CN116715768A - 一种抗EGFRvIII单克隆抗体及CAR-T细胞及其应用 - Google Patents
一种抗EGFRvIII单克隆抗体及CAR-T细胞及其应用 Download PDFInfo
- Publication number
- CN116715768A CN116715768A CN202310431051.9A CN202310431051A CN116715768A CN 116715768 A CN116715768 A CN 116715768A CN 202310431051 A CN202310431051 A CN 202310431051A CN 116715768 A CN116715768 A CN 116715768A
- Authority
- CN
- China
- Prior art keywords
- amino acid
- acid sequence
- chain variable
- seq
- variable region
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 61
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims abstract description 60
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 31
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 27
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 27
- 239000012634 fragment Substances 0.000 claims abstract description 19
- 102000036639 antigens Human genes 0.000 claims abstract description 14
- 108091007433 antigens Proteins 0.000 claims abstract description 14
- 239000000427 antigen Substances 0.000 claims abstract description 13
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 12
- 238000011282 treatment Methods 0.000 claims abstract description 10
- 230000000139 costimulatory effect Effects 0.000 claims abstract description 5
- 108010087914 epidermal growth factor receptor VIII Proteins 0.000 claims abstract 3
- 210000004027 cell Anatomy 0.000 claims description 70
- 150000001413 amino acids Chemical class 0.000 claims description 22
- 238000012217 deletion Methods 0.000 claims description 22
- 230000037430 deletion Effects 0.000 claims description 22
- 238000007792 addition Methods 0.000 claims description 21
- 238000006467 substitution reaction Methods 0.000 claims description 20
- 239000013598 vector Substances 0.000 claims description 20
- 230000006870 function Effects 0.000 claims description 17
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 12
- 239000012620 biological material Substances 0.000 claims description 12
- 210000002865 immune cell Anatomy 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 201000010099 disease Diseases 0.000 claims description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 7
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 150000007523 nucleic acids Chemical class 0.000 claims description 7
- 230000011664 signaling Effects 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 6
- 230000004064 dysfunction Effects 0.000 claims description 6
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 4
- 206010033128 Ovarian cancer Diseases 0.000 claims description 4
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 4
- 201000005202 lung cancer Diseases 0.000 claims description 4
- 208000020816 lung neoplasm Diseases 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- 206010005003 Bladder cancer Diseases 0.000 claims description 3
- 206010006187 Breast cancer Diseases 0.000 claims description 3
- 208000026310 Breast neoplasm Diseases 0.000 claims description 3
- 206010060862 Prostate cancer Diseases 0.000 claims description 3
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 3
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 3
- 208000005017 glioblastoma Diseases 0.000 claims description 3
- 201000002313 intestinal cancer Diseases 0.000 claims description 3
- 201000007270 liver cancer Diseases 0.000 claims description 3
- 208000014018 liver neoplasm Diseases 0.000 claims description 3
- 201000001441 melanoma Diseases 0.000 claims description 3
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 3
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 2
- 201000003741 Gastrointestinal carcinoma Diseases 0.000 claims description 2
- 208000007569 Giant Cell Tumors Diseases 0.000 claims description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 2
- 206010024612 Lipoma Diseases 0.000 claims description 2
- 206010025323 Lymphomas Diseases 0.000 claims description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 2
- 206010038389 Renal cancer Diseases 0.000 claims description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 2
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 2
- 201000010881 cervical cancer Diseases 0.000 claims description 2
- 208000006990 cholangiocarcinoma Diseases 0.000 claims description 2
- 230000004069 differentiation Effects 0.000 claims description 2
- 230000003511 endothelial effect Effects 0.000 claims description 2
- 201000004101 esophageal cancer Diseases 0.000 claims description 2
- 206010016629 fibroma Diseases 0.000 claims description 2
- 206010017758 gastric cancer Diseases 0.000 claims description 2
- 201000010536 head and neck cancer Diseases 0.000 claims description 2
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 2
- 201000010982 kidney cancer Diseases 0.000 claims description 2
- 210000002540 macrophage Anatomy 0.000 claims description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 2
- 206010027191 meningioma Diseases 0.000 claims description 2
- 210000001616 monocyte Anatomy 0.000 claims description 2
- 210000000822 natural killer cell Anatomy 0.000 claims description 2
- 208000008798 osteoma Diseases 0.000 claims description 2
- 201000008968 osteosarcoma Diseases 0.000 claims description 2
- 201000002528 pancreatic cancer Diseases 0.000 claims description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 2
- 206010041823 squamous cell carcinoma Diseases 0.000 claims description 2
- 210000000130 stem cell Anatomy 0.000 claims description 2
- 201000011549 stomach cancer Diseases 0.000 claims description 2
- 201000002510 thyroid cancer Diseases 0.000 claims description 2
- 230000002792 vascular Effects 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims 1
- 230000002147 killing effect Effects 0.000 abstract description 7
- 108010074328 Interferon-gamma Proteins 0.000 abstract description 4
- 230000019491 signal transduction Effects 0.000 abstract description 2
- 238000003745 diagnosis Methods 0.000 abstract 1
- 235000001014 amino acid Nutrition 0.000 description 12
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 9
- 101100112922 Candida albicans CDR3 gene Proteins 0.000 description 8
- 102100035361 Cerebellar degeneration-related protein 2 Human genes 0.000 description 8
- 101000737793 Homo sapiens Cerebellar degeneration-related antigen 1 Proteins 0.000 description 8
- 101000737796 Homo sapiens Cerebellar degeneration-related protein 2 Proteins 0.000 description 8
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 8
- 210000004881 tumor cell Anatomy 0.000 description 8
- 241000713666 Lentivirus Species 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 241000700605 Viruses Species 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 4
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 3
- 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 3
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 3
- 102100037850 Interferon gamma Human genes 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000009169 immunotherapy Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 238000001959 radiotherapy Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 101150039808 Egfr gene Proteins 0.000 description 2
- 102400001368 Epidermal growth factor Human genes 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 208000032612 Glial tumor Diseases 0.000 description 2
- 206010018338 Glioma Diseases 0.000 description 2
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 2
- 229940116977 epidermal growth factor Drugs 0.000 description 2
- 108700021358 erbB-1 Genes Proteins 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000011287 therapeutic dose Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-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 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 238000011357 CAR T-cell therapy Methods 0.000 description 1
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 1
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 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 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 229960000446 abciximab Drugs 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 229960002964 adalimumab Drugs 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 229960002806 daclizumab Drugs 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 230000008303 genetic mechanism Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000019691 hematopoietic and lymphoid cell neoplasm Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000007762 localization of cell Effects 0.000 description 1
- 201000001142 lung small cell carcinoma Diseases 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 235000020004 porter Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000009258 tissue cross reactivity Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 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
- C07K16/2863—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 growth factors, growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001103—Receptors for growth factors
- A61K39/001104—Epidermal growth factor receptors [EGFR]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Gastroenterology & Hepatology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Oncology (AREA)
- Animal Behavior & Ethology (AREA)
- Dermatology (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Virology (AREA)
Abstract
本发明公开了一种抗EGFRvIII单克隆抗体及CAR‑T细胞及其应用;本发明涉及单克隆抗体及其单链可变片段(scFv),单克隆抗体包含具有SEQ ID NO.6的氨基酸序列的重链可变区VH和具有SEQ ID NO.7的氨基酸序列的轻链可变区VL;本发明还涉及一种嵌合抗原受体融合蛋白,其由信号肽、scFv、铰链区、跨膜结构域、共刺激结构域、信号传导结构域串联组成;并且将scFv片段用于构建本发明的CAR‑T细胞——其可针对EGFRvIII阳性靶细胞特异性分泌高水平细胞因子IFN‑γ,并对EGFRvIII抗原阳性靶细胞具有特异性杀伤作用;可广泛应用于CS1抗原阳性的肿瘤的治疗和诊断。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种抗EGFRvIII单克隆抗体及CAR-T细胞及其应用。
背景技术
免疫治疗肿瘤是目前公认的除手术,化疗和放疗之外能够根治肿瘤的有效方法之一。目前有效的免疫治疗方法主要包括抗体和免疫细胞治疗。1891年德国科学家PaulEhrlich首次提出了抗体的概念,并用侧链理论(Side-chain theory)描述了抗原-抗体相互作用的机制,推测抗体可以作为神奇子弹(Magic bullet)用于治疗。自此,抗体研究逐渐获得了人们的重要关注。1962年,Edelman和Porter成功的破解了抗体的化学结构,1976年至1980年,Susumu Tonegawa进一步阐明了抗体具有多样性的遗传机制。然而在很长一段时间内,关于抗体研究进展缓慢,主要原因是很难获得好质量的抗体。直到1975年,Jerne、Kohler,和Milstein成功的实现了杂交瘤技术,研究人员才能获得足够的单抗用于研究。11年后的1986年,第一个鼠源性全长单抗--抗CD3单抗被批准用于治疗急性器官排斥反应"。1994年,第一个嵌合Fab抗体abciximab(糖蛋白Ⅱβ特异性Fab)诞生。1997年,第一个全长嵌合型抗体rituximab(CD20特异性)研制成功。同年,首个人源化抗daclizumab(CD25特异性)诞生。2002年,首个全人源抗体adalimumab(TNF特异性)上市,抗体用于肿瘤和其它疾病的治疗,进展较大。
嵌合抗原受体修饰的T细胞免疫疗法(Chimeric Antigen Receptor T-CellImmunotherapy,CAR-T),是指将编码来源于抗体的用于识别肿瘤细胞抗原的免疫球蛋白的单链可变区抗体(scfv)的重链可变区(VH)和轻链可变区(VL)的重组蛋白基因和T细胞杀伤激活信号CD3ζ的DNA链,通过基因重组技术连接在基因工程表达载体上,并将其表达于杀伤性T细胞膜上形成嵌合抗原受体,从而使得T细胞不依赖MHC-I而识别肿瘤细胞,再经纯化、体外扩增及活化,将CAR-T细胞回输至患者体内,使其特异性行使杀灭肿瘤细胞的功能。CAR载体主要由3部分构成:抗原结合域、跨膜区及胞内信号转导区。嵌合抗原受体(CAR)-T细胞技术的优点:1、单链的CAR能与靶抗原高亲和性结合而不受中枢耐受的影响以非HLA分子限制的模式识别肿瘤抗原,这使得T细胞尤其是CTL绕过DC并克服了肿瘤细胞HLA分子表达下调的免疫逃逸机制直接杀伤肿瘤细胞,并且能够攻击低免疫原性的肿瘤。2、TCR只能识别蛋白抗原,而由于大部分的来源于单克隆抗体,可以设计特异性的以针对生物化学性质不同的肿瘤特异性标记,包括蛋白来源的多肽分子,或者非蛋白质分子如脂类和碳水化合物。扩大了选择的范围,增强了应用型。3、活化后的CAR-T细胞以及其他类型的效应细胞能够分泌很多细胞因子来对抗免疫抑制的肿瘤微环境,提高T细胞的抗肿瘤效应。4、自体或异体的细胞均具有一定的扩增能力,能够在体外迅速扩增至治疗剂量,并在体内能够维持治疗剂量,甚至产生针对特定肿瘤抗原记忆型的T细胞,增强了其临床可行性。5、相同的CAR可用于不同HLA-单倍体的病人,有利于技术的临床应用。
近年来,CAR-T在临床上取得了巨大的成功。例如,应用于B淋巴细胞白血病治疗的CD19 CAR-T疗法,最早进入临床实验,治疗效果也最为突出。它是通过识别肿瘤细胞表面的CD19来清除患者血液中的肿瘤细胞。在一项对晚期急性淋巴细胞白血病(ALL)患者进行的临床实验中:共30位患者,他们已经历了包括化疗,靶向治疗,甚至骨髓移植等各种治疗方法,已经没有其他治疗方案,通常情况下,生存时间不会超过半年。通过CD19 CAR-T治疗以后,27位病人的癌细胞完全消失,20位病人在半年以后复查,仍然没有发现任何癌细胞。在短短的几年时间里,CAR-T的研制和开发取得了飞速的发展,目前已经有20多项靶向不同肿瘤抗原的CAR-T疗法进入了临床试验,展现了极好的应用前景。并且已经逐步从最初的血液系统肿瘤拓展到了胶质瘤,肺癌、前列腺癌等多种实体肿瘤的治疗尝试。
EGFR基因在人类染色体上位于7p11-13,长度为110kb,编码野生型EGFR(Widetype epidermal growth factor receptor,wtEGFR)的氨基酸残基有1186个,其中氨基端的621个氨基酸为胞外区,该区域富含半胱氨酸,是与受体结合的主要区域。EGFRvIII在胞外域有2~7个外显子框内缺失,致其第6~273位氨基酸缺失,因而不具有与表皮生长因子(Epidermal growth factor,EGF)结合的能力。EGFRvIII在多种肿瘤中表达,包括胶质瘤(25-33%)、肺小细胞癌(16-39%)、肝癌(30-60%)、乳腺癌、膀胱癌等。EGFRvIII可通过调控Ras/Raf/MEK/细胞外调节蛋白激酶(Extracellular regulated protein kinases,ERK)等信号通路,影响肿瘤的发生和发展,尤其是在肿瘤的放射治疗(以下简称放疗)和化学药物治疗(以下简称化疗)过程中,发挥类似逃逸的功能。
CAR-T细胞疗法在血液肿瘤中已显示出令人鼓舞的临床结果,然而,在实体瘤中提高CAR-T细胞的功效方面仍然存在许多挑战,究其原因是无法突破肿瘤微环境的抑制作用。实体瘤具备纤维组织的物理屏障,不利于CAR-T细胞的定位和浸润。(1)相较于上述问题,EGFRVIII-CAR-T或许能有不一样的效果。现有临床试验的初步结果显示表皮生长因子受体III型突变体(EGFRvIII),(2)人表皮生长因子受体-2(HER2)等实体瘤中的抗原显示出较好的前景。EGFRvIII具有多种潜在优势:首先它在细胞表面表达,EGFRvIII相较于EGFR基因结构缺失第2至7外显子编码区,对应于EGFR在细胞外域缺失第6至273位的氨基酸残基,产生的突变是肿瘤特异性的,所以表皮生长因子受体变体III(EGFRvIII)仅在肿瘤中表达,在健康组织中并不表达,所以EGFRvIII是治疗的一个很好的靶点。
发明内容
本发明第一方面的目的,在于提供一种抗EGFRvIII单克隆抗体。
本发明第二方面的目的,在于提供一种单链可变片段。
本发明第三方面的目的,在于提供一种嵌合抗原受体(CAR)融合蛋白。
本发明第四方面的目的,在于提供上述单克隆抗体、单链可变片段、嵌合抗原受体(CAR)融合蛋白相关的生物材料。
本发明第五方面的目的,在于提供上述单克隆抗体、单链可变片段、嵌合抗原受体(CAR)融合蛋白或相关的生物材料的应用。
本发明第六方面的目的,在于提供一种产品。
本发明所采取的技术方案是:
本发明的第一方面,提供一种抗EGFRvIII单克隆抗体,所述单克隆抗体包括重链可变区和轻链可变区;
所述重链可变区包含CDR1、CDR2和CDR3;
所述重链可变区CDR1的氨基酸序列为:
a)SEQ ID NO.1;或
b)SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述重链可变区CDR2的氨基酸序列为:
a)SEQ ID NO.2;或
b)SEQ ID NO.2所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述重链可变区CDR3的氨基酸序列为:
a)TAD;或
b)TAD所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述轻链可变区包含CDR1、CDR2和CDR3;
所述轻链可变区CDR1的氨基酸序列为:
a)SEQ ID NO.3;或
b)SEQ ID NO.3所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述轻链可变区CDR2的氨基酸序列为:
a)SEQ ID NO.4;或
b)SEQ ID NO.4所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述轻链可变区CDR3的氨基酸序列为:
a)SEQ ID NO.5;或
b)SEQ ID NO.5所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列。
优选地,所述单克隆抗体的重链可变区的氨基酸序列为:
a)SEQ ID NO.6;或
b)SEQ ID NO.6所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述单克隆抗体的轻链可变区的氨基酸序列为:
a)SEQ ID NO.7;或
b)SEQ ID NO.7所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列。
本发明的第二方面,提供一种单链可变片段(scFv),所述单链可变片段包括本发明第一方面所述的单克隆抗体的轻链可变区和重链可变区。
优选地,所述单链可变片段还包括连接轻链可变区和重链可变区的接头。
优选地,所述单链可变片段的氨基酸序列为:
a)SEQ ID NO.9;或
b)SEQ ID NO.9所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列。
本发明的第三方面,提供一种嵌合抗原受体(CAR)融合蛋白,所述融合蛋白包括:信号肽、本发明第二方面所述的scFv、铰链区、跨膜结构域、共刺激结构域、信号传导结构域。
优选地,所述信号肽包括CD8信号肽。
优选地,所述铰链区包括CD8α铰链区。
优选地,所述跨膜结构域包括CD8α跨膜结构域。
优选地,所述共刺激结构域包括4-1BB。
优选地,所述信号传导结构域包括CD3ζ。
优选地,所述嵌合抗原受体(CAR)融合蛋白由信号肽、本发明第二方面所述的scFv、铰链区、跨膜结构域、共刺激结构域、信号传导结构域串联组成。
优选地,所述嵌合抗原受体(CAR)融合蛋白的氨基酸序列为:
a)SEQ ID NO.15;或
b)SEQ ID NO.15所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列。
本发明的第四方面,提供与本发明第一方面所述单克隆抗体或本发明第二方面所述单链可变片段或本发明第三方面所述嵌合抗原受体(CAR)融合蛋白相关的生物材料,所述生物材料为下述(c)~(f)中的任一种:
(c)编码本发明第一方面所述单克隆抗体或本发明第二方面所述单链可变片段或本发明第三方面所述嵌合抗原受体(CAR)融合蛋白;
(d)含有(c)所述核酸分子的表达盒;
(e)含有(c)所述核酸分子的重组载体、或含有(d)所述表达盒的重组载体;
(f)含有(c)所述核酸分子的重组微生物、或含有(d)所述表达盒的重组微生物、或含有(e)所述重组载体的重组细胞。
优选地,所述载体包括病毒性载体或非病毒性载体。
优选地,所述病毒性载体包括慢病毒载体、腺病毒载体、杆状病毒载体、反转录病毒载体、痘病毒载体、仙台病毒载体、单纯疱疹病毒载体中的至少一种。
优选地,所述重组细胞为哺乳动物细胞。
优选地,所述哺乳动物细胞包括293细胞、293T细胞或293F细胞中的任意一种或至少两种的组合。
优选地,所述哺乳动物细胞为293T细胞。
优选地,所述重组细胞还包含有辅助质粒。
本发明的第五方面,提供一种嵌合抗原受体免疫细胞,所述嵌合抗原受体免疫细胞表达本发明第三方面所述的嵌合抗原受体(CAR)融合蛋白。
优选地,所述嵌合抗原受体免疫细胞的基因组中整合有本发明第三方面所述的嵌合抗原受体(CAR)融合蛋白相关的生物材料。
优选地,所述免疫细胞包括T细胞、干细胞分化产生的免疫细胞、单核细胞、巨噬细胞或NK细胞的任意一种。
本发明第六方面,提供本发明第一方面所述的单克隆抗体或本发明第二方面所述的scFv或本发明第三方面所述的嵌合抗原受体(CAR)融合蛋白或相关生物材料在以下任一项所述的应用;
(g)检测EGFRvIII表达或制备检测EGFRvIII表达的产品或制备诊断EGFRvIII表达或功能异常的疾病的产品;
(h)预防或治疗与EGFRvIII表达或功能异常相关的疾病的产品。
优选地,所述产品包括:试剂盒、芯片、药物。
优选地,所述药物包括药学上可接受的载体。
优选地,所述的与EGFRvIII表达或功能异常相关的疾病为肿瘤。
优选地,所述肿瘤包括但不限于肠癌、肺癌、黑色素瘤、胃癌、鳞状细胞癌、纤维瘤、胰腺癌、脂肪瘤、血管内皮瘤、骨肉瘤、血管球瘤、骨瘤、巨细胞瘤、脑膜瘤、淋巴瘤、甲状腺癌、肝癌、卵巢癌、头颈癌、乳腺癌、宫颈癌、肾癌、膀胱癌、前列腺癌、食管癌、卵巢癌、胶质母细胞瘤和胆管癌。
优选地,所述肿瘤选自肠癌、肺癌、黑色素瘤、胶质母细胞瘤中的至少一种。
本发明的第七方面,提供一种产品,所述产品包括本发明第一方面所述单克隆抗体或本发明第二方面所述的scFv或本发明第三方面所述的嵌合抗原受体(CAR)融合蛋白或相关生物材料。
优选地,所述产品包括:试剂盒、芯片、药物。
本发明的有益效果是:
本发明提供了一种Anti EGFRvIII抗体及其单链可变片段(scFv),单克隆抗体包含具有SEQ ID NO.6的氨基酸序列的重链可变区VH和具有SEQ ID NO.7的氨基酸序列的轻链可变区VL;本发明还涉及一种嵌合抗原受体融合蛋白,其由信号肽、scFv、铰链区、跨膜结构域、共刺激结构域、信号传导结构域串联组成;本发明将SCFV片段用于CAR-T细胞的构建,并提出以EGFRvIII分子为靶抗原,利用Anti EGFRvIII CAR-T细胞杀伤肿瘤细胞,结果显示其可针对EGFRvIII阳性靶细胞特异性分泌高水平细胞因子IFN-γ,并对EGFRvIII抗原阳性靶细胞具有特异性杀伤作用;可作为细胞药物用于高表达EGFRvIII分子肿瘤类疾病的治疗。而且本发明还通过制备Anti EGFRvIII人源化嵌合单克隆抗体,可用于表达EGFRvIII分子肿瘤类疾病的诊断和治疗。
附图说明
图1示出了EGFRvIII CAR分子的结构示意图。
图2示出了慢病毒质粒载体Phage-EGFRvIII/CAR-mthy1.1(Anti EGFRvIII)图。
图3示出了高表达EGFRvIII的工程细胞株A549-EGFRvIII流式检测结果图。
图4示出了Anti EGFRvIII抗体与Jurkat-EGFRvIII阳性细胞株特异性结合的流式检测图。
图5示出了EGFRvIII CAR-T体外杀伤实验结果图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
本发明构建了一种人源化Anti EGFRvIII抗体,包含重链可变区(VH区)和轻链可变区(VL区)。
EGFRvIII VH区包括CDR1、CDR2和CDR3;其中
CDR1序列为:DYYMH(SEQ ID NO.1);
CDR2序列为:WIGWIDPENGKTKYAPKFQG(SEQ ID NO.2);
CDR3序列为:TAD;
EGFRvIII VH区氨基酸序列SEQ ID NO.6为:
QVQTQQSGTELVRPGALVKLSCKPSGFNIKDYYMHWVKQRPEQGLEWIGWIDP ENGKTKYAPKFQGKAIITADRSSNTAYLQLSSLTSEDTAVYYCAGTADWGQGTTVTVS S(划线部分为CDR序列);
人源化EGFRvIII VL区包括CDR1、CDR2和CDR3;其中
CDR1序列为:KSSQSLLHSDGETYLN(SEQ ID NO.3);
CDR2序列为:LVSKLDS(SEQ ID NO.4);
CDR3序列为:WQGKHFPYT(SEQ ID NO.5);
人源化EGFRvIII VL区氨基酸序列SEQ ID NO.7为:
DIQLTQSPLTLSVSIGQPASISCKSSQSLLHSDGETYLNWLLQRPGQSPKRLIYLVS KLDSGVPDRFTGTGSGTDFTLKISRVEAEDLGVYYCWQGKHFPYTFGGGTKLEI(划线部分为CDR序列)。
本发明还构建了一种单链可变区片段(scFv),包含EGFRvIII VL区和EGFRvIII VH区,其中EGFRvIII VL区和EGFRvIII VH区由接头连接,
其中接头序列为:GGGGSGGGGSGGGGS(SEQ ID NO.8);
scFv序列为:QVQTQQSGTELVRPGALVKLSCKPSGFNIKDYYMHWVKQRPEQG LEWIGWIDPENGKTKYAPKFQGKAIITADRSSNTAYLQLSSLTSEDTAVYYCAGTADWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPLTLSVSIGQPASISCKSSQSLLHSDGETYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGTGSGTDFTLKISRVEAEDLGVYYCWQGKHFPYTFGGGTKLEI(SEQ ID NO.9)。
本发明还涉及一种嵌合抗原受体融合蛋白(EGFRvIII SCFV-CD8-4-1BB-CD3),分子结构图如图1所示,其由信号肽、scFv、铰链区、跨膜结构域、共刺激结构域、信号传导结构域串联组成;
其中,信号肽为CD8信号肽,序列为MALPVTALLLPLALLLHAARP(SEQ ID NO.10);铰链区包括CD8α铰链区,序列为TTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACD(SEQ IDNO.11);跨膜结构域包括CD8α跨膜结构域;序列为IYIWAPLAGTCGVLLLSLVITLYC(SEQ IDNO.12);共刺激结构域包括4-1BB,序列为KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.13);信号传导结构域包括CD3ζ,序列为RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.14)。
嵌合抗原受体融合蛋白的氨基酸序列为:MALPVTALLLPLALLLHAARPQVQTQQ SGTELVRPGALVKLSCKPSGFNIKDYYMHWVKQRPEQGLEWIGWIDPENGKTKYAPKFQGKAIITADRSSNTAYLQLSSLTSEDTAVYYCAGTADWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPLTLSVSIGQPASISCKSSQSLLHSDGETYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGTGSGTDFTLKISRVEAEDLGVYYCWQGKHFPYTFGGGTKLEITTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ IDNO.15)。
本发明还提供了嵌合抗原受体(CAR)融合蛋白的编码基因序列:ATGGCCCTCC CTGTCACCGCCCTGCTGCTGCCTCTGGCTCTGCTGCTCCACGCCGCTCGGCCCCAGGTGCAGACCCAGCAGAGCGGCACCGAGCTGGTGCGCCCTGGAGCCCTGGTGAAGCTGAGCTGCAAGCCCAGCGGCTTCAACATCAAGGACTACTACATGCACTGGGTGAAGCAGCGCCCCGAGCAGGGCCTGGAGTGGATCGGCTGGATCGACCCCGAGAACGGCAAGACCAAGTACGCCCCCAAGTTCCAGGGCAAGGCCATCATCACCGCCGACCGCAGCAGCAACACCGCCTACCTGCAGCTGAGCAGCCTGACCAGCGAGGACACCGCCGTGTACTACTGCGCCGGCACCGCCGACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGAGGCGGAGGGAGCGGCGGAGGCGGGAGCGGCGGAGGCGGGAGCGACATCCAGCTGACCCAGAGCCCTCTGACCCTGAGCGTGAGCATCGGCCAGCCCGCCAGCATCAGCTGCAAGAGCAGCCAGAGCCTGCTGCACAGCGACGGCGAGACCTACCTGAACTGGCTGCTGCAGAGACCCGGCCAGAGCCCCAAGCGCCTGATCTACCTGGTGAGCAAGCTGGACAGCGGCGTGCCCGACCGCTTCACCGGCACCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCCGCGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCTGGCAGGGCAAGCACTTCCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCACCACCACACCAGCACCTAGGCCTCCCACCCCTGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGAGACCTGAGGCATGCAGACCCGCAGCTGGCGGAGCCGTGCATACCCGGGGCCTGGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGCACCTGCGGGGTCCTGCTGCTGAGCCTCGTGATCACCCTGTACTGTAAGCGCGGCCGGAAGAAGCTGCTGTACATCTTTAAGCAGCCCTTCATGAGGCCTGTGCAGACCACCCAGGAGGAGGACGGCTGTAGCTGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCTCGCAGAAAGAATCCCCAGGAGGGCCTGTACAACGAGCTCCAGAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGCATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTGCACATGCAGGCCCTGCCTCCTCGGTAA(SEQ IDNO.16)。
实施例1:慢病毒表达载体制备
发明人在慢病毒载体内,经克隆至慢病毒载体(Phage-mthy1.1)的BamHI和MluI限制性内切酶位点,构建了EGFRvIII SCFV-CAR结构体。在Phage-mthy1.1慢病毒CAR构建体中,包含了EGFRvIII SCFV-CD8-4-1BB-CD3片段,其插入位点在BamHI和MluI限制性内切酶位点之间,基因序列如SEQ ID NO:16所示。载体转化Stbl3大肠杆菌菌株,氨苄青霉素筛选,获得阳性克隆,提取质粒,酶切鉴定克隆,获得Phage-EGFRvIII SCFV/CAR-mthy1.1慢病毒转染载体,见图2。
实施例2:慢病毒制备
慢病毒载体通过在293T细胞中扩增生产,显微镜下观察T175细胞培养瓶内细胞铺板密度,密度达到90%-95%最佳;
换液:将密度适宜的细胞放置生物安全柜内,使用25ml移液管吸弃细胞培养液,并更换17ml包装用细胞培养基(DMEM培养基,无血清添加),并放回CO2培养箱内;
配制磷酸钙-DNA沉淀复合物:取4ml CaCl2于50ml离心管内,依次添加表达质粒38.5μg,包装辅助质粒(pCMV-dR8.91)24.5μg,包膜质粒(pCMV-VSVG)11μg,混匀。取4ml 2×HBS(900ml超纯水中加入NaCl 16.3g,KCl 0.74g,Na2HPO4 0.214g,Glucose 2.4g和HEPES10g,调pH至7.05,定容至1L,0.2μm滤膜过滤后储存于4°C备用),逐滴添加至DNA-CaCl2混合液中,边添加,边震荡,室温孵育15-20min;
取出培养箱内待包装细胞,并向其中添加磷酸钙-DNA沉淀复合物,4-6h使用更换新鲜培养基,继续培养,分别在48h和72h后收集含病毒的上清液。4500rpm离心5min后取上清并使用0.45μm滤器过滤;
取出高压灭菌后的高速离心管,加入20ml过滤后的病毒上清。吸取12ml 20%的蔗糖溶液(PBS配制)。将移液管一直插入到离心管的底部,缓慢加入10ml蔗糖溶液,注意不要用大的冲击力打破两液面的分界,否则会影响慢病毒的浓缩效果。4℃离心,19000rpm离心4h。离心完毕后小心取出超速离心管,弃去上清液,管底可见少量的半透明或白色沉淀。将超速离心管倒置在吸水纸上晾干约10min,并用枪头吸去管壁上多余的液体。每管中加入100μl冷PBS重悬沉淀,4℃溶解2h,每隔20min轻轻震荡,避免产生泡沫,50μl/管病毒液进行分装,-80℃冻存。
实施例3:EGFRvIII-PBMC和EGFRvIII-A549工程细胞株的构建及EGFRvIII抗体的特异性检测
(1)构建高表达EGFRvIII PBMC/A549工程细胞株的慢病毒制备(具体制备方法见实施例2中的方法);
(2)PBMC/A549细胞的感染:感染前一天,接种5×105个PBMC/A549细胞于6孔板中,待第二天细胞长到80%时,加入包装好的500μl EGFRvIII慢病毒,同时设置对照细胞(不添加病毒),12-16小时后换液,感染3天后,流式分选EGFRvIII-PBMC和EGFRvIII-A549阳性细胞。其中EGFRvIII-PBMC细胞作为EGFRvIII抗体的特异性检测,EGFRvIII-A549细胞用于后续杀伤实验的靶细胞。结果如图3所示,使用流式抗体检测mthy1.1的阳性比率,EGFRvIII-PBMC阳性细胞的比率为72.1%。
(3)EGFRvIII抗体的特异性检测:取100μl EGFRvIII-PBMC工程细胞株(2*106/ml),加入100μl含anti EGFRvIII抗体上清(阴性对照为不加EGFRvIII抗体上清),混匀后冰上孵育30min,加入2ml PBS清洗并离心,加100μl PBS重悬,每管加入0.3μlanti-mouse-APC-antibody lgG,混匀,冰上染色30min,PBS清洗后流式细胞仪上机检测。如图4所示,使用流式细胞仪检测APC的阳性比率,EGFRvIII-PBMC工程细胞株和anti EGFRvIII抗体上清的结合率高达93.7%。
实施例4:Anti EGFRvIII-CAR-T细胞制备
取1ml血液进行快速的病原微生物检测,排除HBV、HCV、HDV和HEV、HIV-1/2、梅毒螺旋体感染。无菌条件下,用10ml EDTA抗凝采血管收集血液50ml,立即(2-8℃,24h内)送至细胞制备实验室,保证此过程无上述病原微生物污染。到达GMP制备室后,用酒精棉球擦拭EDTA采血管表面进行消毒后放入生物安全柜。室温500g离心10min,收集上层血浆,留下沉淀层。收集的自体血浆经56℃灭活30min,室温放置15min,室温900g离心30min,取上清备用。沉淀层用生理盐水稀释至25ml/管,打开2个新的50ml离心管,每个离心管分别加入20ml人淋巴细胞分离液。用移液管把稀释后的血细胞液缓缓加入到盛有人淋巴分离液的离心管中,调至最小的升降速率,室温600g离心20min。收集离心管的中间层白细胞于一支50ml无菌离心管中,加入生理盐水至50ml,洗两次(500g离心5min),沉淀细胞即为人外周血单核细胞(PBMC)。
将分离得到的PBMC用培养基稀释成2×106/ml,取50ul流式检测PBMC中T细胞的纯度。配置完全生长培养基(X-VIVO15培养基添加2%自体血浆,IL2的终浓度为1000U/mL,IL/7的终浓度为10ng/ml,IL/15的终浓度为10ng/ml,IL/21的终浓度为10ng/ml)。
第一天,按操作说明书清洗人CD3/CD28 beads,将PBMC细胞和beads混合后按2×106PBMC/ml加到合适的培养瓶中(beads与T细胞比例3:1)。第二天按Lentivirus:cell=5:1(MOI=5)比例添加实施例2制备的慢病毒。第三天,补加新鲜的完全培养基并将细胞密度调整至0.3×106/ml继续培养。每隔2-3天半量换液,维持细胞密度0.3-1×106/ml。第5天,用磁力架去掉CD3/CD28 beads。第10-12天,细胞数量达到109级别,500g离心5min后用生理盐水洗涤三遍(500g离心5min)。血球计数板计数,流式细胞仪检测细胞类群以及CAR-T细胞比例(mthy1.1阳性比例)(如图3)。
实施例5:EGFRvIII-CAR-T细胞体外活性检测
本实施例中检测杀伤效应,通过流式分析检测Anti EGFRvIII-CAR-T细胞对工程细胞株A549/EGFRvIII的杀伤作用以及释放IFN-γ的能力。
(1)用X-VIVO15培养基将靶细胞调整到1×107个/ml;
(2)在24孔细胞培养板中加入200μl靶细胞,对照组为A549-WT细胞(野生型A549细胞);
(3)向各孔加1×104个EGFRvIII-CAR-T效应细胞(效应细胞:靶细胞=5:1),共同孵育6小时;
(4)收集并使用生理盐水清洗上述细胞,加入1μl PE-anti-human-CD3流式抗体,4℃避光孵育25-30min后加入500μl 1X固定/破膜缓冲液(BD),4℃过夜后加入1ml1X破膜/清洗缓冲液,2000rpm离心5min,弃去上清液,重复上述步骤一次后加入1μlAPC-anti-human-IFN-γ流式抗体,4℃避光孵育25-30min;
(5)生理盐水清洗细胞后流式细胞仪上机检测。
结果如图5所示,Anti EGFRvIII-CAR-T细胞能够显著杀伤高表达EGFRvIII的靶细胞株A549/EGFRvIII,并分泌IFN-γ。
上述具体实施方式对本发明作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
Claims (10)
1.一种抗EGFRvIII单克隆抗体,其特征在于,所述单克隆抗体包括重链可变区和轻链可变区;
所述重链可变区包含CDR1、CDR2和CDR3;
所述重链可变区CDR1的氨基酸序列为:
a)SEQ ID NO.1;或
b)SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述重链可变区CDR2的氨基酸序列为:
a)SEQ ID NO.2;或
b)SEQ ID NO.2所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述重链可变区CDR3的氨基酸序列为:
a)TAD;或
b)TAD所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述轻链可变区包含CDR1、CDR2和CDR3;
所述轻链可变区CDR1的氨基酸序列为:
a)SEQ ID NO.3;或
b)SEQ ID NO.3所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述轻链可变区CDR2的氨基酸序列为:
a)SEQ ID NO.4;或
b)SEQ ID NO.4所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述轻链可变区CDR3的氨基酸序列为:
a)SEQ ID NO.5;或
b)SEQ ID NO.5所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列。
2.根据权利要求1所述的单克隆抗体,其特征在于,所述单克隆抗体的重链可变区的氨基酸序列为:
a)SEQ ID NO.6;或
b)SEQ ID NO.6所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列;
所述单克隆抗体的轻链可变区的氨基酸序列为:
a)SEQ ID NO.7;或
b)SEQ ID NO.7所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列。
3.一种单链可变片段(scFv),所述单链可变片段包括权利要求1或2所述的单克隆抗体的轻链可变区和重链可变区;优选地,所述单链可变片段还包括连接轻链可变区和重链可变区的接头;
优选地,所述单链可变片段的氨基酸序列为:
a)SEQ ID NO.9;或
b)SEQ ID NO.9所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列。
4.一种嵌合抗原受体(CAR)融合蛋白,所述融合蛋白包括:信号肽、权利要求3所述的scFv、铰链区、跨膜结构域、共刺激结构域、信号传导结构域;
优选地,所述信号肽包括CD8信号肽;
优选地,所述铰链区包括CD8α铰链区;
优选地,所述跨膜结构域包括CD8α跨膜结构域;
优选地,所述共刺激结构域包括4-1BB;
优选地,所述信号传导结构域包括CD3ζ;
优选地,所述嵌合抗原受体(CAR)融合蛋白的氨基酸序列为:
a)SEQ ID NO.15;或
b)SEQ ID NO.15所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列。
5.与权利要求1或2所述的单克隆抗体、权利要求3所述的单链可变片段或权利要求4所述的嵌合抗原受体(CAR)融合蛋白相关的生物材料,所述生物材料为下述(c)~(f)中的任一种:
(c)编码权利要求1或2所述的单克隆抗体、权利要求3所述的单链可变片段或权利要求4所述的嵌合抗原受体(CAR)融合蛋白的核酸分子;
(d)含有(c)所述核酸分子的表达盒;
(e)含有(c)所述核酸分子的重组载体、或含有(d)所述表达盒的重组载体;
(f)含有(c)所述核酸分子的重组微生物、或含有(d)所述表达盒的重组微生物、或含有(e)所述重组载体的重组细胞。
6.一种嵌合抗原受体免疫细胞,所述嵌合抗原受体免疫细胞表达权利要求4所述的嵌合抗原受体(CAR)融合蛋白;
优选地,所述嵌合抗原受体免疫细胞的基因组中整合有权利要求4所述的嵌合抗原受体(CAR)融合蛋白相关的生物材料。
7.根据权利要求6所述的嵌合抗原受体免疫细胞,其特征在于,所述免疫细胞包括T细胞、干细胞分化产生的免疫细胞、单核细胞、巨噬细胞或NK细胞中的任意一种。
8.权利要求1或2所述的单克隆抗体或权利要求3所述的scFv或权利要求5所述的嵌合抗原受体(CAR)融合蛋白或相关生物材料在以下任一项中的应用;
(g)检测EGFRvIII表达或制备检测EGFRvIII表达的产品或制备诊断EGFRvIII表达或功能异常的疾病的产品;
(h)制备预防或治疗与EGFRvIII表达或功能异常相关的疾病的产品中的应用。
9.根据权利要求8所述的应用,其特征在于,所述与EGFRvIII表达或功能异常相关的疾病为肿瘤;
优选地,所述肿瘤包括但不限于肠癌、肺癌、黑色素瘤、胃癌、鳞状细胞癌、纤维瘤、胰腺癌、脂肪瘤、血管内皮瘤、骨肉瘤、血管球瘤、骨瘤、巨细胞瘤、脑膜瘤、淋巴瘤、甲状腺癌、肝癌、卵巢癌、头颈癌、乳腺癌、宫颈癌、肾癌、膀胱癌、前列腺癌、食管癌、卵巢癌、胶质母细胞瘤和胆管癌。
10.一种产品,所述产品包括权利要求1或2所述的单克隆抗体或权利要求3所述的scFv或权利要求4所述的嵌合抗原受体(CAR)融合蛋白或相关生物材料。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310431051.9A CN116715768A (zh) | 2023-04-20 | 2023-04-20 | 一种抗EGFRvIII单克隆抗体及CAR-T细胞及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310431051.9A CN116715768A (zh) | 2023-04-20 | 2023-04-20 | 一种抗EGFRvIII单克隆抗体及CAR-T细胞及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116715768A true CN116715768A (zh) | 2023-09-08 |
Family
ID=87874109
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310431051.9A Pending CN116715768A (zh) | 2023-04-20 | 2023-04-20 | 一种抗EGFRvIII单克隆抗体及CAR-T细胞及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116715768A (zh) |
-
2023
- 2023-04-20 CN CN202310431051.9A patent/CN116715768A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110214025A (zh) | 携带双特异性T细胞衔接器(BiTE)的腺病毒 | |
CN108373504A (zh) | Cd24特异性抗体和抗cd24-car-t细胞 | |
WO2023046110A1 (zh) | 联合表达CCR2b的工程化免疫细胞及其制备和应用 | |
CN108864307A (zh) | 信号肽优化靶向cd19的嵌合抗原受体、表达该嵌合抗原受体的t细胞及制备方法和应用 | |
CN110055269B (zh) | 人间皮素嵌合抗原受体、其t细胞及其制备方法和用途 | |
CN110317822B (zh) | Trop2嵌合抗原受体、其t细胞及其制备方法和用途 | |
CN113755448B (zh) | 联合表达CCR2b和CD40L的工程化免疫细胞及其制备和应用 | |
CN113248616B (zh) | 靶向gpc3的嵌合抗原受体及其用途 | |
CN112426526B (zh) | 一种nk细胞的制备方法及其在治疗癌症中的应用 | |
CN109021114B (zh) | 联合两种单链抗体的双特异性嵌合抗原受体及表达载体 | |
WO2021057866A1 (zh) | 一种单域抗体及包含抗体结构的嵌合抗原受体 | |
CN111944053B (zh) | 抗bcma的car及其表达载体和应用 | |
CN113045675B (zh) | 一种抗cd22蛋白分子的抗体及其应用 | |
CN116120465B (zh) | 一种靶向bcma和/或fcrh5的嵌合抗原受体及其应用 | |
CN110078830A (zh) | 一种在共刺激结构域上携带重复活化基序的嵌合抗原受体t细胞 | |
CN116143943B (zh) | 一种靶向baffr嵌合抗原受体、car-t细胞及应用 | |
WO2021139755A1 (zh) | 工程改造的t细胞、其制备及应用 | |
CN116715768A (zh) | 一种抗EGFRvIII单克隆抗体及CAR-T细胞及其应用 | |
CN109970859A (zh) | Glypican-3特异性抗体及其特异性CAR-T细胞 | |
CN114853893A (zh) | 一种靶向gpc3嵌合抗原受体t细胞及其应用 | |
CN111286512A (zh) | 靶向人源化酪氨酸激酶孤儿受体1的嵌合抗原受体及其用途 | |
CN117247462B (zh) | 一种靶向ror1的嵌合抗原受体、car-t细胞及其用途 | |
CN116063569B (zh) | Epha2嵌合抗原受体及其用途 | |
CN113549157B (zh) | 双靶向嵌合抗原受体及其应用 | |
CN114891123B (zh) | 一种基于CD79b人源化抗体的嵌合抗原受体及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |