CN107793481A - 一种靶向人cd123的嵌合受体配体及其应用 - Google Patents
一种靶向人cd123的嵌合受体配体及其应用 Download PDFInfo
- Publication number
- CN107793481A CN107793481A CN201610795955.XA CN201610795955A CN107793481A CN 107793481 A CN107793481 A CN 107793481A CN 201610795955 A CN201610795955 A CN 201610795955A CN 107793481 A CN107793481 A CN 107793481A
- Authority
- CN
- China
- Prior art keywords
- seq
- amino acid
- chimerical receptor
- people
- receptor part
- 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
- 101000998120 Homo sapiens Interleukin-3 receptor subunit alpha Proteins 0.000 title claims abstract description 206
- 102100033493 Interleukin-3 receptor subunit alpha Human genes 0.000 title claims abstract description 204
- 108020003175 receptors Proteins 0.000 title claims abstract description 79
- 230000008685 targeting Effects 0.000 claims abstract description 49
- 230000027455 binding Effects 0.000 claims abstract description 34
- 230000003834 intracellular effect Effects 0.000 claims abstract description 30
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 25
- 239000003814 drug Substances 0.000 claims abstract description 6
- 102000005962 receptors Human genes 0.000 claims description 75
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 65
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 34
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims description 31
- 230000014509 gene expression Effects 0.000 claims description 27
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 20
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 20
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 17
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 claims description 17
- 239000002773 nucleotide Substances 0.000 claims description 17
- 125000003729 nucleotide group Chemical group 0.000 claims description 17
- 230000009466 transformation Effects 0.000 claims description 14
- 238000010353 genetic engineering Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 108091026890 Coding region Proteins 0.000 claims description 11
- 230000000139 costimulatory effect Effects 0.000 claims description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 7
- 108010064548 Lymphocyte Function-Associated Antigen-1 Proteins 0.000 claims description 6
- 125000000539 amino acid group Chemical group 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 230000019491 signal transduction Effects 0.000 claims description 6
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 claims description 5
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 claims description 5
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 claims description 5
- 230000009870 specific binding Effects 0.000 claims description 5
- 210000000130 stem cell Anatomy 0.000 claims description 5
- -1 CD28 Proteins 0.000 claims description 4
- 230000031146 intracellular signal transduction Effects 0.000 claims description 4
- 102100027207 CD27 antigen Human genes 0.000 claims description 3
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 claims description 3
- 102100040678 Programmed cell death protein 1 Human genes 0.000 claims description 3
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 claims description 3
- 102100038078 CD276 antigen Human genes 0.000 claims description 2
- 101710185679 CD276 antigen Proteins 0.000 claims description 2
- 101150013553 CD40 gene Proteins 0.000 claims description 2
- 102100035793 CD83 antigen Human genes 0.000 claims description 2
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 claims description 2
- 101001109503 Homo sapiens NKG2-C type II integral membrane protein Proteins 0.000 claims description 2
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 claims description 2
- 102100022683 NKG2-C type II integral membrane protein Human genes 0.000 claims description 2
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 claims description 2
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims description 2
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 claims description 2
- 238000004113 cell culture Methods 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 230000004069 differentiation Effects 0.000 claims description 2
- 210000000822 natural killer cell Anatomy 0.000 claims description 2
- 108010002386 Interleukin-3 Proteins 0.000 claims 7
- 208000002250 Hematologic Neoplasms Diseases 0.000 claims 2
- 230000000118 anti-neoplastic effect Effects 0.000 claims 2
- 210000001671 embryonic stem cell Anatomy 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 137
- 239000003446 ligand Substances 0.000 abstract description 18
- 239000012642 immune effector Substances 0.000 abstract description 8
- 229940121354 immunomodulator Drugs 0.000 abstract description 8
- 210000004881 tumor cell Anatomy 0.000 abstract description 7
- 230000001665 lethal effect Effects 0.000 abstract description 5
- 238000011398 antitumor immunotherapy Methods 0.000 abstract 1
- 108090000176 Interleukin-13 Proteins 0.000 description 53
- 102000003816 Interleukin-13 Human genes 0.000 description 52
- 239000005089 Luciferase Substances 0.000 description 28
- 108060001084 Luciferase Proteins 0.000 description 26
- 108091028043 Nucleic acid sequence Proteins 0.000 description 21
- 230000002147 killing effect Effects 0.000 description 19
- 230000000694 effects Effects 0.000 description 16
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 108091007047 SCF complex Proteins 0.000 description 12
- 108091007045 Cullin Ring E3 Ligases Proteins 0.000 description 11
- 102000036364 Cullin Ring E3 Ligases Human genes 0.000 description 11
- 108010002350 Interleukin-2 Proteins 0.000 description 11
- 102100040066 Interleukin-27 receptor subunit alpha Human genes 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 11
- 238000001514 detection method Methods 0.000 description 11
- 238000000338 in vitro Methods 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 108010074328 Interferon-gamma Proteins 0.000 description 10
- 239000000427 antigen Substances 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 9
- 102100037850 Interferon gamma Human genes 0.000 description 9
- 238000005119 centrifugation Methods 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 108010038452 Interleukin-3 Receptors Proteins 0.000 description 8
- 102000010790 Interleukin-3 Receptors Human genes 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 6
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 6
- 108090000331 Firefly luciferases Proteins 0.000 description 6
- 230000022534 cell killing Effects 0.000 description 6
- 230000003013 cytotoxicity Effects 0.000 description 6
- 231100000135 cytotoxicity Toxicity 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 238000009169 immunotherapy Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- 230000011664 signaling Effects 0.000 description 5
- 238000010361 transduction Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 102100024633 Carbonic anhydrase 2 Human genes 0.000 description 4
- 102100024650 Carbonic anhydrase 3 Human genes 0.000 description 4
- 101000760643 Homo sapiens Carbonic anhydrase 2 Proteins 0.000 description 4
- 101000760630 Homo sapiens Carbonic anhydrase 3 Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 239000000370 acceptor Substances 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 230000026683 transduction Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102100024263 CD160 antigen Human genes 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 102100038497 Cytokine receptor-like factor 2 Human genes 0.000 description 3
- 101000761938 Homo sapiens CD160 antigen Proteins 0.000 description 3
- 101000994375 Homo sapiens Integrin alpha-4 Proteins 0.000 description 3
- 101001033279 Homo sapiens Interleukin-3 Proteins 0.000 description 3
- 101000633786 Homo sapiens SLAM family member 6 Proteins 0.000 description 3
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 3
- 102100032818 Integrin alpha-4 Human genes 0.000 description 3
- 102100032816 Integrin alpha-6 Human genes 0.000 description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 108091007110 SCF2 complex Proteins 0.000 description 3
- 102100029197 SLAM family member 6 Human genes 0.000 description 3
- 102100029215 Signaling lymphocytic activation molecule Human genes 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 208000014951 hematologic disease Diseases 0.000 description 3
- 102000055276 human IL3 Human genes 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 206010000830 Acute leukaemia Diseases 0.000 description 2
- 208000025321 B-lymphoblastic leukemia/lymphoma Diseases 0.000 description 2
- 208000019838 Blood disease Diseases 0.000 description 2
- 102100038077 CD226 antigen Human genes 0.000 description 2
- 101000884298 Homo sapiens CD226 antigen Proteins 0.000 description 2
- 101001078158 Homo sapiens Integrin alpha-1 Proteins 0.000 description 2
- 101000994365 Homo sapiens Integrin alpha-6 Proteins 0.000 description 2
- 101001035237 Homo sapiens Integrin alpha-D Proteins 0.000 description 2
- 101001046687 Homo sapiens Integrin alpha-E Proteins 0.000 description 2
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 2
- 101000935040 Homo sapiens Integrin beta-2 Proteins 0.000 description 2
- 101001019600 Homo sapiens Interleukin-17 receptor B Proteins 0.000 description 2
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 2
- 101001043817 Homo sapiens Interleukin-31 receptor subunit alpha Proteins 0.000 description 2
- 101000971538 Homo sapiens Killer cell lectin-like receptor subfamily F member 1 Proteins 0.000 description 2
- 101000633780 Homo sapiens Signaling lymphocytic activation molecule Proteins 0.000 description 2
- 101000946860 Homo sapiens T-cell surface glycoprotein CD3 epsilon chain Proteins 0.000 description 2
- 102100025323 Integrin alpha-1 Human genes 0.000 description 2
- 102100039904 Integrin alpha-D Human genes 0.000 description 2
- 102100022341 Integrin alpha-E Human genes 0.000 description 2
- 102100022338 Integrin alpha-M Human genes 0.000 description 2
- 102100022297 Integrin alpha-X Human genes 0.000 description 2
- 102100025304 Integrin beta-1 Human genes 0.000 description 2
- 102100025390 Integrin beta-2 Human genes 0.000 description 2
- 102100035014 Interleukin-17 receptor B Human genes 0.000 description 2
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 2
- 102100021594 Interleukin-31 receptor subunit alpha Human genes 0.000 description 2
- 102100021458 Killer cell lectin-like receptor subfamily F member 1 Human genes 0.000 description 2
- 101100497331 Oryza sativa subsp. japonica CRL5 gene Proteins 0.000 description 2
- 102000014128 RANK Ligand Human genes 0.000 description 2
- 108010025832 RANK Ligand Proteins 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 102100027744 Semaphorin-4D Human genes 0.000 description 2
- 102100035794 T-cell surface glycoprotein CD3 epsilon chain Human genes 0.000 description 2
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 201000009277 hairy cell leukemia Diseases 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 208000017426 precursor B-cell acute lymphoblastic leukemia Diseases 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- OJHZNMVJJKMFGX-RNWHKREASA-N (4r,4ar,7ar,12bs)-9-methoxy-3-methyl-1,2,4,4a,5,6,7a,13-octahydro-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one;2,3-dihydroxybutanedioic acid Chemical compound OC(=O)C(O)C(O)C(O)=O.O=C([C@@H]1O2)CC[C@H]3[C@]4([H])N(C)CC[C@]13C1=C2C(OC)=CC=C1C4 OJHZNMVJJKMFGX-RNWHKREASA-N 0.000 description 1
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 1
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 1
- 108010056102 CD100 antigen Proteins 0.000 description 1
- 108010017009 CD11b Antigen Proteins 0.000 description 1
- 208000016778 CD4+/CD56+ hematodermic neoplasm Diseases 0.000 description 1
- 108010062802 CD66 antigens Proteins 0.000 description 1
- 102100024533 Carcinoembryonic antigen-related cell adhesion molecule 1 Human genes 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 1
- 102100027816 Cytotoxic and regulatory T-cell molecule Human genes 0.000 description 1
- 102000016607 Diphtheria Toxin Human genes 0.000 description 1
- 108010053187 Diphtheria Toxin Proteins 0.000 description 1
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010021468 Fc gamma receptor IIA Proteins 0.000 description 1
- 102100022086 GRB2-related adapter protein 2 Human genes 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 102100029360 Hematopoietic cell signal transducer Human genes 0.000 description 1
- 102100022132 High affinity immunoglobulin epsilon receptor subunit gamma Human genes 0.000 description 1
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 1
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 1
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 1
- 101000900690 Homo sapiens GRB2-related adapter protein 2 Proteins 0.000 description 1
- 101000990188 Homo sapiens Hematopoietic cell signal transducer Proteins 0.000 description 1
- 101000824104 Homo sapiens High affinity immunoglobulin epsilon receptor subunit gamma Proteins 0.000 description 1
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 1
- 101001046683 Homo sapiens Integrin alpha-L Proteins 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 101001046668 Homo sapiens Integrin alpha-X Proteins 0.000 description 1
- 101001015037 Homo sapiens Integrin beta-7 Proteins 0.000 description 1
- 101001076430 Homo sapiens Interleukin-13 Proteins 0.000 description 1
- 101001043809 Homo sapiens Interleukin-7 receptor subunit alpha Proteins 0.000 description 1
- 101000777628 Homo sapiens Leukocyte antigen CD37 Proteins 0.000 description 1
- 101001047640 Homo sapiens Linker for activation of T-cells family member 1 Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 101001090688 Homo sapiens Lymphocyte cytosolic protein 2 Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 description 1
- 101000589305 Homo sapiens Natural cytotoxicity triggering receptor 2 Proteins 0.000 description 1
- 101000873418 Homo sapiens P-selectin glycoprotein ligand 1 Proteins 0.000 description 1
- 101000702132 Homo sapiens Protein spinster homolog 1 Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101000633778 Homo sapiens SLAM family member 5 Proteins 0.000 description 1
- 101000633784 Homo sapiens SLAM family member 7 Proteins 0.000 description 1
- 101000934346 Homo sapiens T-cell surface antigen CD2 Proteins 0.000 description 1
- 101000596234 Homo sapiens T-cell surface protein tactile Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 101000809875 Homo sapiens TYRO protein tyrosine kinase-binding protein Proteins 0.000 description 1
- 101000795169 Homo sapiens Tumor necrosis factor receptor superfamily member 13C Proteins 0.000 description 1
- 101000648507 Homo sapiens Tumor necrosis factor receptor superfamily member 14 Proteins 0.000 description 1
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 1
- 101000679857 Homo sapiens Tumor necrosis factor receptor superfamily member 3 Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 102100022339 Integrin alpha-L Human genes 0.000 description 1
- 108010041100 Integrin alpha6 Proteins 0.000 description 1
- 108010030465 Integrin alpha6beta1 Proteins 0.000 description 1
- 102100033016 Integrin beta-7 Human genes 0.000 description 1
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 1
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 102100030703 Interleukin-22 Human genes 0.000 description 1
- 108010052781 Interleukin-3 Receptor alpha Subunit Proteins 0.000 description 1
- 102000018883 Interleukin-3 Receptor alpha Subunit Human genes 0.000 description 1
- 101710123866 Interleukin-3 receptor subunit alpha Proteins 0.000 description 1
- 102100021593 Interleukin-7 receptor subunit alpha Human genes 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 102100031586 Leukocyte antigen CD37 Human genes 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102100024032 Linker for activation of T-cells family member 1 Human genes 0.000 description 1
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 102100034709 Lymphocyte cytosolic protein 2 Human genes 0.000 description 1
- 208000028018 Lymphocytic leukaemia Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 101100236305 Mus musculus Ly9 gene Proteins 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 description 1
- 108010004217 Natural Cytotoxicity Triggering Receptor 1 Proteins 0.000 description 1
- 108010004222 Natural Cytotoxicity Triggering Receptor 3 Proteins 0.000 description 1
- 102100032870 Natural cytotoxicity triggering receptor 1 Human genes 0.000 description 1
- 102100032851 Natural cytotoxicity triggering receptor 2 Human genes 0.000 description 1
- 102100032852 Natural cytotoxicity triggering receptor 3 Human genes 0.000 description 1
- 102100038082 Natural killer cell receptor 2B4 Human genes 0.000 description 1
- 101710141230 Natural killer cell receptor 2B4 Proteins 0.000 description 1
- 102100034925 P-selectin glycoprotein ligand 1 Human genes 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 208000009052 Precursor T-Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 102100029216 SLAM family member 5 Human genes 0.000 description 1
- 102100029198 SLAM family member 7 Human genes 0.000 description 1
- 108010074687 Signaling Lymphocytic Activation Molecule Family Member 1 Proteins 0.000 description 1
- 206010041047 Slow virus infection Diseases 0.000 description 1
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 description 1
- 102100035268 T-cell surface protein tactile Human genes 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- 102100038717 TYRO protein tyrosine kinase-binding protein Human genes 0.000 description 1
- 102100029690 Tumor necrosis factor receptor superfamily member 13C Human genes 0.000 description 1
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 description 1
- 102100033733 Tumor necrosis factor receptor superfamily member 1B Human genes 0.000 description 1
- 101710187830 Tumor necrosis factor receptor superfamily member 1B Proteins 0.000 description 1
- 102100022156 Tumor necrosis factor receptor superfamily member 3 Human genes 0.000 description 1
- 101001038499 Yarrowia lipolytica (strain CLIB 122 / E 150) Lysine acetyltransferase Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 108700010039 chimeric receptor Proteins 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 108010072917 class-I restricted T cell-associated molecule Proteins 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000004940 costimulation Effects 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 229960003983 diphtheria toxoid Drugs 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical group O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 208000018706 hematopoietic system disease Diseases 0.000 description 1
- 238000002868 homogeneous time resolved fluorescence Methods 0.000 description 1
- 102000052088 human IL3RA Human genes 0.000 description 1
- 102000019207 human interleukin-13 Human genes 0.000 description 1
- 102000006426 human interleukin-13 receptor Human genes 0.000 description 1
- 108010044118 human interleukin-13 receptor Proteins 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 108010074109 interleukin-22 Proteins 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 239000002085 irritant Substances 0.000 description 1
- 231100000021 irritant Toxicity 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 208000003747 lymphoid leukemia Diseases 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 230000036259 sexual stimuli Effects 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 108010078373 tisagenlecleucel Proteins 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70517—CD8
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated 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
- A61K39/464416—Receptors for cytokines
- A61K39/464419—Receptors for interleukins [IL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5403—IL-3
-
- 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
-
- 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
- C07K14/70507—CD2
-
- 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
- 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
- C07K14/70521—CD28, CD152
-
- 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
- C07K14/70546—Integrin superfamily
-
- 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
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- 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
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/867—Retroviral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
-
- 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
本发明提供了一种靶向人CD123的嵌合受体配体,包含基于IL‑3分子的CD123结合结构域、跨膜区及胞内信号结构域。本发明还提供了一种靶向人CD123的嵌合受体配体修饰的T细胞,能够特异地与肿瘤细胞表面的CD123结合,进而对肿瘤细胞产生特异的杀伤作用。本发明还涉及所述靶向人CD123的嵌合受体配体及其免疫效应细胞在制备抗肿瘤免疫治疗药物中的应用。
Description
技术领域
本发明属于生物医学或生物制药技术领域,涉及一种靶向人CD123的嵌合受体配体及其应用。
背景技术
CD123,即人白介素3受体α链(Interleukin-3receptor subunit alpha,IL-3Rα),全长378个氨基酸(NP_002174.1),其中信号肽包含18个氨基酸(1-18),胞外区包含287个氨基酸(19-305),跨膜区包含20个氨基酸(306-325),胞内区包含53个氨基酸(326-378)。过去20年的研究表明,CD123在许多血液肿瘤中过量表达,包括急性髓系白血病(AML),急性B细胞型淋巴细胞白血病(B-ALLs),多毛细胞白血病(Hairy Cell Leukemia),母细胞性浆细胞树突细胞瘤(BPDCN)。 L等人检测64例急性白血病病例,发现其中的45例AML病人中有43例为CD123阳性,2例M7的AML病人为CD123阴性,其中的13例B细胞急性白血病均为CD123阳性,而其中的6例T细胞急性白血病均为CD123阴性(Haematologica.2001,Dec;86(12):1261-9)。计雪强等人报道健康儿童骨髓淋巴细胞CD123为阴性表达,91例儿童B-ALL患者中65例骨髓淋巴细胞CD123阳性表达(阳性率71.43%),且表达水平与白血病细胞成熟度呈负相关(江苏大学学报:医学版,2012)。A Ehninger等人也报道79%(232/298)的AML病人细胞表达CD123分子。这些报道均显示CD123是AML的一个良好的血液病治疗靶标(BloodCancer Journal,2014)。
人白介素3(IL-3)是CD123的天然配体。人IL-3基因位于人的五号染色体(5q23-31),分子量约15-17kDa。IL-3分子在进化上并不保守,人的IL-3分子和小鼠的IL-3分子仅有29%的氨基酸同源性。IL-3与其受体CD123(IL-3R)具有极高的亲和力(Kd=0.1~1nM)。
DT388IL-3是一种IL-3分子和白喉类毒素(diphtheria toxin)的融合蛋白。在体外研究中,DT388IL-3显示出与CD123具有良好的亲和力(Kd=3nM)和良好的CD123抗原选择性细胞杀伤作用(IC50=5~10pM)(Protein Eng.2000,Aug;13(8):575-81),并且对正常细胞无显著的杀伤作用(Cancer Res.2002,Mar 15;62(6):1730-6)。在小鼠的体内实验中,DT388IL-3显著提高荷载IL-3受体阳性AML细胞小鼠的生存期,且未出现明显的副作用(Leukemia.2003Jan;17(1):155-9)。在食蟹猴的安全性评估中,DT388IL-3也显示出良好的安全性(Leuk Lymphoma.2004,Aug;45(8):1647-56)。DT388IL-3良好的安全性也揭示了利用IL-3和CD123之间的配体和受体相互作用进行疾病治疗的安全性。
嵌合抗原受体T细胞免疫疗法(Chimeric Antigen Receptor Modified T celltherapy,CAR-T)近年来在血液性疾病治疗临床试验取得了令人鼓舞的进展。从2013年Carl.H June小组报道第一例使用靶向CD19的CAR-T治疗患有急性B淋巴细胞性白血病的儿童(Emily Whitehead)得到完全缓解开始,短短几年时间CAR-T细胞免疫疗法进入了飞速发展的阶段。以诺华/宾夕法尼亚大学为代表开发的以CD19为靶点的CTL019在复发性/难治性急性淋巴细胞白血病(r/r ALL)儿童及年轻成人患者中完全缓解率达到90%以上。截至到2016年8月,美国临床试验注册网站(https://clinicaltrials.gov/)检索信息显示,全世界有142项CAR-T注册临床试验,其中美国注册71项,而中国也注册了51项。目前,CAR-T治疗在如急性髓系白血病(AML),多发性骨髓瘤(MM),神经胶质瘤,结肠癌,***癌等疾病中也正得到全面的研究与产品开发。
嵌合抗原受体(CAR)是由一个肿瘤相关抗原结合区或抗原结合结构域、胞外铰链区、跨膜区以及胞内信号转导区等结构组成。一般CAR的抗原结合结构域由不同形式的抗体组成。目前绝大多数开发的嵌合抗原受体T细胞免疫疗法,其抗原识别序列均为基于scFv抗体片段的形式。例如,美国宾夕法尼亚大学Abramson Cancer Center以及美国City ofHope相关研究组使用的抗人CD123鼠源抗体为克隆32716(clone32716)、克隆26292(clone26292)。其中克隆32716显示出比克隆26292更好的体外和体内杀伤CD123阳性细胞抗肿瘤活性,可在两周内清除小鼠模型中的CD123阳性肿瘤细胞(J Immunother.2007,Sep;30(6):607-13;US20140271582A1;Blood.2013;122(18):3138-3148)。法国CELECTIS公司也开发了CD123 CAR-T载体,其中所使用的CD123抗体为Klon43(WO2015193406A1)。在美国注册临床试验网站注册的针对AML的CAR-T治疗临床试验主要是City of Hope的NCT02159495以及Abramson Cancer Center of the University ofPennsylvania的NCT02623582。这些基于scFv抗体设计的嵌合抗原受体T细胞及其免疫疗法均存在因选用抗体本身可能具有交叉反应而导致嵌合抗原受体技术常有的脱靶效应的可能,因此都需要在临床试验中谨慎验证其安全性。由于以上CD123 CAR-T均使用鼠源单克隆抗体作为CD123的靶向分子,因此在应用到人体中时还可能具有潜在的免疫原性。
发明内容
本发明利用配体和受体间的相互作用原理,提供了一种靶向人CD123的嵌合受体配体(Chimeric Receptor Ligand,CRL),不同于传统基于抗体scFv片段的嵌合抗原受体的构造,是一种全新的、非抗体依赖性的、靶向人CD123的类似嵌合抗原受体的结构。本发明提供的靶向人CD123的嵌合受体配体(CD123 CRL)可以包含基于自然IL-3分子的CD123特异结合结构域、铰链区或无铰链区、跨膜区、以及胞内信号传导结构域等。本发明进一步提供了一种由嵌合受体配体修饰的免疫细胞,即CD123嵌合受体配体修饰的T细胞(ChimericReceptor Ligand Modified T cell,CRL-T)。本发明提供的靶向人CD123的嵌合受体配体及其免疫细胞具有比传统的基于scFv抗体的CD123 CAR-T更好的CD123靶点特异性肿瘤杀伤效果及安全性;使用基于天然的人IL-3分子靶向CD123分子进行免疫疗法,不会产生异源免疫原性;在治疗CD123阳性的血液疾病和其他疾病有极高的临床应用价值。
本发明的一个目的是提供一种靶向人CD123的嵌合受体配体。
本发明的另一目的是提供一种基因工程改造的免疫效应细胞。
本发明的另一目的是提供靶向人CD123的嵌合受体配体及其免疫效应细胞的应用。
本发明的又一目的是提供一种制备靶向人CD123嵌合受体配体及其免疫效应细胞的方法。
本发明的目的可通过以下技术方案实现:
一种靶向人CD123的嵌合受体配体,包含基于IL-3分子的CD123特异结合结构域、跨膜区及胞内信号结构域。
在本发明的实施方案中,所述靶向人CD123的嵌合受体配体,其CD123特异结合结构域包含SEQ ID NO:1或SEQ ID NO:2所示的特异性结合CD123的氨基酸序列,或与其具有85%~99%或90%~99%或95%~99%同一性的氨基酸序列。
在本发明的实施方案中,所述靶向人CD123的嵌合受体配体,其胞内信号结构域包括信号传导结构域,和/或共刺激信号结构域。
所述的胞内信号结构域,其信号传导结构域可选自CD3zeta,CD3gamma,CD3delta,CD3epsilon,common FcR gamma(FCER1G),FcR beta(Fc Epsilon R1b),CD79a,CD79b,Fc gamma RIIa,DAP10,and DAP12分子,优选CD3zeta分子。
所述的胞内信号结构域,其共刺激信号结构域可选自以下信号分子的细胞内结构域:CD27、CD28、4-1BB、OX40、CD30、CD40、CD2、淋巴细胞功能相关抗原-1(LFA-1)、LIGHT、NKG2C、B7-H3、PD-1、ICOS、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、CD7、NKp80(KLRF1)、CD160、CD19、CD4、CD8alpha、CD8凸eta、IL2R凸eta、IL2R gamma、IL7R alpha、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244,2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A,Ly108)、SLAM(SLAMFI,CD150,IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、CD83特异性结合的配体或其任意组合。
所述胞内信号结构域进一步优选以下分子的细胞内信号结构域:CD3zeta、4-1BB和/或CD28。
所述靶向人CD123的嵌合受体配体,其跨膜结构域可选自TCRα、TCRβ、TCRγ、CD3ζ、CD3ε、CD5、CD9、CD16、CD22、CD27、CD28、CD33、CD37、CD45、CD64、CD80、CD86、CD134、CD154、CD152、CD4、CD8α、CD28、PD1和4-1BB跨膜区,优选CD8α、CD28和/或4-1BB跨膜区。
在本发明的实施方案中,所述靶向人CD123的嵌合受体配体包含蛋白质分泌相关信号肽序列,可选自CD8α信号肽,GM-CSFRα信号肽,CD4信号肽或IL-3信号肽,优选IL-3信号肽。
所述靶向人CD123的嵌合受体配体,可包含铰链区结构,或不包含铰链区结构,铰链区可选自CD8α、CD28分子。
所述靶向人CD123的嵌合受体配体,其胞外区和跨膜区之间可以包含Linker结构,或不包含Linker结构。
本发明所述的靶向人CD123的嵌合受体配体,可包含两个或两个以上重复CD123嵌合受体配体串联,或是多个基于IL-3分子的CD123结合结构域相互串联,或基于IL-3分子的CD123结合结构域与基于抗体的抗原结合结构域串联形成的多价的靶点特异性的嵌合受体配体组合,重复单元间可由一个或多个Linker结构连接。
在本发明的实施方案中,所述靶向人CD123的嵌合受体配体,其特征在于编码的氨基酸序列为SEQ ID NO:14~SEQ ID NO:19所示,或与其具有85%~99%或90%~99%或95%~99%同一性的改造氨基酸序列。
一种核酸分子,其特征在于编码以上所述靶向人CD123的嵌合受体配体的核苷酸序列。
在本发明的实施方案中,所述的核酸分子,其特征在于核苷酸编码序列如SEQ IDNO:33~SEQ ID NO:38所示。
一种载体,其特征在于包含以上编码所述靶向人CD123的嵌合受体配体的核酸序列。
一种基因工程改造的免疫效应细胞,包含编码本发明所述的靶向人CD123的嵌合受体配体的基因序列。
所述的免疫效应细胞优选T淋巴细胞、NK细胞、造血干细胞、多能干细胞或胚胎干细胞培养分化的免疫细胞,进一步优选T淋巴细胞。
所述的基因工程改造的免疫细胞,其特征在于表达的靶向人CD123的嵌合受体配体包含基于IL-3分子的CD123特异结合结构域、跨膜区及胞内信号结构域。
在本发明的实施方案中,所述的基因工程改造的免疫细胞,其特征在于表达的靶向人CD123的嵌合受体配体具有SEQ ID NO:14所示氨基酸序列,或与其具有85%~99%或90%~99%或95%~99%同一性的氨基酸序列。
在本发明的实施方案中,所述的基因工程改造的免疫细胞,其特征在于表达的靶向人CD123的嵌合受体配体具有SEQ ID NO:15或SEQ ID NO:16或SEQ IDNO:17或SEQ IDNO:18或SEQ ID NO:19所示氨基酸序列,或与其具有85%~99%或90%~99%或95%~99%同一性的氨基酸序列。
本发明提供一种方法,其特征在于制备上述靶向人CD123的嵌合受体配体及其修饰的免疫效应细胞。
本发明涉及所述的靶向人CD123的嵌合受体配体及其免疫效应细胞在制备抗肿瘤免疫治疗药物中的应用,优选在制备治疗急性髓细胞白血病药物中的应用。
有益效果:
本发明提供了一种靶向人CD123的嵌合受体配体(CD123 CRL),创造性地使用人IL-3分子作为胞外特异性靶向CD123分子的结合区,通过配体/受体特异性地识别肿瘤相关表面抗原CD123,然后通过胞内共刺激信号分子将识别的信号传递到细胞内,激活免疫细胞的杀伤性,进而减少和消除肿瘤细胞。本发明同时提供了一种靶向人CD123的嵌合受体配体修饰的T细胞(CD123 CRL-T),能够特异地与肿瘤细胞表面的CD123结合,进而对肿瘤细胞产生特异的杀伤作用。与传统基于抗体如scFv片段的CD123 CAR-T相比,CD123 CRL-T显示出更好的CD123靶点特异性的肿瘤杀伤效果以及细胞因子释放量,提高了靶向治疗的特异性及安全性。
附图说明
图1为本发明构建的靶向人CD123的嵌合受体配体结构示意图。图1a为本发明提供的基于IL-3的CD123嵌合受体配体结构示意图,其中信号肽部分结构可选自IL-3、CD8α分子。共刺激信号结构域以4-1BB和/或CD28信号分子为例。图1b为传统基于scFv抗体的CD123CAR-T结构图。
图2为本发明构建的靶向人CD123嵌合受体配体载体示意图。以CRL1为例展示本发明构建的CD123 CRL载体结构图,所示CRL1包含IL-3胞外信号肽及CD123结合结构域,CD8α铰链区(Hinge)及跨膜区(TM),CD137胞内共刺激结构域(Cyto)及CD3z信号传导结构域。
图3为K562.CD123.Luc细胞系的验证。构建的clone 8细胞系的CD123分子表达水平最高,Luciferase表达水平也较高,clone 8被命名为K562.CD123.Luc。
图4为靶向CD123嵌合受体配体体外活性功能研究。如图4a所示,纵坐标显示的是共培养结束后,反应孔中所剩余的荧光素酶相对活性RLU,对应孔中活细胞的相对量。CAR1-T、CAR2-T、CAR3-T及CRL1-T细胞对RPMI8226.Luc细胞均有较好的体外杀伤活性。图4b显示CAR1-T、CAR2-T、CAR3-T及CRL1-T细胞均能在RPMI8226.Luc细胞存在的情况下释放细胞因子IFNγ。而在没有RPMI8226.Luc靶细胞存在的情况下,CRL1-T显示出比CAR1-T、CAR2-T及CAR3-T更低的细胞因子IFNγ释放量。
图5为基于IL-3分子的CD123 CRL-T的细胞因子IFN-γ释放水平检测试验。图5a显示在过表达及低表达CD123分子的K562.CD123.Luc存在的情况下,CRL1-T细胞释放的IFN-γ水平;图5b为在低表达CD123分子的RPMI8226.Luc细胞刺激下,CRL1-T细胞释放出的IFN-γ水平;图5c为在CD123阴性的A549.Luc细胞刺激的情况下,CRL1-T细胞释放的IFN-γ水平。
图6为基于IL-3分子的CD123 CRL-T的细胞因子IL-2释放水平检测试验。图6a显示在过表达及低表达CD123分子的K562.CD123.Luc存在的情况下,CRL1-T细胞释放的IL-2水平;图6b为在低表达CD123分子的RPMI8226.Luc细胞刺激下,CRL1-T细胞释放出的IL-2水平;图6c为在CD123阴性的A549.Luc细胞刺激的情况下,CRL1-T细胞释放的IL-2水平。
图7为基于IL-3分子的CD123 CRL-T功能性和特异性评估。图7a显示与阴性对照UnT细胞相比,CRL1-T对过表达CD123的K562.CD123.Luc细胞出良好的体外杀伤作用;图7b显示CRL1-T对CD123阴性的K562.CD19.Luc细胞则几乎没有体外杀伤作用;图7c显示CRL1-T细胞对低表达CD123分子的RPMI8226.Luc细胞系具有良好的体外杀伤作用;图7d显示CRL1-T细胞对不表达CD123分子的肺癌细胞系A549.Luc则完全没有体外杀伤活性。
具体实施方式
本发明提供了一种靶向人CD123的嵌合受体配体,包含:基于IL-3分子的CD123结合结构域、跨膜区及胞内信号结构域。
本文所用的术语“基于IL-3分子的CD123结合结构域”是指来源于天然人IL-3氨基酸序列的CD123结合结构域。
天然人IL-3的氨基酸序列如SEQ ID NO:40所示,共含有152个氨基酸残基。天然人IL-3分子N端的19个氨基酸残基为IL-3信号肽,其氨基酸序列如SEQ ID NO:3所示。
如果从天然人IL-3分子中除去信号肽部分(SEQ ID NO:3),即可得到SEQ ID NO:1序列(含有133个氨基酸)。SEQ ID NO:1可以用作IL-3受体结合域(也可称为CD123结合结构域),具有特异结合CD123的能力。
如果将SEQ ID NO:1的C端9个氨基酸去除,即可获得SEQ ID NO:2(含有124个氨基酸)。只包含SEQ ID NO:2序列的IL-3片段也能结合CD123,可以用作IL-3受体结合域。
本文所述的“基于IL-3分子的CD123结合结构域”具有特异结合CD123的能力。例如,在本发明的实施方案中,基于IL-3分子的CD123结合结构域可以包含SEQ ID NO:1的至少100个连续氨基酸残基,例如102、105、110、115或120个连续氨基酸残基。
在本发明的优选实施方案中,基于IL-3分子的CD123结合结构域可以为SEQ IDNO:1或SEQ IDNO:2。
本发明的靶向人CD123的嵌合受体配体中,可以包含一个基于IL-3分子的CD123结合结构域,也可以包含两个或更多个基于IL-3分子的CD123结合结构域。这些CD123结合结构域可以彼此相同或不同。例如,可以包含两个或两个以上重复CD123嵌合受体配体串联,或是多个基于IL-3分子的CD123结合结构域相互串联,或基于IL-3分子的CD123结合结构域与基于抗体的抗原结合结构域串联形成的多价的靶点特异性的嵌合受体配体组合,重复单元间可由一个或多个Linker结构连接。
此处所用的术语“Linker”也称连接肽或接头,是用于连接两个结构域的柔性氨基酸序列。连接肽的选择和制备对本领域技术人员来说是容易的。
本文所用的术语“跨膜区”(简称TM)可以与“跨膜结构域”互换使用,指的是锚定在细胞膜内具有热力学稳定的蛋白质结构区域。跨膜区可以从天然蛋白质中获得,可选自CD8α分子或CD28分子。例如,SEQ IDNO:6或7所示氨基酸序列。
本文所用的术语“胞内信号结构域”指的是能够传导细胞效应功能信号并指导细胞执行特定功能的蛋白质结构区域。胞内信号结构域可以包括信号传导结构域和/或共刺激信号结构域。
本文所用的术语氨基酸序列的“同一性”(identity)可以与“相同性”互换使用,指的是氨基酸序列之间通过序列比对软件例如BLAST确定的相似程度。氨基酸序列比对的方法和软件对于本领域技术人员是公知的。
可以通过对已知氨基酸序列进行一个或几个(例如1-15个,例如2、3、5、8、10或12个)氨基酸残基的取代、缺失和/或添加而获得经改造的氨基酸序列。
例如,通过常规蛋白质工程手段(例如氨基酸保守取代等),对本发明SEQ ID NO:1所示的CD123结合结构域进行改造,可以获得与SEQ ID NO:1具有至少85%(例如85%~99%或90%~99%或95%~99%)序列同一性,并且具有基本相同的结合CD123能力的变体序列(例如保留至少80%,例如至少85%、90%甚至95%结合能力)。
本文所用的术语“scFv”或“scFv抗体”是指单链抗体(single chain antibodyfragment,scFv),是由抗体重链可变区和轻链可变区通过15~20个氨基酸的短肽连接而成。
优选地,本发明的靶向人CD123的嵌合受体配体还可以包含胞外信号肽结构。例如,IL-3信号肽或CD8α信号肽。
本发明通过对人IL-3自然分子与共刺激因子等嵌合受体配体结构结合进行设计,下面结合具体实施例,进一步阐述本发明。
实施例1构建表达CD123的K562细胞系K562.CD123.Luc
K562细胞几乎不表达CD123。本发明首先基因合成编码人CD123分子的核苷酸序列,由南京金斯瑞生物科技有限公司提供基因合成技术服务。合成的CD123核苷酸序列在T4连接酶的作用下,20℃过夜连接到预先进行BamH1和XbaI限制酶切位点酶切后的pLVX-Puro(Clontech,货号#632164)慢病毒载体上。将连接产物转化到DH5α感受态细胞中并涂布细菌平板,挑取多个克隆斑进行质粒的提取(Qiagen Endofree Mega kit)。经酶切鉴定、测序比对,构建成功的载体命名为pLVX-CD123-Puro。
将提取的pLVX-CD123-Puro表达质粒和pCMV-ΔR-8.74及pMD2.G辅助质粒,按一定比例混合,共转染293FT细胞。转染96h后,收集含有病毒的细胞培养上清,4℃、3000rpm离心5min。上清经0.45μm滤器过滤后,经4℃、25000rpm高速离心120min。弃上清重悬溶解后即获得病毒浓缩液,-80℃保存备用。携带萤火虫荧光素酶(Firefly luciferase/Luciferase)报告基因的慢病毒载体也采用同样的方法进行制备。
K562细胞系购自ATCC(货号CCL-243),使用90%IMDM(Life technology,货号A10491-01)+10%FBS(Life technology,货号10099-141)+1%青霉素/链霉素(Lifetechnology,货号15140-122)进行常规培养。将构建的携带CD123基因的慢病毒载体与携带萤火虫荧光素酶(Firefly luciferase/Luciferase)报告基因的慢病毒载体加入到培养的K562细胞上清中,共同转导K562细胞系。转导后24h,加入终浓度为10μg/ml的Puromycin,每三天换一次培养液并添加相同浓度的Puromycin,进一步进行单克隆的筛选获得一些单克隆细胞。
利用流式细胞术对上述获得的单克隆细胞进行CD123的表达鉴定。设置未转导CD123基因的K562细胞作为阴性对照。分别吸取5x105细胞数的细胞悬液至15ml离心管中,室温离心300g、10min后弃上清,用DPBS重悬洗一次后重悬到200μl DPBS中,再加入2μlanti-CD123-FITC抗体(Miltenyi,货号130-098-886),室温孵育45min。加入1ml DPBS,室温离心300g、10min后弃上清。再次加入1ml DPBS,室温离心300g、10min后弃上清,最后加入200μl的DPBS重悬细胞,并在FACScalibur流式细胞仪(BD公司)上检测FITC的信号。荧光素酶的检测,采用One-Glo Luciferase assay试剂盒(Promega,货号E6110):把每个克隆的细胞密度调整到2000个细胞/20μl,然后加入到384-孔微孔检测板中,然后每孔加入20μlOne-Glo Luciferase assay检测试剂,室温离心300g、1min后静置10min,然后在酶标仪PHERAStar(购自BMG公司)上检测化学发光信号。
通过上述过程,获得9个高表达CD123分子和Luciferase的稳定细胞系。根据表1所示,所构建的细胞系表达CD123分子的阳性率和Luciferase相对酶活性(RLU,relativelight unit,相对发光值)检测结果表明,所构建的不同克隆号的细胞系分别表达不同水平的CD123分子,以及不同水平的Luciferase。克隆8的CD123分子表达水平最高,92.9%的细胞为CD123阳性细胞,而未转导的K562细胞CD123阳性率仅为3.31%。同时,克隆8的Luciferase表达水平也较高,其信号值是未转导的阴性K562细胞对照的659倍(相对酶活性761623/1156)。综合起来克隆8是一个比较好的细胞系,clone 8被命名为K562.CD123.Luc,作为后续的实验工具细胞。
表1构建表达CD123的K562细胞系K562.CD123.Luc
实施例2基于人IL-3分子的靶向人CD123嵌合受体配体的构建
本发明通过将人IL-3分子的核苷酸序列进行密码子优化,以利于在人细胞中优化表达。密码子优化利用南京金斯瑞生物科技有限公司OptimumGeneTM密码子优化技术完成。密码子优化后的IL-3核苷酸序列如SEQ ID NO:39所示,其编码的氨基酸序列如SEQ ID NO:40所示,由该基因表达的IL-3分子包括IL-3信号肽及IL-3受体结合域。
本发明按以下编码基因的顺序设计CD123 CRL融合基因片段:胞外信号肽,基于IL-3的CD123结合结构域,铰链区(Hinge),跨膜区(TM),胞内共刺激信号结构域及胞内信号结构域。融合基因合成由南京金斯瑞生物科技有限公司提供技术服务。本发明所提供的基于IL-3的靶向人CD123的嵌合受体配体的基本结构如表2所示,包括CRL1~CRL6所示嵌合受体配体结构,其对应的嵌合受体配体氨基酸序列分别为SEQ ID NO:14~SEQ ID NO:19所示。
本发明同时设计传统CAR作为实验对照,分别以CD8α信号肽、anti-CD123scFv、CD8α铰链区(Hinge)、CD8α跨膜区(TM)、4-1BB胞内共刺激信号结构域及CD3ζ胞内信号结构域顺序通过基因合成技术直接合成该融合基因,如表2所示CAR1、CAR2及CAR3结构。
本发明设计的CD123 CRL主要结构成分及序列如下:
CD123结合结构域:CD123结合结构域包含SEQ ID NO:1所示氨基酸序列中至少100个连续氨基酸序列。在本发明的实施例中,基于IL-3分子的CD123结合结构域包含如SEQ IDNO:1或SEQ ID NO:2所示氨基酸序列。其核苷酸编码序列如SEQ ID NO:20或SEQ ID NO:21所示。
胞外信号肽:可选自IL-3信号肽或CD8α信号肽。IL-3信号肽,其氨基酸序列如SEQID NO:3所示,其核苷酸编码序列如SEQ ID NO:22所示;CD8α信号肽,其氨基酸序列如SEQID NO:4所示,其核苷酸编码序列如SEQ ID NO:23所示。
铰链区:可选自多种分子的铰链区,CRL1选用CD8α铰链区,其氨基酸序列如SEQ IDNO:5所示,其核苷酸编码序列如SEQ ID NO:24所示。
跨膜区:可选自CD8α跨膜区或CD28跨膜区。CD8α跨膜区,其氨基酸序列如SEQ IDNO:6所示,其核苷酸编码序列如SEQ ID NO:25所示;CD28跨膜区,其氨基酸序列如SEQ IDNO:7所示,其核苷酸编码序列如SEQ ID NO:26所示。
胞内信号传导结构域:CD3zeta,其氨基酸序列如SEQ ID NO:8所示,其核苷酸编码序列如SEQ ID NO:27所示。
胞内共刺激信号结构域:选自4-1BB信号分子或CD28信号分子。4-1BB信号分子,其氨基酸序列如SEQ ID NO:9所示,其核苷酸编码序列如SEQ ID NO:28所示。CD28信号分子,其氨基酸序列如SEQ ID NO:10所示,其核苷酸编码序列如SEQ ID NO:29所示。
表2本发明涉及的主要CRL及CAR组合结构说明
实施例3 CD123特异性嵌合受体配体修饰的T细胞的制备
(一)CD123嵌合受体配体慢病毒表达载体的构建
本发明将购自Clontech公司的pLVX-Puro载体经ClaI和EcoRI限制酶的酶切反应,将CMV启动子敲除,并将人EF1α启动子(GenBank:J04617.1)克隆到经酶切后的载体上,从而获得pLVX-hEF1α载体。将基因合成的CD123 CRL融合基因序列克隆到表达质粒pLVX-hEF1α上形成重组CRL表达质粒。提取重组CRL表达质粒(pLLV-CRL),并与pCMV-AR-8.74及pMD2.G辅助质粒按一定比例混合,共转染293FT细胞。转染96h后,收集含有病毒的细胞培养上清,4℃、3000rpm离心5min。上清经0.45μm滤器过滤后,经4℃、25000rpm高速离心120min。弃上清重悬溶解后即获得病毒浓缩液,-80℃保存备用。
通过以上方法获得的重组CRL慢病毒表达载体分别被命名为pLLV-CRL1、pLLV-CRL2、pLLV-CRL3……pLLV-CRL6。
作为实验的对照,本发明合成了上述clone 32716、clone 26292和Klon43的scFv抗体序列,并克隆到慢病毒表达载体上,分别命名为pLLV-CAR1、pLLV-CAR2和pLLV-CAR3。
构建上述载体使用的是常规的分子生物学技术即酶切、连接、转化、以及克隆鉴定技术,是本行业从业人员能容易掌握和操作的。
(二)T淋巴细胞的制备
取50mL志愿者新鲜外周血,通过淋巴细胞分离液、密度梯度离心方法分离外周血单核细胞(PBMC)。利用Pan T Cell Isolation Kit(Miltenyi Biotech)对细胞进行磁珠标记,并分离纯化出T淋巴细胞。纯化后的T细胞,再利用CD3/CD28磁珠进行T淋巴细胞激活及增殖。
(三)慢病毒转导T淋巴细胞
收集激活的T淋巴细胞,重悬在RPMI1640培养基中。用慢病毒感染1x106个活化的T淋巴细胞,将细胞悬液加在6孔板中,置37℃、5%CO2培养箱中孵育过夜。第二天,再次离心并换新鲜培养基,每隔2天加入新鲜培养基,继续扩大培养。
(四)荧光定量实时qPCR法检测CRL的转导效率
离心收集制备的CRL-T及CAR-T细胞,分别用DPBS洗3次后利用人基因组提取试剂盒Gentra Puregene Cell Kit(购自Qiagen)制备基因组DNA。制备的DNA利用NanoDrop2000(Thermo Scientific)检测OD260nm和OD280nm吸光度并计算浓度。根据试剂盒SYBR GreenRealtime PCR Master mix plus(购自Toyobo)说明书配置50μl反应体系,然后在荧光定量PCR仪(ABI#7300)上进行基因拷贝数的检测。qPCR检测采用精确定量的包含目的片段的质粒作为阳性对照,并绘制标准曲线。根据qPCR各个不同拷贝数浓度的CT值与对应的拷贝数绘制直线并拟合标准曲线,其他检测样品根据标准曲线的拟合方程计算相对的拷贝数。
本发明中CRL-T细胞表达嵌合受体配体的检测,以CAR-T细胞表达CAR作阳性对照,以未转导的T淋巴细胞(UnT)作为空白对照。
CRL及CAR整合拷贝数检测结果如表3显示。结果显示:CRL1-T细胞基因组中检测到CRL1基因的整合,且其拷贝数(2.34x105拷贝/ng基因组DNA)与CAR-T细胞中转导的CAR基因拷贝数相当(3.1x104到2.77x105拷贝/ng基因组DNA),空白对照UnT检测值极低(16个拷贝/ng基因组DNA),属于检测的背景。
表3 qPCR法检测CRL的细胞转导效率
实施例4靶向CD123分子的嵌合受体配体体外活性功能研究
RPMI8226细胞表达一定水平的CD123受体(http://www.proteinatlas.org/ENSG0000018 5291-IL-3RA/tissue),且有报道靶向CD123分子的CAR-T对RPMI8226细胞有较好的体外杀伤作用(WO2015193406A1)。本发明构建了稳定表达Luciferase的RPMI8226细胞系(RPMI8226.Luc),构建方法可参照实施例1。本发明通过使用RPMI8226.Luc细胞作为模式细胞对CRL-T细胞进行体外功能的研究,并以CAR1、CAR2、CAR3修饰的T细胞作为对照,分别命名为CAR1-T、CAR2-T及CAR3-T。
分别将CRL-T细胞、CAR-T细胞、及UnT细胞与靶细胞按20∶1比例在37℃,5%CO2条件下共培养过夜。共培养结束后,离心后加入ONE-GloTM Luciferase Assay试剂,并使用PHERAStar Plus检测RLU读值,评估体外杀伤效果。如图4a所示,纵坐标显示的是共培养结束后,反应孔中所剩余的荧光素酶相对活性(RLU,relative light unit)对应孔中活细胞的相对量,如果显示的荧光素酶RLU值高,则表示反应孔中剩余的未被杀死的靶细胞多,表明孔中的细胞杀伤作用弱;反之,如果显示的荧光素酶RLU值低,则表示反应孔中的未被杀死的靶细胞少,表明孔中的细胞杀伤作用强。
如图4a所示,CAR1-T、CAR2-T、CAR3-T及CRL1-T细胞对RPMI8226.Luc细胞均有较好的体外杀伤活性,其中CAR1-T处理组剩余活细胞的相对Luciferase信号值为1413±152,CAR2-T处理组剩余活细胞的相对Luciferase信号值为3883±423RLU,CAR3-T处理组剩余活细胞的相对Luciferase信号值为3184±262,CRL1-T处理组剩余活细胞的相对Luciferase信号值为4531±276,相对于UnT处理组剩余活细胞的相对Luciferase信号值为9442±1553,显示出良好的杀伤活性。
抗原特异性刺激下细胞因子(γ-干扰素/IFN-γ和白介素2/IL-2)的释放是评估CAR-T体外活性和应用安全性的指标。良好的CAR-T载体可在靶标抗原存在的情况下释放上述细胞因子,而在靶标抗原缺失的情况下释放水平较低。图4b检测结果显示CAR1-T、CAR2-T、CAR3-T及CRL1-T细胞均能在RPMI8226.Luc细胞存在的情况下释放细胞因子IFNγ。其中,尽管CAR1-T显示较CAR2-T、CAR3-T及CRL1-T较好的体外杀伤作用(图4a),但是CAR1-T在没有RPMI8226.Luc靶细胞存在的情况下能释放出明显高于CAR2-T、CAR3-T及CRL1-T释放的细胞因子IFNγ,具有较高的非特异性。在没有RPMI8226.Luc靶细胞存在的情况下,CRL1-T显示出比CAR1-T、CAR2-T及CAR3-T更低的细胞因子IFNγ释放量,同时CRL1-T对RPMI8226.Luc的杀伤效果与CAR2-T、CAR3-T相当,显示出较好的体外有效性和安全性。
实施例5基于IL-3分子的CD123 CRL-T细胞对肿瘤的细胞因子的释放
本发明优选CRL1载体进行T细胞修饰,并通过对CD123阳性的肿瘤细胞进行细胞因子IFN-γ和IL-2的刺激试验。本发明将CRL-T细胞与不同的靶细胞按比例在37℃、5%CO2条件下共培养过夜。共培养结束后,将实验微孔板离心200g、5min,然后小心取出部分上清,使用实时荧光分辨技术试剂盒(HTRF,Cisbio#64IL2PEB)检测上清中的IFN-γ和IL-2的分泌水平。
如图5a所示,在过表达CD123分子的K562.CD123.Luc存在的情况下,CRL1-T细胞释放出高水平的IFN-γ(4383±236.1pg/ml),而阴性T细胞(UnT)释放水平则很低(108.6±23.97pg/ml),接近基线(29.78±37.52pg/ml);在K562.CD123.Luc缺失的情况下,CRL1-T细胞释放出极低水平的本底IFN-γ(109.3±10.41pg/ml),接近基线(29.78±37.52pg/ml)。而在低表达CD123分子的RPMI8226.Luc细胞刺激下,CRL1-T细胞也能比UnT细胞释放出更高水平的IFN-γ(697.4±0.00pg/ml,261.6±3.1pg/ml)(图5b)。在CD123阴性的A549.Luc细胞刺激的情况下,CRL1-T细胞和UnT细胞对照类似,释放出低水平的IFN-γ(315±0.00pg/ml,58.54±11.53pg/ml)(图5c)。
如图6a所示,在过表达CD123分子的K562.CD123.Luc存在的情况下,CRL1-T细胞释放出高水平的IL-2(2.41±0.06IU/ml),而UnT细胞释放水平则很低(0.60±0.00IU/ml),接近基线(0.59±0.00IU/ml);在缺少K562.CD123.Luc细胞刺激的情况下,CRL1-T细胞释放出极低水平的IL-2(0.59±0.00IU/ml),与基线(0.59±0.00IU/ml)一样。而在低表达CD123分子的RPMI8226.Luc细胞刺激下,CRL1-T细胞释放出的IL-2水平也较低(0.61±0.00IU/ml)(图6b)。在CD123阴性的A549.Luc细胞刺激的情况下,CRL1-T细胞和UnT细胞阴性对照类似,释放出极低水平的IL-2(0.60±0.00IU/ml,0.60±0.00IU/ml)(图6c)。
实施例6基于IL-3分子的CD123 CRL-T细胞功能性和特异性评估
分别将CRL-T细胞、CAR-T细胞、及UnT细胞与靶细胞按20∶1比例在37℃,5%CO2条件下共培养过夜。共培养结束后,离心后加入ONE-GloTM Luciferase Assay试剂,并使用PHERAStar Plus检测RLU读值,评估体外杀伤效果。如图7所示,同实施例4,纵坐标显示的是共培养结束后,反应孔中所剩余的荧光素酶相对活性RLU对应孔中活细胞的相对量。如果显示的荧光素酶RLU值高,则表示反应孔中剩余的未被杀死的靶细胞多,进而表明孔中的细胞杀伤作用弱;反之,如果显示的荧光素酶RLU值低,则表示反应孔中的未被杀死的靶细胞少,进而表明孔中的细胞杀伤作用强。
如图7所示,CRL1-T对过表达CD123的K562.CD123.Luc细胞显示出良好的体外杀伤作用,97.5%的K562.CD123.Luc细胞被CRL1-T杀伤,而不被阴性对照UnT细胞杀伤:CRL1杀伤组剩余细胞Luciferase相对发光值为7307±3639,而阴性对照UnT细胞杀伤组剩余细胞Luciferase相对发光值为292420±19102(图7a)。而CRL1-T对CD123阴性的K562.CD19.Luc细胞则几乎没有体外杀伤作用:CRL1杀伤组剩余细胞Luciferase相对发光值为411629±14399,而阴性对照UnT细胞杀伤组剩余细胞Luciferase相对发光值为458030±11222(图7b)。图7a和图7b显示出CRLL1-T的细胞杀伤具有严格的靶点特异性。
本发明进一步在低表达CD123分子的多发性骨髓瘤细胞系RPMI8226.Luc细胞和不表达CD123分子的肺癌细胞系A549.Luc进行体外的杀伤实验。如图7c所示,同实施例4,CRL1-T细胞对RPMI8226.Luc细胞系具有良好的体外杀伤作用:CRL1杀伤组剩余细胞Luciferase相对发光值为2467±239.6,而阴性对照UnT细胞杀伤组剩余细胞Luciferase相对发光值为13248±331.3;而对不表达CD123分子的肺癌细胞系A549.Luc则完全没有体外杀伤活性:CRL1杀伤组剩余细胞Luciferase相对发光值为577341±17365,而阴性对照UnT细胞杀伤组剩余细胞Luciferase相对发光值为522895±14198(图7d)。图7c和图7d进一步显示出CRL1-T细胞的杀伤具有严格的靶点特异性及安全性。
本申请文件中未详细记载的实验方法均为本领域的常规技术,可通过申请日以前的文献或技术手段实现。
序列
IL-3受体结合域氨基酸序列:SEQ ID NO:1 1-133aa
APMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVK
SLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF
IL-3受体结合域氨基酸序列:SEQ ID NO:2 1-124aa
APMTQTTPLKTSWVNCSNMIDEI ITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVK
SLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQ
IL-3信号肽氨基酸序列:SEQ ID NO:3
MSRLPVLLLLQLLVRPGLQ
CD8α信号肽氨基酸序列:SEQ ID NO:4
MALPVTALLLPLALLLHAARP
CD8α铰链区氨基酸序列:SEQ ID NO:5
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
CD8α跨膜区氨基酸序列:SEQ ID NO:6
IYIWAPLAGTCGVLLLSLVITLYC
CD28跨膜区氨基酸序列:SEQ ID NO:7
FWVLVVVGGVLACYSLLVTVAFIIFWV
CD3z氨基酸序列:SEQ ID NO:8
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CD137(4-1BB)胞内区氨基酸序列:SEQ ID NO:9
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD28胞内区氨基酸序列:SEQ ID NO:10
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CAR1氨基酸序列:SEQ ID NO:11
MALPVTALLLPLALLLHAARPDIVLTQSPASLAVSLGQRATISCRASESVDNYGNTFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPPTFGAGTKLELKGGGGSGGGGSSGGGSQIQLVQSGPELKKPGETVKISCKASGYIFTNYGMNWVKQAPGKSFKWMGWINTYTGESTYSADFKGRFAFSLETSASTAYLHINDLKNEDTATYFCARSGGYDPMDYWGQGTSVTVSSTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CAR2氨基酸序列:SEQ ID NO:12
MALPVTALLLPLALLLHAARPDVQITQSPSYLAASPGETITINCRASKSISKDLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNKYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPDQGLEWIGRIDPYDSETHYNQKFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCARGNWDDYWGQGTTLTVSSTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CAR3氨基酸序列:SEQ ID NO:13
MALPVTALLLPLALLLHAARPMADYKDIVMTQSHKFMSTSVGDRVNITCKASQNVDSAVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGRGSGTDFTLTISSVQAEDLAVYYCQQYYSTPWTFGGGTKLEIKRGGGGSGGGGSGGGGSEVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYMSWVRQPPGKALEWLALIRSKADGYTTEYSASVKGRFTLSRDDSQSILYLQMNALRPEDSATYYCARDAAYYSYYSPEGAMDYWGQGTSVTVSSTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CRL1氨基酸序列:SEQ ID NO:14
MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIFTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CRL2氨基酸序列:SEQ ID NO:15
MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIFTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CRL3氨基酸序列:SEQ ID NO:16
MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIFTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CRL4氨基酸序列:SEQ ID NO:17
MALPVTALLLPLALLLHAARP
APMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIFTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CRL5氨基酸序列:SEQ ID NO:18
MALPVTALLLPLALLLHAARPAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIFTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CRL6氨基酸序列:SEQ ID NO:19
MALPVTALLLPLALLLHAARPAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIFTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
IL-3受体结合域核苷酸序列:SEQ ID NO:201-399
GCCCCAATGACACAGACAACCCCACTGAAAACCTCTTGGGTGAACTGCAGCAATATGATCGACGAGATCATCACACACCTGAAGCAGCCCCCTCTGCCCCTGCTGGATTTCAACAATCTGAACGGCGAGGACCAGGATATCCTGATGGAGAACAATCTGAGACGGCCCAACCTGGAGGCCTTTAATCGGGCCGTGAAGAGCCTGCAGAACGCCAGCGCCATCGAGTCCATCCTGAAGAATCTGCTGCCATGTCTGCCACTGGCAACCGCAGCACCTACAAGGCACCCAATCCACATCAAGGACGGCGATTGGAATGAGTTCAGGCGCAAGCTGACCTTTTACCTGAAAACACTGGAAAACGCTCAGGCACAGCAGACCACACTGTCACTGGCAATCTTC
IL-3受体结合域核苷酸序列:SEQ ID NO:211-372
GCCCCAATGACACAGACAACCCCACTGAAAACCTCTTGGGTGAACTGCAGCAATATGATCGACGAGATCATCACACACCTGAAGCAGCCCCCTCTGCCCCTGCTGGATTTCAACAATCTGAACGGCGAGGACCAGGATATCCTGATGGAGAACAATCTGAGACGGCCCAACCTGGAGGCCTTTAATCGGGCCGTGAAGAGCCTGCAGAACGCCAGCGCCATCGAGTCCATCCTGAAGAATCTGCTGCCATGTCTGCCACTGGCAACCGCAGCACCTACAAGGCACCCAATCCACATCAAGGACGGCGATTGGAATGAGTTCAGGCGCAAGCTGACCTTTTACCTGAAAACACTGGAAAACGCTCAGGCACAG
IL-3信号肽核苷酸序列:SEQ ID NO:22
ATGAGTAGACTGCCCGTGCTGCTGCTGCTGCAGCTGCTGGTGCGACCTGGACTGCAG
CD8α信号肽核苷酸序列:SEQ ID NO:23
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG
CD8α铰链区核苷酸序列:SEQ ID NO:24
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT
CD8α跨膜区核苷酸序列:SEQ ID NO:25
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC
CD28跨膜区核苷酸序列:SEQ ID NO:26
TTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGG
CD3z核苷酸序列:SEQ ID NO:27
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
CD137(4-1BB)胞内区核苷酸序列:SEQ ID NO:28
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG
CD28胞内区核苷酸序列:SEQ ID NO:29
CCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC
CAR1核苷酸序列:SEQ ID NO:30
ATGGCTCTGCCCGTGACCGCACTGCTGCTGCCCCTGGCTCTGCTGCTGCACGCCGCCCGACCTGGAAGCGACATCGTCCTGACACAGAGCCCAGCATCCCTGGCCGTGTCCCTGGGACAGCGGGCCACCATCTCTTGCAGAGCCTCTGAGAGCGTGGACAACTACGGCAATACATTCATGCACTGGTATCAGCAGAAGCCCGGCCAGCCCCCTAAGCTGCTGATCTACCGGGCCTCCAACCTGGAGTCTGGCATCCCCGCAAGGTTCTCCGGATCTGGCAGCCGCACCGACTTTACCCTGACAATCAACCCTGTGGAGGCCGACGATGTGGCCACATACTATTGCCAGCAGAGCAATGAGGATCCACCCACCTTTGGCGCCGGCACAAAGCTGGAGCTGAAGGGAGGAGGAGGATCCGGAGGAGGAGGAAGCTCCGGAGGAGGCTCTCAGATCCAGCTGGTGCAGAGCGGCCCTGAGCTGAAGAAGCCAGGCGAGACAGTGAAGATCAGCTGTAAGGCCTCCGGCTACATCTTCACAAACTATGGCATGAATTGGGTGAAGCAGGCCCCTGGCAAGTCTTTTAAGTGGATGGGCTGGATCAATACCTACACAGGCGAGTCTACCTATAGCGCCGATTTCAAGGGCCGGTTCGCCTTTAGCCTGGAGACAAGCGCCTCTACAGCCTACCTGCACATCAACGACCTGAAGAATGAGGATACCGCCACATATTTTTGTGCCAGGTCAGGGGGGTATGATCCTATGGACTATTGGGGGCAGGGGACCTCCGTGACCGTCTCAAGCACTAGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
CAR2核苷酸序列:SEQ ID NO:31
ATGGCCCTGCCCGTCACTGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCAAGACCCGATGTCCAGATTACTCAGAGCCCATCCTACCTGGCCGCCTCTCCCGGCGAGACAATCACAATCAACTGCAGGGCCTCCAAGTCTATCAGCAAGGACCTGGCCTGGTACCAGGAGAAGCCCGGCAAGACCAATAAGCTGCTGATCTATTCCGGCTCTACACTGCAGTCTGGCATCCCTAGCAGGTTCAGCGGATCCGGATCTGGAACCGACTTTACCCTGACAATCAGCTCCCTGGAGCCTGAGGATTTCGCCATGTACTATTGCCAGCAGCACAACAAGTACCCATATACCTTTGGCGGCGGCACAAAGCTGGAGATCAAGGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGGCGGCGGCTCTCAGGTGCAGCTGCAGCAGCCCGGCGCCGAGCTGGTGCGGCCTGGAGCATCCGTGAAGCTGTCTTGTAAGGCCAGCGGCTACACCTTCACATCCTATTGGATGAACTGGGTGAAGCAGCGGCCAGACCAGGGCCTGGAGTGGATCGGCAGAATCGACCCCTACGATAGCGAGACACACTATAATCAGAAGTTTAAGGACAAGGCCATCCTGACCGTGGATAAGTCTAGCTCCACAGCCTATATGCAGCTGTCTAGCCTGACAAGCGAGGATTCCGCCGTGTACTATTGTGCTCGGGGAAACTGGGATGACTATTGGGGACAGGGGACAACTCTGACCGTCTCAAGCACTAGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
CAR3核苷酸序列:SEQ ID NO:32
ATGGCTCTGCCTGTGACCGCACTGCTGCTGCCCCTGGCTCTGCTGCTGCACGCTGCCCGCCCTATGGCCGACTATAAAGACATTGTGATGACCCAGTCTCACAAGTTCATGTCTACAAGCGTGGGCGACCGGGTGAACATCACCTGCAAGGCCTCCCAGAATGTGGATTCTGCCGTGGCCTGGTACCAGCAGAAGCCAGGCCAGTCCCCCAAGGCCCTGATCTATTCCGCCTCTTACCGGTATTCTGGAGTGCCTGACAGGTTCACCGGAAGAGGAAGCGGCACAGATTTTACCCTGACAATCAGCTCCGTGCAGGCAGAGGACCTGGCAGTGTACTATTGCCAGCAGTACTATAGCACCCCATGGACATTTGGCGGCGGCACCAAGCTGGAGATCAAGAGGGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGGCGGCGGCTCTGAGGTGAAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAGCCAGGAGGCAGCCTGTCCCTGTCTTGTGCCGCCAGCGGCTTCACCTTTACAGACTACTATATGTCCTGGGTCAGGCAGCCACCTGGAAAGGCACTGGAGTGGCTGGCACTGATCAGGAGCAAGGCCGATGGCTACACCACAGAGTATAGCGCCTCCGTGAAGGGCAGGTTCACCCTGTCCCGCGACGATTCTCAGAGCATCCTGTACCTGCAGATGAACGCACTGCGGCCTGAGGACTCCGCAACATACTATTGTGCCAGAGATGCCGCCTACTATTCTTACTATTCACCAGAAGGGGCTATGGATTATTGGGGGCAGGGGACAAGCGTCACCGTCTCATCATCAACTAGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
CRL1核苷酸序列:SEQ ID NO:33
ATGAGTAGACTGCCCGTGCTGCTGCTGCTGCAGCTGCTGGTGCGACCTGGACTGCAGGCCCCAATGACACAGACAACCCCACTGAAAACCTCTTGGGTGAACTGCAGCAATATGATCGACGAGATCATCACACACCTGAAGCAGCCCCCTCTGCCCCTGCTGGATTTCAACAATCTGAACGGCGAGGACCAGGATATCCTGATGGAGAACAATCTGAGACGGCCCAACCTGGAGGCCTTTAATCGGGCCGTGAAGAGCCTGCAGAACGCCAGCGCCATCGAGTCCATCCTGAAGAATCTGCTGCCATGTCTGCCACTGGCAACCGCAGCACCTACAAGGCACCCAATCCACATCAAGGACGGCGATTGGAATGAGTTCAGGCGCAAGCTGACCTTTTACCTGAAAACACTGGAAAACGCTCAGGCACAGCAGACCACACTGTCACTGGCAATCTTCACTAGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
CRL2核苷酸序列:SEQ ID NO:34
ATGAGTAGACTGCCCGTGCTGCTGCTGCTGCAGCTGCTGGTGCGACCTGGACTGCAGGCCCCAATGACACAGACAACCCCACTGAAAACCTCTTGGGTGAACTGCAGCAATATGATCGACGAGATCATCACACACCTGAAGCAGCCCCCTCTGCCCCTGCTGGATTTCAACAATCTGAACGGCGAGGACCAGGATATCCTGATGGAGAACAATCTGAGACGGCCCAACCTGGAGGCCTTTAATCGGGCCGTGAAGAGCCTGCAGAACGCCAGCGCCATCGAGTCCATCCTGAAGAATCTGCTGCCATGTCTGCCACTGGCAACCGCAGCACCTACAAGGCACCCAATCCACATCAAGGACGGCGATTGGAATGAGTTCAGGCGCAAGCTGACCTTTTACCTGAAAACACTGGAAAACGCTCAGGCACAGCAGACCACACTGTCACTGGCAATCTTCACTAGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
CRL3核苷酸序列:SEQ ID NO:35
ATGAGTAGACTGCCCGTGCTGCTGCTGCTGCAGCTGCTGGTGCGACCTGGACTGCAGGCCCCAATGACACAGACAACCCCACTGAAAACCTCTTGGGTGAACTGCAGCAATATGATCGACGAGATCATCACACACCTGAAGCAGCCCCCTCTGCCCCTGCTGGATTTCAACAATCTGAACGGCGAGGACCAGGATATCCTGATGGAGAACAATCTGAGACGGCCCAACCTGGAGGCCTTTAATCGGGCCGTGAAGAGCCTGCAGAACGCCAGCGCCATCGAGTCCATCCTGAAGAATCTGCTGCCATGTCTGCCACTGGCAACCGCAGCACCTACAAGGCACCCAATCCACATCAAGGACGGCGATTGGAATGAGTTCAGGCGCAAGCTGACCTTTTACCTGAAAACACTGGAAAACGCTCAGGCACAGCAGACCACACTGTCACTGGCAATCTTCACTAGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
CRL4核苷酸序列:SEQ ID NO:36
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCCCCAATGACACAGACAACCCCACTGAAAACCTCTTGGGTGAACTGCAGCAATATGATCGACGAGATCATCACACACCTGAAGCAGCCCCCTCTGCCCCTGCTGGATTTCAACAATCTGAACGGCGAGGACCAGGATATCCTGATGGAGAACAATCTGAGACGGCCCAACCTGGAGGCCTTTAATCGGGCCGTGAAGAGCCTGCAGAACGCCAGCGCCATCGAGTCCATCCTGAAGAATCTGCTGCCATGTCTGCCACTGGCAACCGCAGCACCTACAAGGCACCCAATCCACATCAAGGACGGCGATTGGAATGAGTTCAGGCGCAAGCTGACCTTTTACCTGAAAACACTGGAAAACGCTCAGGCACAGCAGACCACACTGTCACTGGCAATCTTCACTAGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
CRL5核苷酸序列:SEQ ID NO:37
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCCCCAATGACACAGACAACCCCACTGAAAACCTCTTGGGTGAACTGCAGCAATATGATCGACGAGATCATCACACACCTGAAGCAGCCCCCTCTGCCCCTGCTGGATTTCAACAATCTGAACGGCGAGGACCAGGATATCCTGATGGAGAACAATCTGAGACGGCCCAACCTGGAGGCCTTTAATCGGGCCGTGAAGAGCCTGCAGAACGCCAGCGCCATCGAGTCCATCCTGAAGAATCTGCTGCCATGTCTGCCACTGGCAACCGCAGCACCTACAAGGCACCCAATCCACATCAAGGACGGCGATTGGAATGAGTTCAGGCGCAAGCTGACCTTTTACCTGAAAACACTGGAAAACGCTCAGGCACAGCAGACCACACTGTCACTGGCAATCTTCACTAGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
CRL6核苷酸序列:SEQ ID NO:38
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCCCCAATGACACAGACAACCCCACTGAAAACCTCTTGGGTGAACTGCAGCAATATGATCGACGAGATCATCACACACCTGAAGCAGCCCCCTCTGCCCCTGCTGGATTTCAACAATCTGAACGGCGAGGACCAGGATATCCTGATGGAGAACAATCTGAGACGGCCCAACCTGGAGGCCTTTAATCGGGCCGTGAAGAGCCTGCAGAACGCCAGCGCCATCGAGTCCATCCTGAAGAATCTGCTGCCATGTCTGCCACTGGCAACCGCAGCACCTACAAGGCACCCAATCCACATCAAGGACGGCGATTGGAATGAGTTCAGGCGCAAGCTGACCTTTTACCTGAAAACACTGGAAAACGCTCAGGCACAGCAGACCACACTGTCACTGGCAATCTTCACTAGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
密码子优化后的IL-3核苷酸序列:SEQ ID NO:39
ATGAGTAGACTGCCCGTGCTGCTGCTGCTGCAGCTGCTGGTGCGACCTGGACTGCAGGCCCCAATGACACAGACAACCCCACTGAAAACCTCTTGGGTGAACTGCAGCAATATGATCGACGAGATCATCACACACCTGAAGCAGCCCCCTCTGCCCCTGCTGGATTTCAACAATCTGAACGGCGAGGACCAGGATATCCTGATGGAGAACAATCTGAGACGGCCCAACCTGGAGGCCTTTAATCGGGCCGTGAAGAGCCTGCAGAACGCCAGCGCCATCGAGTCCATCCTGAAGAATCTGCTGCCATGTCTGCCACTGGCAACCGCAGCACCTACAAGGCACCCAATCCACATCAAGGACGGCGATTGGAATGAGTTCAGGCGCAAGCTGACCTTTTACCTGAAAACACTGGAAAACGCTCAGGCACAGCAGACCACACTGTCACTGGCAATCTTC
IL-3氨基酸序列:SEQ ID NO:40
MSRLPVLLLL QLLVRPGLQA PMTQTTPLKT SWVNCSNMID EIITHLKQPP
LPLLDFNNLN GEDQDILMEN NLRRPNLEAF NRAVKSLQNA SAIESILKNL
LPCLPLATAA PTRHPIHIKD GDWNEFRRKL TFYLKTLENA QAQQTTLSLAIF
Claims (33)
1.一种靶向人CD123的嵌合受体配体,包含:基于IL-3分子的CD123结合结构域、跨膜区及胞内信号结构域。
2.根据权利要求1所述的靶向人CD123的嵌合受体配体,其中CD123结合结构域包含SEQID NO:1的至少100个连续氨基酸残基。
3.根据权利要求1所述的靶向人CD123的嵌合受体配体,其中编码CD123结合结构域的氨基酸序列为SEQ ID NO:1或2所示,或与其具有85%~99%或90%~99%或95%~99%同一性的改造的氨基酸序列。
4.根据权利要求1或2所述的靶向人CD123的嵌合受体配体,其中所述嵌合受体配体包含两个或更多个基于IL-3分子的CD123结合结构域。
5.根据权利要求1~4任一项所述的靶向人CD123的嵌合受体配体,其中所述跨膜区选自CD4、CD8α、CD28、PD1和/或4-1BB跨膜区。
6.根据权利要求5所述的靶向人CD123的嵌合受体配体,其特征在于跨膜区为CD8α跨膜区。
7.根据权利要求1~6任一项所述的靶向人CD123的嵌合受体配体,其特征在于胞内信号结构域包括信号传导结构域和/或共刺激信号结构域,选自以下信号分子的细胞内结构域:CD27、CD28、4-1BB、OX40、CD30、CD40、CD3、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、CD83特异性结合的配体和其任意组合。
8.根据权利要求7所述的靶向人CD123的嵌合受体配体,其特征在于胞内信号结构域包括信号传导结构域和/或共刺激信号结构域,进一步优选CD3、4-1BB和/或CD28信号结构域。
9.根据权利要求1~8任一项所述的靶向人CD123的嵌合受体配体,其特征在于包含胞外信号肽结构,优选CD8α信号肽、GM-CSFRα信号肽、CD4信号肽或IL-3信号肽。
10.根据权利要求9所述的靶向人CD123的嵌合受体配体,其特征在于胞外信号肽优选CD8α信号肽或IL-3信号肽,更优选为IL-3信号肽。
11.根据权利要求9~10任一项所述的靶向人CD123的嵌合受体配体,其胞外信号肽包含SEQ ID NO:3所示的IL-3信号肽氨基酸序列。
12.根据权利要求9~10任一项所述的靶向人CD123的嵌合受体配体,其胞外信号肽包含SEQ ID NO:4所示的CD8α信号肽氨基酸序列。
13.根据权利要求1~12任一项所述的靶向人CD123的嵌合受体配体,其特征在于不包含铰链区结构。
14.根据权利要求1~12任一项所述的靶向人CD123的嵌合受体配体,其特征在于包括铰链区结构,优选CD8α分子。
15.根据权利要求14所述的靶向人CD123的嵌合受体配体,其铰链区包含SEQ ID NO:5所示的CD8α铰链区氨基酸序列。
16.根据权利要求1~15任一项所述的靶向人CD123的嵌合受体配体,其特征在于跨膜区结构包含SEQ ID NO:6所示的CD8α跨膜区氨基酸序列,或SEQ ID NO:7所示的CD28跨膜区氨基酸序列。
17.根据权利要求1~16任一项所述的靶向人CD123的嵌合受体配体,其特征在于胞内信号传导结构域包含SEQ ID NO:8所示的CD3zeta氨基酸序列。
18.根据权利要求1~17任一项所述的靶向人CD123的嵌合受体配体,其特征在于胞内共刺激信号结构域包含SEQ ID NO:9所示的4-1BB氨基酸序列,和/或SEQ ID NO:10所示的CD28氨基酸序列。
19.根据权利要求1~18任一项所述的靶向人CD123的嵌合受体配体,其特征在于编码的氨基酸序列为SEQ ID NO:14所示,或与其具有95%~99%同一性的氨基酸序列。
20.根据权利要求1~18任一项所述的靶向人CD123的嵌合受体配体,其特征在于编码的氨基酸序列为SEQ ID NO:15或SEQ ID NO:16或SEQ ID NO:17或SEQ ID NO:18或SEQ IDNO:19所示,或与其具有95%~99%同一性的氨基酸序列。
21.一种核酸分子,其特征在于编码权利要求1~20任一项所述靶向人CD123的嵌合受体配体的核苷酸序列。
22.根据权利要求21所述的核酸分子,其特征在于核苷酸编码序列如SEQ ID NO:33或SEQ ID NO:34或SEQ ID NO:35或SEQ ID NO:36或SEQ ID NO:37或SEQ IDNO:38所示。
23.一种载体,其特征在于包含权利要求21或22所述的核酸分子。
24.一种基因工程改造的免疫细胞,其特征在于包含权利要求21或22所述的核酸分子。
25.根据权利要求24所述的基因工程改造的免疫细胞,其特征在于包含权利要求23所述的载体。
26.根据权利要求24~25任一项所述的基因工程改造的免疫细胞,其特征在于可选自T淋巴细胞、NK细胞、造血干细胞、多能干细胞或胚胎干细胞培养分化的免疫细胞,进一步优选T淋巴细胞。
27.根据权利要求24~26任一项所述的基因工程改造的免疫细胞,其特征在于表达的靶向人CD123的嵌合受体配体包含胞外信号肽、基于IL-3分子的CD123结合结构域、跨膜区及胞内信号结构域。
28.根据权利要求24-27任一项所述的基因工程改造的免疫细胞,其特征在于表达的靶向人CD123的嵌合受体配体具有SEQ ID NO:14所示氨基酸序列,或与其具有95%~99%同一性的氨基酸序列。
29.根据权利要求24-27任一项所述的基因工程改造的免疫细胞,其特征在于表达的靶向人CD123的嵌合受体配体具有SEQ ID NO:15或SEQ ID NO:16或SEQ ID NO:17或SEQ IDNO:18或SEQ ID NO:19所示氨基酸序列,或与其具有95%~99%同一性的氨基酸序列。
30.一种方法,其特征在于制备权利要求1~20中任一项所述靶向人CD123的嵌合受体配体。
31.根据权利要求30所述的方法,其特征在于制备权利要求24~29所述的基因工程改造的免疫细胞。
32.权利要求1~20中任一项所述靶向人CD123的嵌合受体配体在制备抗肿瘤药物中的应用;优选在制备抗血液恶性肿瘤药物中的应用。
33.权利要求24~29中任一项所述的基因工程改造的免疫细胞在制备抗肿瘤药物中的应用;优选在制备抗血液恶性肿瘤药物中的应用。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610795955.XA CN107793481A (zh) | 2016-08-31 | 2016-08-31 | 一种靶向人cd123的嵌合受体配体及其应用 |
JP2019531525A JP6990244B2 (ja) | 2016-08-31 | 2017-08-31 | ヒトcd123を標的とするキメラ受容体リガンド及びその応用 |
US16/329,739 US11401317B2 (en) | 2016-08-31 | 2017-08-31 | Human-CD123-targeting chimeric receptor ligand and application thereof |
PCT/CN2017/099941 WO2018041220A1 (zh) | 2016-08-31 | 2017-08-31 | 一种靶向人cd123的嵌合受体配体及其应用 |
EP17845522.6A EP3508504A4 (en) | 2016-08-31 | 2017-08-31 | AGAINST HUMAN-CD123 CHIMERAL RECIPE LIGAND AND APPLICATION THEREOF |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610795955.XA CN107793481A (zh) | 2016-08-31 | 2016-08-31 | 一种靶向人cd123的嵌合受体配体及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN107793481A true CN107793481A (zh) | 2018-03-13 |
Family
ID=61300124
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610795955.XA Pending CN107793481A (zh) | 2016-08-31 | 2016-08-31 | 一种靶向人cd123的嵌合受体配体及其应用 |
Country Status (5)
Country | Link |
---|---|
US (1) | US11401317B2 (zh) |
EP (1) | EP3508504A4 (zh) |
JP (1) | JP6990244B2 (zh) |
CN (1) | CN107793481A (zh) |
WO (1) | WO2018041220A1 (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109970869A (zh) * | 2019-04-12 | 2019-07-05 | 南京卡提医学科技有限公司 | 一种靶向人trail死亡受体的嵌合受体配体及其应用 |
CN110157678A (zh) * | 2018-02-12 | 2019-08-23 | 深圳宾德生物技术有限公司 | 一种靶向性t淋巴细胞及其制备方法和应用 |
CN110904048A (zh) * | 2019-12-04 | 2020-03-24 | 合肥中科干细胞再生医学有限公司 | 一种car-cd123t2嵌合抗原受体t细胞及其应用 |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20210031898A (ko) * | 2018-07-13 | 2021-03-23 | 난징 레전드 바이오테크 씨오., 엘티디. | 전염병을 치료하기 위한 공동수용체 시스템 |
WO2023000036A1 (en) * | 2021-07-22 | 2023-01-26 | The Westmead Institute for Medical Research | Chimeric antigen receptor useful in treating cancer |
WO2023010126A2 (en) * | 2021-07-29 | 2023-02-02 | Vor Biopharma Inc. | Chimeric antigen receptors for treatment of cancer |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104769103A (zh) * | 2012-09-04 | 2015-07-08 | 塞勒克提斯公司 | 多链嵌合抗原受体和其用途 |
WO2016028896A1 (en) * | 2014-08-19 | 2016-02-25 | Novartis Ag | Anti-cd123 chimeric antigen receptor (car) for use in cancer treatment |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK2063907T3 (da) * | 2006-09-07 | 2017-11-13 | Arthur E Frankel | Fremgangsmåder og sammensætninger baseret på difteritoksin-interleukin-3-konjugater |
US8822647B2 (en) * | 2008-08-26 | 2014-09-02 | City Of Hope | Method and compositions using a chimeric antigen receptor for enhanced anti-tumor effector functioning of T cells |
US9657105B2 (en) * | 2013-03-15 | 2017-05-23 | City Of Hope | CD123-specific chimeric antigen receptor redirected T cells and methods of their use |
TR201910814T4 (tr) * | 2014-03-19 | 2019-08-21 | Cellectis | Kanser immünoterapisi için cd123 spesifik kimerik antijen reseptörleri. |
WO2015193406A1 (en) * | 2014-06-17 | 2015-12-23 | Cellectis | Cd123 specific multi-chain chimeric antigen receptor |
-
2016
- 2016-08-31 CN CN201610795955.XA patent/CN107793481A/zh active Pending
-
2017
- 2017-08-31 US US16/329,739 patent/US11401317B2/en active Active
- 2017-08-31 EP EP17845522.6A patent/EP3508504A4/en active Pending
- 2017-08-31 JP JP2019531525A patent/JP6990244B2/ja active Active
- 2017-08-31 WO PCT/CN2017/099941 patent/WO2018041220A1/zh unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104769103A (zh) * | 2012-09-04 | 2015-07-08 | 塞勒克提斯公司 | 多链嵌合抗原受体和其用途 |
WO2016028896A1 (en) * | 2014-08-19 | 2016-02-25 | Novartis Ag | Anti-cd123 chimeric antigen receptor (car) for use in cancer treatment |
Non-Patent Citations (4)
Title |
---|
ARMEN MARDIROS, ET AL.: ""T cells expressing CD123 chimeric antigen receptors for treatment of acute myeloid leukemia"", 《CURR OPIN HEMATOL》 * |
BAXTER,E.W., ET AL.: ""interleukin-3 precursor [Homo sapiens],NP_000579.2,152 aa"", 《NCBI GENBANK》 * |
RADHIKA THOKALA, ET AL.: ""Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors"", 《PLOS ONE》 * |
张砚君 等: ""IL-3-LDM融合蛋白对CD123+白血病细胞的靶向杀伤作用"", 《中国肿瘤生物治疗杂质》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110157678A (zh) * | 2018-02-12 | 2019-08-23 | 深圳宾德生物技术有限公司 | 一种靶向性t淋巴细胞及其制备方法和应用 |
CN109970869A (zh) * | 2019-04-12 | 2019-07-05 | 南京卡提医学科技有限公司 | 一种靶向人trail死亡受体的嵌合受体配体及其应用 |
CN110904048A (zh) * | 2019-12-04 | 2020-03-24 | 合肥中科干细胞再生医学有限公司 | 一种car-cd123t2嵌合抗原受体t细胞及其应用 |
Also Published As
Publication number | Publication date |
---|---|
EP3508504A4 (en) | 2020-04-22 |
EP3508504A1 (en) | 2019-07-10 |
WO2018041220A1 (zh) | 2018-03-08 |
JP6990244B2 (ja) | 2022-01-12 |
US20190225668A1 (en) | 2019-07-25 |
JP2019532668A (ja) | 2019-11-14 |
US11401317B2 (en) | 2022-08-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2018121712A1 (zh) | 一种新型嵌合抗原受体及其应用 | |
CN107793481A (zh) | 一种靶向人cd123的嵌合受体配体及其应用 | |
CN106554414B (zh) | 抗cd19全人抗体以及靶向cd19的免疫效应细胞 | |
JP7303749B2 (ja) | Tim-1を標的とするキメラ抗原受容体 | |
CN109385403B (zh) | 靶向gpc3的car nk细胞 | |
JP2024045111A (ja) | ガイダンス及びナビゲーションコントロール蛋白質の生産及び使用方法 | |
CN109400713A (zh) | 新型嵌合抗原受体修饰的t细胞治疗癌症的用途 | |
CN103502438A (zh) | 用于细胞免疫治疗的方法和组合物 | |
US20220064255A1 (en) | Anti-tcr antibody molecules and uses thereof | |
AU2020418199A1 (en) | Modified immune effector cell and preparation method therefor | |
WO2018111340A1 (en) | Methods for determining potency and proliferative function of chimeric antigen receptor (car)-t cells | |
US20230048244A1 (en) | Anti-tcr antibody molecules and uses thereof | |
WO2020181164A1 (en) | Compositions and methods for treating cancer with self-driving chimeric antigen receptors | |
US20240033357A1 (en) | Chimeric antigen receptors and uses thereof | |
JP2023515364A (ja) | キメラ抗原受容体及びその適用 | |
CN116789849B (zh) | 一种嵌合抗原受体及其应用 | |
CN110078833A (zh) | 基于亲合体靶向her2的嵌合抗原受体及其应用 | |
CN117210410A (zh) | 一种t细胞、其用途及药物组合物 | |
TW202300520A (zh) | 特異性識別baff-r的嵌合抗原受體分子及其應用 | |
CN115463155A (zh) | 间充质干细胞的用途 | |
CN117946284A (zh) | 一种iNKT细胞在癌症治疗中的应用 | |
CN117924519A (zh) | 靶向cd138的嵌合抗原受体 |
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 |