcaaga tgcccgactg gcacttcatc 600
cagcacaagc tgacccgcga ggaccgcagc gacgccaaga accagaagtg gcacctgacc 660
gagcacgcca tcgcctccgg ctccgccttg ccctga 696
<210> 12
<211> 575
<212> DNA
<213>Artificial sequence
<400> 12
gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60
gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120
ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180
gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240
aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300
tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360
acgttgtgag ttggatagtt gtggaaagag tcaaatggct cacctcaagc gtattcaaca 420
aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480
gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc cgaaccacgg 540
ggacgtggtt ttcctttgaa aaacacgatg ataat 575
<210> 13
<211> 592
<212> DNA
<213>Artificial sequence
<400> 13
aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60
ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120
atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180
tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240
ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300
attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360
ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420
gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480
aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540
cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tg 592
<210> 14
<211> 1178
<212> DNA
<213>Artificial sequence
<400> 14
gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60
gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120
atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180
tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240
tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300
cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360
agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420
ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480
tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540
aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600
cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660
cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720
gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780
ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840
cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900
cctcagccgt cgcttcatgt gactccactg agtaccgggc gccgtccagg cacctcgatt 960
agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020
agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080
tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140
tggttcaaag tttttttctt ccatttcagg tgtcgtga 1178
<210> 15
<211> 63
<212> DNA
<213>Artificial sequence
<400> 15
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 16
<211> 333
<212> DNA
<213>Artificial sequence
<400> 16
gatattgtgc tgacccagag cccgccgagc ctggcgatga gcctgggcaa acgcgcgacc 60
attagctgcc gcgcgagcga aagcgtgacc attctgggca gccatctgat tcattggtat 120
cagcagaaac cgggccagcc gccgaccctg ctgattcagc tggcgagcaa cgtgcagacc 180
ggcgtgccgg cgcgctttag cggcagcggc agccgcaccg attttaccct gaccattgat 240
ccggtggaag aagatgatgt ggcggtgtat tattgcctgc agagccgcac cattccgcgc 300
acctttggcg gcggcaccaa actggaaatt aaa 333
<210> 17
<211> 351
<212> DNA
<213>Artificial sequence
<400> 17
cagattcagc tggtgcagag cggcccggaa ctgaaaaaac cgggcgaaac cgtgaaaatt 60
agctgcaaag cgagcggcta tacctttacc gattatagca ttaactgggt gaaacgcgcg 120
ccgggcaaag gcctgaaatg gatgggctgg attaacaccg aaacccgcga accggcgtat 180
gcgtatgatt ttcgcggccg ctttgcgttt agcctggaaa ccagcgcgag caccgcgtat 240
ctgcagatta acaacctgaa atatgaagat accgcgacct atttttgcgc gctggattat 300
agctatgcga tggattattg gggccagggc accagcgtga ccgtgagcag c 351
<210> 18
<211> 324
<212> DNA
<213>Artificial sequence
<400> 18
gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgca aagcgagcca ggatgtgggc attgcggtgg cgtggtatca gcagaaaccg 120
ggcaaagtgc cgaaactgct gatttattgg gcgagcaccc gccataccgg cgtgccggat 180
cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
gaagatgtgg cgacctatta ttgccagcag tatagcagct atccgtatac ctttggccag 300
ggcaccaaag tggaaattaa acgc 324
<210> 19
<211> 357
<212> DNA
<213>Artificial sequence
<400> 19
gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60
agctgcgcgg cgagcggctt tgattttagc cgctattgga tgagctgggt gcgccaggcg 120
ccgggcaaag gcctggaatg gattggcgaa attaacccgg atagcagcac cattaactat 180
gcgccgagcc tgaaagataa atttattatt agccgcgata acgcgaaaaa cagcctgtat 240
ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gcgcccggat 300
ggcaactatt ggtattttga tgtgtggggc cagggcaccc tggtgaccgt gagcagc 357
<210> 20
<211> 321
<212> DNA
<213>Artificial sequence
<400> 20
gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120
ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180
aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240
gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctccgac gttcggccaa 300
gggaccaagg tggaaatcaa a 321
<210> 21
<211> 366
<212> DNA
<213>Artificial sequence
<400> 21
gaagtgcagc tgctggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60
agctgcgcgg tgagcggctt tacctttaac agctttgcga tgagctgggt gcgccaggcg 120
ccgggcaaag gcctggaatg ggtgagcgcg attagcggca gcggcggcgg cacctattat 180
gcggatagcg tgaaaggccg ctttaccatt agccgcgata acagcaaaaa caccctgtat 240
ctgcagatga acagcctgcg cgcggaagat accgcggtgt atttttgcgc gaaagataaa 300
attctgtggt ttggcgaacc ggtgtttgat tattggggcc agggcaccct ggtgaccgtg 360
agcagc 366
<210> 22
<211> 333
<212> DNA
<213>Artificial sequence
<400> 22
gatattgtgc tgacccagag cccggcgagc ctggcggtga gcccgggcca gcgcgcgacc 60
attacctgcc gcgcgagcca gagcgtgagc accagcagca gcagctttat gcattggtat 120
cagcagaaac cgggccagcc gccgaaactg ctgattaaat atgcgagcaa cctggaaagc 180
ggcgtgccgg cgcgctttag cggcagcggc agcggcaccg attttaccct gaccattaac 240
ccggtggaag cgaacgatac cgcgaactat tattgccagc atagctggga aattccgtat 300
acctttggcc agggcaccaa actggaaatt aaa 333
<210> 23
<211> 348
<212> DNA
<213>Artificial sequence
<400> 23
gaagtgcagc tggtggaaag cggcggcggc ctggtgaaac cgggcggcag cctgcgcctg 60
agctgcgcgg cgagcggctt tatttttcgc agctatggca tgagctgggt gcgccaggcg 120
ccgggcaaag gcctggaatg ggtggcgagc attagcagcg gcggcagcac ctattatccg 180
gatagcgtga aaggccgctt taccattagc cgcgataacg cgaaaaacag cctgtatctg 240
cagatgaaca gcctgcgcgc ggaagatacc gcggtgtatg attgcgcgcg cggctatgat 300
agcggctttg cgtattgggg ccagggcacc ctggtgaccg tgagcagc 348
<210> 24
<211> 324
<212> DNA
<213>Artificial sequence
<400> 24
gatattgtga tgacccagag cccgagcagc gtgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgcc gcgcgagcca gaacgtggat agcgcggtgg cgtggtatca gcagaaaccg 120
ggcaaagcgc cgaaagcgct gatttatagc gcgagctatc gctatagcgg cgtgccgagc 180
cgctttagcg gccgcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
gaagattttg cgacctatta ttgccagcag tattatagca ccccgtggac ctttggccag 300
ggcaccaaag tggaaattaa acgc 324
<210> 25
<211> 381
<212> DNA
<213>Artificial sequence
<400> 25
gaagtgaaac tggtggaaag cggcggcggc ctggtgcagc cgggccgcag cctgcgcctg 60
agctgcaccg cgagcggctt tacctttacc gattattata tgagctgggt gcgccaggcg 120
ccgggcaaag gcctggaatg ggtgggcctg attcgcagca aagcggatgg ctataccacc 180
gaatatagcg cgagcgtgaa aggccgcttt accattagcc gcgatgatag caaaagcatt 240
ctgtatctgc agatgaacag cctgaaaacc gaagataccg cggtgtatta ttgcgcgcgc 300
gatgcggcgt attatagcta ttatagcccg gaaggcgcga tggattattg gggccagggc 360
accctggtga ccgtgagcag c 381
<210> 26
<211> 45
<212> DNA
<213>Artificial sequence
<400> 26
ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45
<210> 27
<211> 57
<212> DNA
<213>Artificial sequence
<400> 27
gcctccacca agggcccatc tgtcttcccc ctggccccca gctcctctgg ctccgga 57
<210> 28
<211> 141
<212> DNA
<213>Artificial sequence
<400> 28
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgatatcta c 141
<210> 29
<211> 66
<212> DNA
<213>Artificial sequence
<400> 29
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 60
tactgc 66
<210> 30
<211> 123
<212> DNA
<213>Artificial sequence
<400> 30
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 123
<210> 31
<211> 111
<212> DNA
<213>Artificial sequence
<400> 31
cgccgcgatc agcgcctgcc gccggatgcg cataaaccgc cgggcggcgg cagctttcgc 60
accccgattc aggaagaaca ggcggatgcg catagcaccc tggcgaaaat t 111
<210> 32
<211> 336
<212> DNA
<213>Artificial sequence
<400> 32
cgcgtgaaat ttagccgcag cgcggatgcg ccggcgtatc agcagggcca gaaccagctg 60
tataacgaac tgaacctggg ccgccgcgaa gaatatgatg tgctggataa acgccgcggc 120
cgcgatccgg aaatgggcgg caaaccgcgc cgcaaaaacc cgcaggaagg cctgtataac 180
gaactgcaga aagataaaat ggcggaagcg tatagcgaaa ttggcatgaa aggcgaacgc 240
cgccgcggca aaggccatga tggcctgtat cagggcctga gcaccgcgac caaagatacc 300
tatgatgcgc tgcatatgca ggcgctgccg ccgcgc 336
<210> 33
<211> 1557
<212> DNA
<213>Artificial sequence
<400> 33
atgaactcct tctccacaag cgccttcggt ccagttgcct tctccctggg gctgctcctg 60
gtgttgcctg ctgccttccc tgccccagat attgtgctga cccagagccc ggcgagcctg 120
gcggtgagcc cgggccagcg cgcgaccatt acctgccgcg cgagccagag cgtgagcacc 180
agcagcagca gctttatgca ttggtatcag cagaaaccgg gccagccgcc gaaactgctg 240
attaaatatg cgagcaacct ggaaagcggc gtgccggcgc gctttagcgg cagcggcagc 300
ggcaccgatt ttaccctgac cattaacccg gtggaagcga acgataccgc gaactattat 360
tgccagcata gctgggaaat tccgtatacc tttggccagg gcaccaaact ggaaattaaa 420
ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgaagt gcagctggtg 480
gaaagcggcg gcggcctggt gaaaccgggc ggcagcctgc gcctgagctg cgcggcgagc 540
ggctttattt ttcgcagcta tggcatgagc tgggtgcgcc aggcgccggg caaaggcctg 600
gaatgggtgg cgagcattag cagcggcggc agcacctatt atccggatag cgtgaaaggc 660
cgctttacca ttagccgcga taacgcgaaa aacagcctgt atctgcagat gaacagcctg 720
cgcgcggaag ataccgcggt gtatgattgc gcgcgcggct atgatagcgg ctttgcgtat 780
tggggccagg gcaccctggt gaccgtgagc agcggtggcg gtggctcggg cggtggtggg 840
tcgggtggcg gcggatctga accgaaaagc tgcgacaaaa ctcacacatg cccaccgtgc 900
ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 960
accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1020
gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1080
aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1140
caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1200
gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1260
accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1320
aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1380
aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1440
ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcac 1500
gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga 1557
<210> 34
<211> 36
<212> DNA
<213>Artificial sequence
<400> 34
attcaaaatt ttatcgatgc tccggtgccc gtcagt 36
<210> 35
<211> 22
<212> DNA
<213>Artificial sequence
<400> 35
tcacgacacc tgaaatggaa ga 22
<210> 36
<211> 46
<212> DNA
<213>Artificial sequence
<400> 36
catttcaggt gtcgtgagga tccgccacca tggcgctgcc ggtgac 46
<210> 37
<211> 33
<212> DNA
<213>Artificial sequence
<400> 37
ggggagggag aggggcttag cgcggcggca gcg 33
<210> 38
<211> 17
<212> DNA
<213>Artificial sequence
<400> 38
gcccctctcc ctccccc 17
<210> 39
<211> 25
<212> DNA
<213>Artificial sequence
<400> 39
attatcatcg tgtttttcaa aggaa 25
<210> 40
<211> 44
<212> DNA
<213>Artificial sequence
<400> 40
aaaacacgat gataatgcca ccatgaactc cttctccaca agcg 44
<210> 41
<211> 46
<212> DNA
<213>Artificial sequence
<400> 41
aatccagagg ttgattgtcg acgaattctc atttgcccgg gctcag 46
<210> 42
<211> 21
<212> DNA
<213>Artificial sequence
<400> 42
cctttccggg actttcgctt t 21
<210> 43
<211> 20
<212> DNA
<213>Artificial sequence
<400> 43
gcagaatcca ggtggcaaca 20
<210> 44
<211> 21
<212> DNA
<213>Artificial sequence
<400> 44
catgtacgtt gctatccagg c 21
<210> 45
<211> 21
<212> DNA
<213>Artificial sequence
<400> 45
ctccttaatg tcacgcacga t 21
Claims (11)
1. the double targeting Chimeric antigen receptors of a kind of OCTS-CAR, it is characterised in that including the CD8 leader being sequentially connected in series
Membrane receptor signal peptide, double antigen binding domains, CD8 Hinge Chimerical receptors hinge, CD8 Transmembrane Chimerical receptor cross-films
Area, CD28 Chimerical receptors costimulating factor, CD134 Chimerical receptors costimulating factor and TCR Chimerical receptor t cell activations domain,
Wherein, double antigen binding domains include the heavy chain VH and light chain of two single-chain antibodies connected in a manner of series connection or corner
Hinge Inter-Linker between VL, antibody inner hinge Inner-Linker and single-chain antibody,
Described two single-chain antibodies refer to that BCMA single-chain antibodies, CD319 single-chain antibodies, CD38 single-chain antibodies, PDL1 are single-stranded anti-
Any combination of two between body, CD123 single-chain antibodies.
2. encode the gene of the double targeting Chimeric antigen receptors of OCTS-CAR described in claim 1, it is characterised in that:
The gene order of the CD8 leader membrane receptor signal peptides is encoded as shown in SEQ ID NO.15;It is mono- to encode the BCMA
Chain antibody light chain VL gene order encodes the gene order of the BCMA single-chain antibodies heavy chain VH as shown in SEQ ID NO.16
As shown in SEQ ID NO.17;The gene order of the CD319 single-chain antibodies light chain VL is encoded as shown in SEQ ID NO.18, is compiled
Code CD319 single-chain antibodies heavy chain VH gene order is as shown in SEQ ID NO.19;It is light to encode the CD38 single-chain antibodies
Chain VL gene order encodes the gene order such as SEQ ID of the CD38 chain antibodies heavy chain VH as shown in SEQ ID NO.20
Shown in NO.21;The gene order of the PDL1 single-chain antibodies light chain VL is encoded as shown in SEQ ID NO.22, encodes the PDL1
Single-chain antibody heavy chain VH gene order is as shown in SEQ ID NO.23;Encode the gene of the CD123 single-chain antibodies light chain VL
Sequence encodes the gene order such as SEQ ID NO.25 institutes of the CD123 single-chain antibodies heavy chain VH as shown in SEQ ID NO.24
Show;Encoding antibody inner hinge Inner-Linker gene order is as shown in SEQ ID NO.26;Hinge between coding single-chain antibody
Inter-Linker gene order is as shown in SEQ ID NO.27;Encode the gene sequence of the CD8Hinge Chimerical receptors hinge
Row are as shown in SEQ ID NO.28;Encode the gene order such as SEQ of the CD8 Transmembrane Chimerical receptor transmembrane regions
Shown in ID NO.29;The gene order of the CD28 Chimerical receptors costimulating factor is encoded as shown in SEQ ID NO.30;Coding
The gene order of the CD134 Chimerical receptors costimulating factor is as shown in SEQ ID NO.31;Encode the TCR Chimerical receptors T
The gene order in cell-stimulating domain is as shown in SEQ ID NO.32.
A kind of 3. OCTS-CAR recombinant expression carriers, it is characterised in that including:
The gene of expression vector, people's EF1 α promoters and the double targeting Chimeric antigen receptors of coding OCTS-CAR,
Wherein, the gene order of people EF1 α promoters is as shown in SEQ ID NO.14, the double targeting chimeric antigens of coding OCTS-CAR
The gene of acceptor is as claimed in claim 2.
A kind of 4. OCTS-CAR recombinant expression carriers, it is characterised in that including:
Expression vector, people EF1 α promoters, the gene of the double targeting Chimeric antigen receptors of coding OCTS-CAR and coding PDL1 are mono-
The gene of chain antibody,
Wherein, the gene order of people EF1 α promoters is as shown in SEQ ID NO.14, the double targeting chimeric antigens of coding OCTS-CAR
The gene of acceptor is as claimed in claim 2, encodes the gene order of PDL1 single-chain antibodies as shown in SEQ ID NO.33.
5. the OCTS-CAR recombinant expression carriers according to claim 3 or 4, it is characterised in that:
Wherein, the expression vector is Lentiviral, retrovirus expression vector, adenovirus expression carrier, adenopathy
Malicious associated virus expression vector or plasmid.
6. OCTS-CAR recombinant expression carriers according to claim 5, it is characterised in that:
Wherein, the expression vector is third generation Lentiviral pLenti-3G basic, the Lentiviral bag
Include the sequences of AmpR containing ampicillin resistance gene, prokaryotic replions pUC Ori sequences, Viral Replicon SV40 Ori sequences,
RSV promoters, terminal LTR of slow virus 5, terminal Self-Inactivating LTR of slow virus 3, Gag are cis
Element, RRE cis elements, env cis elements, cPPT cis elements, ZsGreen1 green fluorescent proteins, IRES ribosomes combine
The enhanced marmot hepatitis B posttranscriptional regulatory element of sequence, eWPRE.
7. a kind of construction method of OCTS-CAR recombinant expression carriers as claimed in claim 3, it is characterised in that including following
Step:
A. ampicillin resistance gene AmpR sequences, prokaryotic replions pUC Ori sequences, Viral Replicon SV40 Ori will be contained
Sequence, RSV promoters, terminal LTR of slow virus 5, terminal Self-Inactivating LTR of slow virus 3,
Gag cis elements, RRE cis elements, env cis elements, cPPT cis elements, ZsGreen1 green fluorescent proteins, IRES cores
The enhanced marmot hepatitis B posttranscriptional regulatory element of sugared body binding sequence, eWPRE is stored in third generation slow virus skeleton matter
On grain pLenti-3G basic;
B. will coding CD8 leader membrane receptors signal peptide, double antigen binding domains, CD8Hinge Chimerical receptors hinge, CD8
Transmembrane Chimerical receptors transmembrane region, CD28 Chimerical receptors costimulating factor, CD134 Chimerical receptors costimulating factor with
And the gene in TCR Chimerical receptor t cell activations domain is cloned into slow virus skeleton plasmid by digestion, connection, recombining reaction
In pLenti-3G basic, the recombinant slow virus plasmid that the third generation is designed based on OCTS is obtained;
C. recombinant slow virus plasmid step B obtained respectively with slow virus packaging plasmid pPac-GP, pPac-R and film
Albumen plasmid pEnv-G transfects HEK293T/17 cells jointly, after carrying out gene transcript expression in HEK293T/17 cells, bag
Dressing up work(recombined lentivirus vector can be discharged into cells and supernatant, collect the supernatant of the recombined lentivirus vector included;
D. obtained recombinant slow virus supernatant is purified using the post way of purification for filtering, adsorbing, eluting, respectively obtained
CAR-T recombinant slow virus expression vectors.
8. a kind of construction method of OCTS-CAR recombinant expression carriers as claimed in claim 4, it is characterised in that including following
Step:
A. ampicillin resistance gene AmpR sequences, prokaryotic replions pUC Ori sequences, Viral Replicon SV40 Ori will be contained
Sequence, RSV promoters, terminal LTR of slow virus 5, terminal Self-Inactivating LTR of slow virus 3,
Gag cis elements, RRE cis elements, env cis elements, cPPT cis elements, ZsGreen1 green fluorescent proteins, IRES cores
The enhanced marmot hepatitis B posttranscriptional regulatory element of sugared body binding sequence, eWPRE is stored in third generation slow virus skeleton matter
On grain pLenti-3G basic;
B. will coding CD8 leader membrane receptors signal peptide, double antigen binding domains, CD8 Hinge Chimerical receptors hinge, CD8
Transmembrane Chimerical receptors transmembrane region, CD28 Chimerical receptors costimulating factor, CD134 Chimerical receptors costimulating factor,
The gene of TCR Chimerical receptor t cell activation domains and coding PDL1 single-chain antibodies is cloned into by digestion, connection, recombining reaction
In slow virus skeleton plasmid pLenti-3G basic, the recombinant slow virus plasmid that the third generation is designed based on OCTS is obtained;
C. recombinant slow virus plasmid step B obtained respectively with slow virus packaging plasmid pPac-GP, pPac-R and film
Albumen plasmid pEnv-G transfects HEK293T/17 cells jointly, after carrying out gene transcript expression in HEK293T/17 cells, bag
Dressing up work(recombined lentivirus vector can be discharged into cells and supernatant, collect the supernatant of the recombined lentivirus vector included;
D. obtained recombinant slow virus supernatant is purified using the post way of purification for filtering, adsorbing, eluting, respectively obtained
CAR-T recombinant slow virus expression vectors.
9. a kind of OCTS-CAR-T cells, it is that the OCTS-CAR-T recombination expressions described in claim 3 or 4 are imported with genome
Carrier or the T lymphocytes through the double targeting Chimeric antigen receptor modifications of CAR described in claim 1.
10. application of the OCTS-CAR-T cells in treating malignant tumor medicine is prepared described in claim 9.
11. application of the OCTS-CAR-T cells according to claim 10 in treating malignant tumor medicine is prepared, it is special
Sign is that the treating malignant tumor medicine is treatment malignant plasma cell tumour, acute myeloid leukemia, melanoma, mammary gland
Cancer, glioma, lymthoma, urological cancer, the medicine of tumor in digestive tract or genital system.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710418260.4A CN107337736B (en) | 2017-06-06 | 2017-06-06 | The bis- targeting Chimeric antigen receptors of OCTS-CAR, encoding gene, recombinant expression carrier and its building and application |
PCT/CN2017/110674 WO2018223600A1 (en) | 2017-06-06 | 2017-11-13 | Octs-car dual targeting chimeric antigen receptor, encoding gene, recombinant expression vector and construction and use thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710418260.4A CN107337736B (en) | 2017-06-06 | 2017-06-06 | The bis- targeting Chimeric antigen receptors of OCTS-CAR, encoding gene, recombinant expression carrier and its building and application |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107337736A true CN107337736A (en) | 2017-11-10 |
CN107337736B CN107337736B (en) | 2019-07-26 |
Family
ID=60221293
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710418260.4A Active CN107337736B (en) | 2017-06-06 | 2017-06-06 | The bis- targeting Chimeric antigen receptors of OCTS-CAR, encoding gene, recombinant expression carrier and its building and application |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN107337736B (en) |
WO (1) | WO2018223600A1 (en) |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107974460A (en) * | 2017-11-30 | 2018-05-01 | 山东兴瑞生物科技有限公司 | Chimeric antigen receptor gene for HIV-1, plasmid, T cell, kit and application with the gene |
CN108314738A (en) * | 2018-01-29 | 2018-07-24 | 山东兴瑞生物科技有限公司 | A kind of bispecific chimeric antigen receptor, plasmid, CIK cell and the MM disease applications of coexpression cell factor IL-21 |
CN108641000A (en) * | 2018-04-26 | 2018-10-12 | 上海怡豪生物科技有限公司 | The double target spot CAR-T therapy vectors and its construction method of liver cancer and application |
CN108641001A (en) * | 2018-04-26 | 2018-10-12 | 上海怡豪生物科技有限公司 | The double target spot CAR-T therapy vectors and its construction method of colon cancer and application |
CN108659133A (en) * | 2018-04-26 | 2018-10-16 | 上海怡豪生物科技有限公司 | The double target spot CAR-T therapy vectors and its construction method of lung cancer and application |
CN108864288A (en) * | 2018-04-26 | 2018-11-23 | 上海怡豪生物科技有限公司 | The double target spot CAR-T therapy vectors and its construction method of breast cancer and application |
CN108864289A (en) * | 2018-04-26 | 2018-11-23 | 上海怡豪生物科技有限公司 | The double target spot CAR-T therapy vectors and its construction method of gastric cancer and application |
WO2018223600A1 (en) * | 2017-06-06 | 2018-12-13 | 上海优卡迪生物医药科技有限公司 | Octs-car dual targeting chimeric antigen receptor, encoding gene, recombinant expression vector and construction and use thereof |
CN109265551A (en) * | 2018-09-25 | 2019-01-25 | 华东师范大学 | CD38 antibody, Chimeric antigen receptor and drug |
CN109971715A (en) * | 2017-12-28 | 2019-07-05 | 深圳华大生命科学研究院 | A kind of cultural method of specific amplification CAR-T cell |
CN110129369A (en) * | 2018-02-09 | 2019-08-16 | 上海交通大学医学院附属上海儿童医学中心 | A kind of Chimeric antigen receptor engineering carrier, immunocyte and its application |
CN110606886A (en) * | 2019-08-12 | 2019-12-24 | 陕西脉元生物科技有限公司 | Detection material for anti-CASPR 2 autoantibody in human body fluid, preparation method and application |
CN111944850A (en) * | 2020-08-28 | 2020-11-17 | 澳门大学 | Preparation method of cell for expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, expression vector and application |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105949316A (en) * | 2016-04-12 | 2016-09-21 | 上海优卡迪生物医药科技有限公司 | Anti-EGFRvIII chimeric antigen receptor, encoding gene, recombinant expression vector, construction method of recombinant expression vector, and application |
WO2016149578A1 (en) * | 2015-03-19 | 2016-09-22 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Dual specific anti-cd22-anti-cd19 chimeric antigen receptors |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102593475B1 (en) * | 2013-02-26 | 2023-10-26 | 메모리얼 슬로안 케터링 캔서 센터 | Compositions and methods for immunotherapy |
CA2968412A1 (en) * | 2014-12-19 | 2016-06-23 | Dana-Farber Cancer Institute, Inc. | Chimeric antigen receptors and methods of use thereof |
CN105384825B (en) * | 2015-08-11 | 2018-06-01 | 南京传奇生物科技有限公司 | A kind of bispecific chimeric antigen receptor and its application based on single domain antibody |
CN105331585A (en) * | 2015-11-13 | 2016-02-17 | 科济生物医药(上海)有限公司 | Chimeric antigen receptor-modified immunologic effector cell with PD-L1 blocking agent |
CN105602992B (en) * | 2016-03-17 | 2019-06-21 | 上海优卡迪生物医药科技有限公司 | It is a kind of based on the CAR-T transgene carrier and its construction method of replication defective recombinant slow virus and application |
CN105777911B (en) * | 2016-04-12 | 2019-07-02 | 上海优卡迪生物医药科技有限公司 | Anti- BCMA Chimeric antigen receptor, encoding gene, recombinant expression carrier and its construction method and application |
CN105950664B (en) * | 2016-05-17 | 2019-03-29 | 上海优卡迪生物医药科技有限公司 | A kind of replication defective recombinant slow virus CAR-T transgene carrier targeting CD123 and its construction method and application |
CN107337736B (en) * | 2017-06-06 | 2019-07-26 | 上海优卡迪生物医药科技有限公司 | The bis- targeting Chimeric antigen receptors of OCTS-CAR, encoding gene, recombinant expression carrier and its building and application |
-
2017
- 2017-06-06 CN CN201710418260.4A patent/CN107337736B/en active Active
- 2017-11-13 WO PCT/CN2017/110674 patent/WO2018223600A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016149578A1 (en) * | 2015-03-19 | 2016-09-22 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Dual specific anti-cd22-anti-cd19 chimeric antigen receptors |
CN105949316A (en) * | 2016-04-12 | 2016-09-21 | 上海优卡迪生物医药科技有限公司 | Anti-EGFRvIII chimeric antigen receptor, encoding gene, recombinant expression vector, construction method of recombinant expression vector, and application |
Non-Patent Citations (1)
Title |
---|
朱童等: "嵌合抗原受体修饰 T 细胞在实体肿瘤中应用的研究进展", 《临床肿瘤学杂志》 * |
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018223600A1 (en) * | 2017-06-06 | 2018-12-13 | 上海优卡迪生物医药科技有限公司 | Octs-car dual targeting chimeric antigen receptor, encoding gene, recombinant expression vector and construction and use thereof |
CN107974460B (en) * | 2017-11-30 | 2021-06-01 | 山东兴瑞生物科技有限公司 | Chimeric antigen receptor gene aiming at HIV-1, plasmid, T cell, kit and application with gene |
CN107974460A (en) * | 2017-11-30 | 2018-05-01 | 山东兴瑞生物科技有限公司 | Chimeric antigen receptor gene for HIV-1, plasmid, T cell, kit and application with the gene |
CN109971715A (en) * | 2017-12-28 | 2019-07-05 | 深圳华大生命科学研究院 | A kind of cultural method of specific amplification CAR-T cell |
CN108314738A (en) * | 2018-01-29 | 2018-07-24 | 山东兴瑞生物科技有限公司 | A kind of bispecific chimeric antigen receptor, plasmid, CIK cell and the MM disease applications of coexpression cell factor IL-21 |
CN108314738B (en) * | 2018-01-29 | 2020-09-08 | 山东兴瑞生物科技有限公司 | Bispecific chimeric antigen receptor co-expressing cytokine IL-21, plasmid, CIK cell and MM disease application |
CN110129369B (en) * | 2018-02-09 | 2023-10-13 | 上海交通大学医学院附属上海儿童医学中心 | Chimeric antigen receptor gene engineering vector, immune cell and application thereof |
CN110129369A (en) * | 2018-02-09 | 2019-08-16 | 上海交通大学医学院附属上海儿童医学中心 | A kind of Chimeric antigen receptor engineering carrier, immunocyte and its application |
CN108659133A (en) * | 2018-04-26 | 2018-10-16 | 上海怡豪生物科技有限公司 | The double target spot CAR-T therapy vectors and its construction method of lung cancer and application |
CN108864289A (en) * | 2018-04-26 | 2018-11-23 | 上海怡豪生物科技有限公司 | The double target spot CAR-T therapy vectors and its construction method of gastric cancer and application |
CN108864288A (en) * | 2018-04-26 | 2018-11-23 | 上海怡豪生物科技有限公司 | The double target spot CAR-T therapy vectors and its construction method of breast cancer and application |
CN108641001A (en) * | 2018-04-26 | 2018-10-12 | 上海怡豪生物科技有限公司 | The double target spot CAR-T therapy vectors and its construction method of colon cancer and application |
CN108641000A (en) * | 2018-04-26 | 2018-10-12 | 上海怡豪生物科技有限公司 | The double target spot CAR-T therapy vectors and its construction method of liver cancer and application |
CN109265551A (en) * | 2018-09-25 | 2019-01-25 | 华东师范大学 | CD38 antibody, Chimeric antigen receptor and drug |
CN110606886A (en) * | 2019-08-12 | 2019-12-24 | 陕西脉元生物科技有限公司 | Detection material for anti-CASPR 2 autoantibody in human body fluid, preparation method and application |
CN110606886B (en) * | 2019-08-12 | 2021-06-01 | 陕西脉元生物科技有限公司 | Detection material for anti-CASPR 2 autoantibody in human body fluid, preparation method and application |
CN111944850A (en) * | 2020-08-28 | 2020-11-17 | 澳门大学 | Preparation method of cell for expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, expression vector and application |
Also Published As
Publication number | Publication date |
---|---|
CN107337736B (en) | 2019-07-26 |
WO2018223600A1 (en) | 2018-12-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107337736B (en) | The bis- targeting Chimeric antigen receptors of OCTS-CAR, encoding gene, recombinant expression carrier and its building and application | |
CN107325185B (en) | The bis- targeting Chimeric antigen receptors of anti-PSCA and PDL1, encoding gene and expression vector based on OCTS-CAR | |
CN107267555B (en) | Malignant glioma CAR-T therapeutic vector based on OCTS technology and construction method and application thereof | |
CN107058315B (en) | Strike the siRNA for subtracting people PD-1, recombinant expression CAR-T carrier and its construction method and application | |
CN107299110B (en) | A kind of cancer of pancreas based on OCTS technology, malignant mesothelioma CAR-T therapy vector and its construction method and application | |
CN108148862B (en) | The CAR-T transgene carrier for inhibiting immune escape of closing PDL1 a kind of and its construction method and application | |
CN107245500B (en) | A kind of leaching based on OCTS technology is leukaemia CAR-T therapy vector and its construction method and application | |
CN107287207B (en) | A kind of label and application for tracing in vivo and artificial removing CAR-T cell | |
CN105777911B (en) | Anti- BCMA Chimeric antigen receptor, encoding gene, recombinant expression carrier and its construction method and application | |
CN108148863A (en) | A kind of CAR-T transgene carriers for being used to alleviate CRS for closing IL6R and its construction method and application | |
CN106636090B (en) | SiRNA, recombinant expression CAR-T carrier and its construction method of human leukocyte interleukin 6 and application | |
CN109678965B (en) | Chimeric antigen receptor, gene and recombinant expression vector thereof, CD22-CD19 dual-targeting T cell and application thereof | |
CN104788573A (en) | Chimeric antigen receptor hCD19scFv-CD8a-CD-28-CD3zata and application thereof | |
CN108018312A (en) | A kind of CAR-T therapy vectors of T lymphocytic leukemias and its construction method and application | |
CN105820255B (en) | Anti-CD 33 Chimeric antigen receptor, encoding gene, recombinant expression carrier and its construction method and application | |
CN105820254B (en) | Anti- CD138 Chimeric antigen receptor, encoding gene, recombinant expression carrier and its construction method and application | |
CN108641000A (en) | The double target spot CAR-T therapy vectors and its construction method of liver cancer and application | |
CN110257429A (en) | The T cell and their application of recombinant expression carrier, targeting | |
CN116419927A (en) | Bispecific antibody CAR cell immunotherapy | |
CN108203720A (en) | Her2, which can be targeted, and close PD-L1 reduces the CAR-T carriers of tumor immune escape and its construction method and application | |
CN107058232A (en) | Cholesterol turns repressed CAR T cells of lipase SOAT1 and its preparation method and application | |
CN107164410B (en) | It is a kind of based on the prostate cancer CAR-T therapy vector and its construction method of OCTS technology and application | |
CN107177632B (en) | OCTS technology-based myeloid leukemia CAR-T treatment vector and construction method and application thereof | |
CN108203717A (en) | Her2 can be targeted and PD-1 is interfered to reduce the CAR-T carriers of tumor immune escape and its construction method and application | |
CN116199766B (en) | Methods of screening TCRs and isolated TCRs thereof |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |