CN112852740A - Car-t细胞和检测方法 - Google Patents
Car-t细胞和检测方法 Download PDFInfo
- Publication number
- CN112852740A CN112852740A CN201911181105.0A CN201911181105A CN112852740A CN 112852740 A CN112852740 A CN 112852740A CN 201911181105 A CN201911181105 A CN 201911181105A CN 112852740 A CN112852740 A CN 112852740A
- Authority
- CN
- China
- Prior art keywords
- antibody
- car
- cell
- cancer
- target
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims description 12
- 210000004027 cell Anatomy 0.000 claims description 54
- 102100040678 Programmed cell death protein 1 Human genes 0.000 claims description 33
- 101710089372 Programmed cell death protein 1 Proteins 0.000 claims description 30
- 108090000623 proteins and genes Proteins 0.000 claims description 19
- 108010074708 B7-H1 Antigen Proteins 0.000 claims description 17
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 claims description 17
- 230000003248 secreting effect Effects 0.000 claims description 17
- 206010028980 Neoplasm Diseases 0.000 claims description 16
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 13
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 13
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 11
- 208000003950 B-cell lymphoma Diseases 0.000 claims description 10
- 239000000427 antigen Substances 0.000 claims description 9
- 102000036639 antigens Human genes 0.000 claims description 9
- 108091007433 antigens Proteins 0.000 claims description 9
- 239000012634 fragment Substances 0.000 claims description 9
- 230000011664 signaling Effects 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 6
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 claims description 6
- 230000003197 catalytic effect Effects 0.000 claims description 6
- 230000000139 costimulatory effect Effects 0.000 claims description 5
- 229920001184 polypeptide Polymers 0.000 claims description 5
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 5
- 102100038080 B-cell receptor CD22 Human genes 0.000 claims description 4
- 102100032937 CD40 ligand Human genes 0.000 claims description 4
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 claims description 4
- 101000868215 Homo sapiens CD40 ligand Proteins 0.000 claims description 4
- 101000934341 Homo sapiens T-cell surface glycoprotein CD5 Proteins 0.000 claims description 4
- 101000679851 Homo sapiens Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 claims description 4
- 102100025244 T-cell surface glycoprotein CD5 Human genes 0.000 claims description 4
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims description 4
- 239000012228 culture supernatant Substances 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 208000019691 hematopoietic and lymphoid cell neoplasm Diseases 0.000 claims description 4
- 230000003834 intracellular effect Effects 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 230000000903 blocking effect Effects 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000019491 signal transduction Effects 0.000 claims description 3
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 claims description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 claims description 2
- 208000031261 Acute myeloid leukaemia Diseases 0.000 claims description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 2
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 claims description 2
- 206010005949 Bone cancer Diseases 0.000 claims description 2
- 208000018084 Bone neoplasm Diseases 0.000 claims description 2
- 206010006187 Breast cancer Diseases 0.000 claims description 2
- 208000026310 Breast neoplasm Diseases 0.000 claims description 2
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 claims description 2
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 claims description 2
- 102100037904 CD9 antigen Human genes 0.000 claims description 2
- 102100025466 Carcinoembryonic antigen-related cell adhesion molecule 3 Human genes 0.000 claims description 2
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 2
- 206010009944 Colon cancer Diseases 0.000 claims description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 2
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 claims description 2
- 101000914337 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 3 Proteins 0.000 claims description 2
- 101000777628 Homo sapiens Leukocyte antigen CD37 Proteins 0.000 claims description 2
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 claims description 2
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 claims description 2
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 claims description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 2
- 101000934346 Homo sapiens T-cell surface antigen CD2 Proteins 0.000 claims description 2
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 claims description 2
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 2
- 102100031586 Leukocyte antigen CD37 Human genes 0.000 claims description 2
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 claims description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 2
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 claims description 2
- 208000034578 Multiple myelomas Diseases 0.000 claims description 2
- 101710135898 Myc proto-oncogene protein Proteins 0.000 claims description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 claims description 2
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 claims description 2
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 claims description 2
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 claims description 2
- 206010061306 Nasopharyngeal cancer Diseases 0.000 claims description 2
- 206010033128 Ovarian cancer Diseases 0.000 claims description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 2
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 claims description 2
- 206010060862 Prostate cancer Diseases 0.000 claims description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 2
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 claims description 2
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 claims description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 claims description 2
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 claims description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 2
- 208000024447 adrenal gland neoplasm Diseases 0.000 claims description 2
- 201000007983 brain glioma Diseases 0.000 claims description 2
- 201000011510 cancer Diseases 0.000 claims description 2
- 210000000170 cell membrane Anatomy 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 claims description 2
- 201000010881 cervical cancer Diseases 0.000 claims description 2
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 claims description 2
- 238000011161 development Methods 0.000 claims description 2
- 206010017758 gastric cancer Diseases 0.000 claims description 2
- 201000011061 large intestine cancer Diseases 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 201000007270 liver cancer Diseases 0.000 claims description 2
- 208000014018 liver neoplasm Diseases 0.000 claims description 2
- 201000005202 lung cancer Diseases 0.000 claims description 2
- 208000020816 lung neoplasm Diseases 0.000 claims description 2
- 210000002751 lymph Anatomy 0.000 claims description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 2
- 201000002314 small intestine cancer Diseases 0.000 claims description 2
- 201000011549 stomach cancer Diseases 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 210000005260 human cell Anatomy 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 7
- 238000012544 monitoring process Methods 0.000 abstract description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 13
- 239000013598 vector Substances 0.000 description 13
- 241000713666 Lentivirus Species 0.000 description 9
- 150000001413 amino acids Chemical group 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 210000004881 tumor cell Anatomy 0.000 description 6
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 4
- HKZAAJSTFUZYTO-LURJTMIESA-N (2s)-2-[[2-[[2-[[2-[(2-aminoacetyl)amino]acetyl]amino]acetyl]amino]acetyl]amino]-3-hydroxypropanoic acid Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O HKZAAJSTFUZYTO-LURJTMIESA-N 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 2
- 108010005327 CD19-specific chimeric antigen receptor Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 2
- OYTPNWYZORARHL-XHNCKOQMSA-N Gln-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N OYTPNWYZORARHL-XHNCKOQMSA-N 0.000 description 2
- YOOAQCZYZHGUAZ-KATARQTJSA-N Thr-Leu-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YOOAQCZYZHGUAZ-KATARQTJSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 108010010147 glycylglutamine Proteins 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 108010044292 tryptophyltyrosine Proteins 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XQNRANMFRPCFFW-GCJQMDKQSA-N Ala-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C)N)O XQNRANMFRPCFFW-GCJQMDKQSA-N 0.000 description 1
- ZDILXFDENZVOTL-BPNCWPANSA-N Ala-Val-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZDILXFDENZVOTL-BPNCWPANSA-N 0.000 description 1
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 1
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 1
- VKKYFICVTYKFIO-CIUDSAMLSA-N Arg-Ala-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N VKKYFICVTYKFIO-CIUDSAMLSA-N 0.000 description 1
- AOHKLEBWKMKITA-IHRRRGAJSA-N Arg-Phe-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N AOHKLEBWKMKITA-IHRRRGAJSA-N 0.000 description 1
- UVTGNSWSRSCPLP-UHFFFAOYSA-N Arg-Tyr Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccc(O)cc1)C(=O)O UVTGNSWSRSCPLP-UHFFFAOYSA-N 0.000 description 1
- MKJBPDLENBUHQU-CIUDSAMLSA-N Asn-Ser-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O MKJBPDLENBUHQU-CIUDSAMLSA-N 0.000 description 1
- UGXYFDQFLVCDFC-CIUDSAMLSA-N Asn-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O UGXYFDQFLVCDFC-CIUDSAMLSA-N 0.000 description 1
- JBDLMLZNDRLDIX-HJGDQZAQSA-N Asn-Thr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O JBDLMLZNDRLDIX-HJGDQZAQSA-N 0.000 description 1
- ANRZCQXIXGDXLR-CWRNSKLLSA-N Asn-Trp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CC(=O)N)N)C(=O)O ANRZCQXIXGDXLR-CWRNSKLLSA-N 0.000 description 1
- VZNOVQKGJQJOCS-SRVKXCTJSA-N Asp-Asp-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VZNOVQKGJQJOCS-SRVKXCTJSA-N 0.000 description 1
- JSHWXQIZOCVWIA-ZKWXMUAHSA-N Asp-Ser-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O JSHWXQIZOCVWIA-ZKWXMUAHSA-N 0.000 description 1
- MNQMTYSEKZHIDF-GCJQMDKQSA-N Asp-Thr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O MNQMTYSEKZHIDF-GCJQMDKQSA-N 0.000 description 1
- 208000025324 B-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000025321 B-lymphoblastic leukemia/lymphoma Diseases 0.000 description 1
- 108010024755 CTL019 chimeric antigen receptor Proteins 0.000 description 1
- SZQCDCKIGWQAQN-FXQIFTODSA-N Cys-Arg-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O SZQCDCKIGWQAQN-FXQIFTODSA-N 0.000 description 1
- JXFLPKSDLDEOQK-JHEQGTHGSA-N Gln-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O JXFLPKSDLDEOQK-JHEQGTHGSA-N 0.000 description 1
- FQCILXROGNOZON-YUMQZZPRSA-N Gln-Pro-Gly Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O FQCILXROGNOZON-YUMQZZPRSA-N 0.000 description 1
- JILRMFFFCHUUTJ-ACZMJKKPSA-N Gln-Ser-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O JILRMFFFCHUUTJ-ACZMJKKPSA-N 0.000 description 1
- ZFBBMCKQSNJZSN-AUTRQRHGSA-N Gln-Val-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZFBBMCKQSNJZSN-AUTRQRHGSA-N 0.000 description 1
- AVZHGSCDKIQZPQ-CIUDSAMLSA-N Glu-Arg-Ala Chemical compound C[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O AVZHGSCDKIQZPQ-CIUDSAMLSA-N 0.000 description 1
- PAQUJCSYVIBPLC-AVGNSLFASA-N Glu-Asp-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PAQUJCSYVIBPLC-AVGNSLFASA-N 0.000 description 1
- INGJLBQKTRJLFO-UKJIMTQDSA-N Glu-Ile-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O INGJLBQKTRJLFO-UKJIMTQDSA-N 0.000 description 1
- SCWYHUQOOFRVHP-MBLNEYKQSA-N Gly-Ile-Thr Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SCWYHUQOOFRVHP-MBLNEYKQSA-N 0.000 description 1
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 1
- RVGMVLVBDRQVKB-UWVGGRQHSA-N Gly-Met-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)CN RVGMVLVBDRQVKB-UWVGGRQHSA-N 0.000 description 1
- NVTPVQLIZCOJFK-FOHZUACHSA-N Gly-Thr-Asp Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O NVTPVQLIZCOJFK-FOHZUACHSA-N 0.000 description 1
- ZZWUYQXMIFTIIY-WEDXCCLWSA-N Gly-Thr-Leu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O ZZWUYQXMIFTIIY-WEDXCCLWSA-N 0.000 description 1
- KSOBNUBCYHGUKH-UWVGGRQHSA-N Gly-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN KSOBNUBCYHGUKH-UWVGGRQHSA-N 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- NZGTYCMLUGYMCV-XUXIUFHCSA-N Ile-Lys-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N NZGTYCMLUGYMCV-XUXIUFHCSA-N 0.000 description 1
- IITVUURPOYGCTD-NAKRPEOUSA-N Ile-Pro-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IITVUURPOYGCTD-NAKRPEOUSA-N 0.000 description 1
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 1
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 208000031671 Large B-Cell Diffuse Lymphoma Diseases 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- HXWALXSAVBLTPK-NUTKFTJISA-N Leu-Ala-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC(C)C)N HXWALXSAVBLTPK-NUTKFTJISA-N 0.000 description 1
- FGNQZXKVAZIMCI-CIUDSAMLSA-N Leu-Asp-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N FGNQZXKVAZIMCI-CIUDSAMLSA-N 0.000 description 1
- AXZGZMGRBDQTEY-SRVKXCTJSA-N Leu-Gln-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(O)=O AXZGZMGRBDQTEY-SRVKXCTJSA-N 0.000 description 1
- FEHQLKKBVJHSEC-SZMVWBNQSA-N Leu-Glu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 FEHQLKKBVJHSEC-SZMVWBNQSA-N 0.000 description 1
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 1
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 1
- LCNASHSOFMRYFO-WDCWCFNPSA-N Leu-Thr-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O LCNASHSOFMRYFO-WDCWCFNPSA-N 0.000 description 1
- AIMGJYMCTAABEN-GVXVVHGQSA-N Leu-Val-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIMGJYMCTAABEN-GVXVVHGQSA-N 0.000 description 1
- UWKNTTJNVSYXPC-CIUDSAMLSA-N Lys-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN UWKNTTJNVSYXPC-CIUDSAMLSA-N 0.000 description 1
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 description 1
- PDIDTSZKKFEDMB-UWVGGRQHSA-N Lys-Pro-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PDIDTSZKKFEDMB-UWVGGRQHSA-N 0.000 description 1
- UGCIQUYEJIEHKX-GVXVVHGQSA-N Lys-Val-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O UGCIQUYEJIEHKX-GVXVVHGQSA-N 0.000 description 1
- MHQXIBRPDKXDGZ-ZFWWWQNUSA-N Met-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)[C@@H](N)CCSC)C(O)=O)=CNC2=C1 MHQXIBRPDKXDGZ-ZFWWWQNUSA-N 0.000 description 1
- YMIZSYUAZJSOFL-SRVKXCTJSA-N Phe-Ser-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O YMIZSYUAZJSOFL-SRVKXCTJSA-N 0.000 description 1
- FGWUALWGCZJQDJ-URLPEUOOSA-N Phe-Thr-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FGWUALWGCZJQDJ-URLPEUOOSA-N 0.000 description 1
- KLYYKKGCPOGDPE-OEAJRASXSA-N Phe-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O KLYYKKGCPOGDPE-OEAJRASXSA-N 0.000 description 1
- QEDMOZUJTGEIBF-FXQIFTODSA-N Ser-Arg-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O QEDMOZUJTGEIBF-FXQIFTODSA-N 0.000 description 1
- OBXVZEAMXFSGPU-FXQIFTODSA-N Ser-Asn-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)CN=C(N)N OBXVZEAMXFSGPU-FXQIFTODSA-N 0.000 description 1
- INCNPLPRPOYTJI-JBDRJPRFSA-N Ser-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CO)N INCNPLPRPOYTJI-JBDRJPRFSA-N 0.000 description 1
- FMDHKPRACUXATF-ACZMJKKPSA-N Ser-Gln-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O FMDHKPRACUXATF-ACZMJKKPSA-N 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 1
- QYSFWUIXDFJUDW-DCAQKATOSA-N Ser-Leu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYSFWUIXDFJUDW-DCAQKATOSA-N 0.000 description 1
- ZIFYDQAFEMIZII-GUBZILKMSA-N Ser-Leu-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZIFYDQAFEMIZII-GUBZILKMSA-N 0.000 description 1
- NNFMANHDYSVNIO-DCAQKATOSA-N Ser-Lys-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NNFMANHDYSVNIO-DCAQKATOSA-N 0.000 description 1
- ADJDNJCSPNFFPI-FXQIFTODSA-N Ser-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO ADJDNJCSPNFFPI-FXQIFTODSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- BIBYEFRASCNLAA-CDMKHQONSA-N Thr-Phe-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 BIBYEFRASCNLAA-CDMKHQONSA-N 0.000 description 1
- SVGAWGVHFIYAEE-JSGCOSHPSA-N Trp-Gly-Gln Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O)=CNC2=C1 SVGAWGVHFIYAEE-JSGCOSHPSA-N 0.000 description 1
- MBLJBGZWLHTJBH-SZMVWBNQSA-N Trp-Val-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 MBLJBGZWLHTJBH-SZMVWBNQSA-N 0.000 description 1
- GAYLGYUVTDMLKC-UWJYBYFXSA-N Tyr-Asp-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 GAYLGYUVTDMLKC-UWJYBYFXSA-N 0.000 description 1
- JWHOIHCOHMZSAR-QWRGUYRKSA-N Tyr-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JWHOIHCOHMZSAR-QWRGUYRKSA-N 0.000 description 1
- QOIKZODVIPOPDD-AVGNSLFASA-N Tyr-Cys-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(O)=O QOIKZODVIPOPDD-AVGNSLFASA-N 0.000 description 1
- QUILOGWWLXMSAT-IHRRRGAJSA-N Tyr-Gln-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O QUILOGWWLXMSAT-IHRRRGAJSA-N 0.000 description 1
- MWUYSCVVPVITMW-IGNZVWTISA-N Tyr-Tyr-Ala Chemical compound C([C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 MWUYSCVVPVITMW-IGNZVWTISA-N 0.000 description 1
- 102100033019 Tyrosine-protein phosphatase non-receptor type 11 Human genes 0.000 description 1
- 101710116241 Tyrosine-protein phosphatase non-receptor type 11 Proteins 0.000 description 1
- SLLKXDSRVAOREO-KZVJFYERSA-N Val-Ala-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N)O SLLKXDSRVAOREO-KZVJFYERSA-N 0.000 description 1
- HQYVQDRYODWONX-DCAQKATOSA-N Val-His-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CO)C(=O)O)N HQYVQDRYODWONX-DCAQKATOSA-N 0.000 description 1
- MYLNLEIZWHVENT-VKOGCVSHSA-N Val-Ile-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](C(C)C)N MYLNLEIZWHVENT-VKOGCVSHSA-N 0.000 description 1
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 1
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 1
- OWFGFHQMSBTKLX-UFYCRDLUSA-N Val-Tyr-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N OWFGFHQMSBTKLX-UFYCRDLUSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000009175 antibody therapy Methods 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000001815 biotherapy Methods 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
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 206010012818 diffuse large B-cell lymphoma Diseases 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 102000048362 human PDCD1 Human genes 0.000 description 1
- 108091008915 immune receptors Proteins 0.000 description 1
- 102000027596 immune receptors Human genes 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 208000017426 precursor B-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
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
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- 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
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
-
- 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
-
- 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
-
- 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
Abstract
本发明公开了一种CAR‑T细胞和检测方法,涉及CAR‑T细胞技术领域。该CAR‑T细胞能够分泌目标抗体,目标抗体的N端或C端融合有检测标签。本发明公开的CAR‑T细胞其分泌的抗体可以被定量检测,为评估与监测CAR‑T分泌抗体的量与其疗效的关系提供了基础和保证。
Description
技术领域
本发明涉及CAR-T细胞技术领域,具体而言,涉及一种CAR-T细胞和检测方法。
背景技术
PD-1(Programmed Cell Death Protein)程序性死亡受体-1,主要表达在B祖细胞,活化的T细胞和B细胞表面,是免疫检查站点家族的成员之一。研究发现,肿瘤细胞会通过表达PD-1的配体PD-L1来逃避免疫系统的监护,当肿瘤细胞通过其表面的PD-L1与T细胞表面的PD-1结合时,PD-1会在免疫受体复合物附近募集大量的酪氨酸磷酸酶SHP2,造成TCR相关信号的去磷酸化从而削弱TCR信号通路,进而造成T细胞的耗竭。
嵌合抗原受体T细胞免疫疗法,简称CAR-T技术,是通过体外改造病人的T细胞,使病人的T细胞具备识别肿瘤细胞的能力,体外扩大培养后回输到病人体内进行治疗的一种方法。目前,以CD19为靶点的CAR-T在治疗B细胞血液肿瘤方面取得了巨大的成果,但根据临床研究结果来看,CD19 CAR-T在治疗B细胞淋巴瘤方面的疗效远远不及在治疗B细胞急性淋巴细胞白血病方面的疗效,这可能是B细胞淋巴瘤表面表达有大量的PD-L1分子造成的,虽然目前尚未有文献对B-ALL病人B细胞上PD-L1分子表达水平报道,但是在针对弥漫性大B细胞淋巴瘤的临床研究中发现病人B细胞淋巴瘤表面的PD-L1分子表达水平与临床疗效直接相关,B细胞淋巴瘤表面PD-L1表达低的病人,在综合疗法、单独化疗和PD-1抗体免疫疗法中均有着更高的生存率,与此同时,再对其他靶点实体肿瘤进行治疗的过程中,肿瘤细胞也会通过过表达PD-L1来逃避CAR-T对其进行杀伤,提示CD19 CAR-T治疗过程中,封闭PD-1/PD-L1信号通路将会最大程度的增加CD19 CAR-T的临床疗效,使患者受益。
运用CAR-T疗法联合PD-1/PD-L1抗体或其他免疫检查站点家族成员抗体疗法在肿瘤治疗过程中疗效明显,但是由于肿瘤微环境的存在,到达肿瘤部位的抗体药物较少,同时由于抗体药物在体内的动态代谢过程,病人治疗成本较大,鉴于此,很多研究者将CAR-T疗法与免疫检查站点疗法联合使用,生产出能分泌PD-1/PD-L1单链抗体的CAR-T,得到了积极的实验结果。
在分泌PD-1/PD-L1单链抗体的CAR-T在应用中,通常需要对其分泌的单链抗体进行定量,以便能够合理地控制用量范围。然而,目前缺乏对这类可分泌抗体的CAR-T细胞类型所分泌的抗体进行有效定量检测的手段。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种CAR-T细胞和检测方法。本发明提供的CAR-T细胞其可以分泌抗体,且分泌的抗体可以被定量检测,本发明提供的检测方法可以有效地定量检测CAR-T细胞分泌的抗体。
本发明是这样实现的:
第一方面,本发明实施例提供一种CAR-T细胞,上述CAR-T细胞能够表达嵌合抗原受体和分泌目标抗体,上述目标抗体的N端或C端融合有检测标签,上述检测标签能够被检测抗体特异性识别和结合,以对上述目标抗体进行定量检测,所述检测抗体标记有信号指示剂。
本发明提供的CAR-T细胞能够表达嵌合抗原受体和分泌抗体,所分泌的抗体的N端或C端融合了检测标签,检测标签可以被检测抗体特异性识别和结合,这样,利用检测抗体与检测标签的特异性结合原理,再通过对检测抗体上的信号指示剂的信号强度检测,将信号强度值代入匹配的标准曲线公式中,即可定量确定CAR-T细胞所分泌的抗体含量。本发明为评估与监测病人血清中或肿瘤微环境中CAR-T分泌抗体的量与疗效的关系提供了基础和保证。
需要说明是,本发明所指的检测标签可以是任何多肽或蛋白,只要其具有被某抗体识别和结合的特异性,即可作为本发明的检测标签,因此,无论选用何种蛋白或多肽,只要其存在对应的特异性抗体,将该类蛋白或多肽作为本发明的检测标签以定量检测目标抗体即属于本发明的保护范围。
在可选的实施方式中,所述检测标签为多肽。
在可选的实施方式中,上述检测标签选自FLAG、HA、HA1、c-Myc、Poly-His、Poly-Arg、Strep-TagII、AU1、EE、T7、4A6、ε、B、gE以及Ty1中的任意一种。
本领域技术人员容易获得针对上述检测标签的特异性抗体,以识别和结合检测标签以对目标抗体进行定量检测。
本发明的目标抗体也可以是任意类别的免疫检查站点家族成员单链抗体。在可选的实施方式中,上述目标抗体为能够封闭PD-1/PD-L1信号通路的抗体。
在可选的实施方式中,上述目标抗体为PD-1抗体的功能性片段。
在可选的实施方式中,上述目标抗体为PD-L1抗体的功能性片段。
在可选的实施方式中,上述功能性片段的结构形式选自F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
在可选的实施方式中,上述目标抗体选自PD-1抗体的scFv。
在可选的实施方式中,上述目标抗体选自PD-L1抗体的scFv。
在可选的实施方式中,上述目标抗体的轻链可变区氨基酸序列如SEQ ID NO.8所示,重链可变区氨基酸序列如SEQ ID NO.4所示。
在可选的实施方式中,上述目标抗体的位于轻链可变区与重链之间可变区的铰接区序列的氨基酸序列如SEQ ID NO.6所示。
在可选的实施方式中,上述目标抗体的C端融合有检测标签。
在可选的实施方式中,上述目标抗体通过如下信号肽实现分泌表达:hIgG SP。
信号肽的作用在于使目标抗体能够被顺利分泌到细胞外,因此,将任何具有该类作用的信号肽均可以应用于本发明,其均属于本发明的保护范围。
在可选的实施方式中,所述检测抗体标记有信号指示剂。
在可选的实施方式中,信号指示剂为荧光基团或者用于催化底物显色的催化酶。
在可选的实施方式中,荧光基团选自BV421。
在可选的实施方式中,催化酶选自辣根过氧化物酶或碱性磷酸酶。
信号指示剂可以指示抗体与抗原的结合,通过对信号指示剂的信号强度检测,可以实现对目标抗体的定量检测。
本发明CAR-T细胞的嵌合抗原受体的靶点可以是多种多样的,可以是正常的组织,也可以是肿瘤组织。
在可选的实施方式中,上述嵌合抗原受体上的抗原结合结构域所识别和靶向的靶点为肿瘤。
在可选的实施方式中,上述肿瘤为血液肿瘤或实体瘤。
在可选的实施方式中,上述血液肿瘤选自B细胞淋巴瘤、慢性淋巴细胞白血病、急性髓细胞白血病、急性淋巴白血病和多发性骨髓瘤中的任意一种。
在可选的实施方式中,上述实体瘤选自胃癌、肝癌、肺癌、小肠癌、前列腺癌、宫颈癌、结直肠癌、大肠癌、乳腺癌、卵巢癌、淋巴癌、鼻咽癌、骨癌、肾上腺肿瘤、脑胶质瘤、非小细胞肺癌和膀胱肿瘤中的任意一种。
在可选的实施方式中,上述靶点为B细胞淋巴瘤。
在可选的实施方式中,上述靶点为B细胞淋巴瘤细胞膜上的CD19抗原。
在可选的实施方式中,上述抗原结合结构域选自CD19抗体的scFv。
在可选的实施方式中,上述CD19抗体为FMC63。
需要说明的是,本领域技术人员可以根据实际需求选择合适的靶点,无论选用何种靶点,均属于本发明的保护范围。
在可选的实施方式中,上述嵌合抗原受体上的跨膜结构域选自如下蛋白分子中的至少一种的跨膜结构域:CD8、CD3ε、CD4、CD9、CD45、CD37、CD5、CD28、CD33、CD16、CD22、CD154、CD134和CD137。
在可选的实施方式中,上述跨膜结构域为CD8跨膜结构域。
在可选的实施方式中,上述嵌合抗原受体上的共刺激信号传导区包含如下共刺激分子中至少一种的细胞内结构域:4-1BB、CD3ζ、CD3δ、CD134、CD79a、CD137、ICD3ε、CD5、CD79b、CD5、CD66d、OX40、CD22、CD2、CD3γ、CD154和CD28。
在可选的实施方式中,上述共刺激信号传导区包含CD3ζ和4-1BB的胞内结构域。
第二方面,本发明实施例提供一种定量检测如前述实施方式任一项所述CAR-T细胞所分泌的抗体的方法,其包括:将所述CAR-T细胞的培养上清液与检测抗体混合,得到混合液。
在可选的实施方式中:
(a)当信号指示剂是荧光基团时,将所述CAR-T细胞的培养上清液与表达所述分泌抗体对应抗原的工程细胞共孵育,之后再与具有荧光基团标记的检测抗体共孵育,得到混合液。
(b)在可选的实施方式中,当信号指示剂是催化酶时,将所述CAR-T细胞的培养上清液与分泌抗体的对应抗原混合,之后再用催化酶标记的检测抗体混合,得到混合液。
在可选的实施方式中,上述方法还包括:
检测所述混合液的信号强度值;
在可选的实施方式中,在检测信号强度值中,如果是上述(a)情形时,检测是的荧光强度值,如果是上述(b)情形时,检测的是吸光度值。
在可选的实施方式中,将检测得到的所述信号强度值代入标准曲线方程并计算得到所述上清液中的由所述CAR-T细胞分泌的所述目标抗体的含量。
在可选的实施方式中,上述方法还包括:上述标准曲线方程是根据不同浓度的目标抗体标准品及其对应的信号强度值建立得到。
第三方面,本发明实施例提供一种细胞药物,其含有前述实施方式任一项所述CAR-T细胞。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为C端融合FLAG标签的分泌型PD-1单链抗体分子模式图;
图2为PD-1 scFv-FLAG标准品SDS-PAGE电泳图;
图3为CHO-PD-1工程细胞株表面抗原PD-1的表达检测;
图4为构建PD-1抗体与CAR19联合表达的慢病毒载体及其对照载体;
图5为慢病毒侵染72h后T细胞的阳性率;
图6为PD-1 scFv-FLAG标准品与CHO-PD-1结合后的标准曲线;
图7为不同效靶比上清中CART的分泌PD-1抗体。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
1.构建PCDNA3.4-PD-1 scFv FLAG表达载体。
基因合成PD-1scFv FLAG核苷酸片段,其结构如图1所示,用限制性内切酶将基因片段连接至PCDNA3.4表达载体中,得到PCDNA3.4-PD-1 scFv FLAG载体,通过基因测序的方式验证载体构建的正确性。
2.转染Expi293表达并纯化PD-1scFv FLAG蛋白。
按照2×106cells/ML密度接种Expi293细胞,接种后24小时将细胞的浓度调整为2.9×106cells/ML,用PEI转染的方法将PCDNA3.4-PD-1scFv FLAG转染至Expi293中,收集转然后7天的上清液,先1000rpm 4℃5min弃去细胞碎片,然后再12000rpm 4℃5min弃去杂质,0.22微米的滤膜进行除菌过滤,过滤后的PD-1 scFv FLAG上清用MMC ImpRes进行捕获层析,之后再经分子筛Surperdex75(GE)再次进行层析,SDS胶分析纯化后的蛋白条分子量大小及其纯度。结果见图2,有单一条带,纯化的PD-1 scFv FLAG蛋白分子量大小约为27KDa,符合预期。
3.构建慢病毒载体PCDHF-PD-1,包装浓缩慢病毒,侵染CHO细胞生产筛选CHO-PD-1单克隆细胞株。
按照NCBI交代的人PD-1核苷酸序列进行基因合成,合成后的基因经过双酶切连接至慢病毒载体PCDHF中,包装慢病毒,25000rpm 4℃2h超速离心浓缩慢病毒,弃上清,加入适量DMEM/F12培养基重悬病毒,置于-80℃冰箱备用。将CHO细胞按照2x105 cells/ML的密度接种于24孔板中,设立对照组,每孔1ML按照MOI=10加入浓缩后的PD-1慢病毒,同时按照8μg/ML的量加入助转剂Polyberen,24h后换成新鲜的DMEM/F12培养基,72h后使用APC Anti-hPD-1抗体流式检测侵染效率,有限稀释法筛选PD-1表达阳性的CHO单克隆细胞株。
结果见图3,以未经染色的CHO细胞作为阴性对照,用APC-Anti hPD-1荧光抗体对CHO细胞与CHO-PD-1单克隆细胞株染色后流式分析发现,所筛选的CHO-PD-1单克隆细胞株PD-1表达率为99.97%。
4.制备分泌PD-1scFv FLAG的CD19 CAR-T细胞
(1)构建PCDHF-CD19 CAR-P2A-PD-1scFv FLAG慢病毒的载体及其对照载体,PCDHF-CD19 CAR-P2A-PD-1scFv FLAG慢病毒载体(图4中2#,其嵌合抗原受体中的抗原结合结构域选自FMC63 scFV)及其对照载体(图4中1#、3#、4#)的基因结构元件如图4所示。
其中,部分基因和对应蛋白的序列如下:
人hIgG信号肽(hIgG SP)核苷酸序列(SEQ ID NO.1):
ATGGGATGGTCTTGTATTATTCTGTTTCTGGTGGCAACTGCTACTGGGGTGCATAGT;
人hIgG信号肽氨基酸序列(SEQ ID NO.2):
MGWSCIILFLVATATGVHS;
抗人PD-1单克隆抗体(PD-1scFV)重链核苷酸序列(SEQ ID NO.3):
CAGGTGCAGCTGGTGGAGAGCGGCGGCGGAGTGGTGCAGCCAGGCAGATCTCTGAGACTGGATTGCAAGGCCAGCGGCATCACCTTCAGCAATTCCGGCATGCACTGGGTGCGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTATGACGGCTCTAAGCGGTACTATGCCGACTCTGTGAAGGGCAGATTCACCATCTCCAGGGACAACTCCAAGAATACCCTGTTCCTGCAGATGAACAGCCTGAGGGCCGAGGATACCGCCGTGTACTATTGCGCCACCAACGACGATTACTGGGGCCAGGGCACACTGGTGACCGTGTCCAGC;
抗人PD-1单克隆抗体(PD-1scFV)重链氨基酸序列(SEQ ID NO.4):
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS;
Linker核苷酸序列(SEQ ID NO.5):
ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct;
Linker氨基酸序列(SEQ ID NO.6):
GGGGSGGGGSGGGGS;
抗人PD-1单克隆抗体(PD1 scFV)轻链核苷酸序列(SEQ ID NO.7):
GAGATCGTGCTGACCCAGTCTCCAGCCACACTGAGCCTGTCTCCTGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGTCCGTGAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCAGGCCAGGCCCCAAGACTGCTGATCTACGACGCCTCCAACAGAGCCACCGGCATCCCAGCCAGATTTTCTGGCTCCGGCTCTGGCACCGACTTCACACTGACCATCAGCTCTCTGGAGCCAGAGGATTTCGCCGTGTATTACTGCCAGCAGAGCTCTAACTGGCCAAGAACATTCGGGCAGGGGACCAAGGTGGAAATCAAGAGG;
抗人PD-1单克隆抗体(PD1 scFV)轻链氨基酸序列(SEQ ID NO.8):
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKR;
FLAG标签核苷酸序列(SEQ ID NO.9):
GACTACAAGGACGACGACGACAAA;
FLAG标签氨基酸序列(SEQ ID NO.10):
DYKDDDDK。
(2)取20ML血,Ficall梯度离心分离PBMC,用Stemcell公司T细胞阴选试剂盒(货号:19051)分离T细胞,分离后的T细胞用含5%人AB血清及300单位/ml的IL-2X-VIVO 15的培养基重悬T细胞至1×106cells/ML,用含1%FBS X-VIVO 15清洗beads,按照磁珠:T细胞=2:1比例加入预先清洗过的磁珠(Cat#40203D,10ML,Life technology),2-3days后用新鲜的培养基重悬T细胞至3-5×106cells/ML,按照MOI=10值加入慢病毒载体步骤(1)制得的PCDHF-CD19 CAR-P2A-PD-1scFv FLAG慢病毒载体或对照载体,同时加入8μg/ML的Polybrene,4-6hours之后,补加培养基稀释细胞至1×106cells/ML,次日更换新鲜培养基,使细胞浓度维持在0.2-0.3×106PBMC/ML,之后每隔2-3天更换一次培养基,病毒侵染完72小时后流式分析细胞阳性率,见图5,各组侵染效率分别为GFP:96.15%,CAR19&PD-1scFv:55.05%,PD-1scFv&GFP:27.4%,GFP:72.6%。
得到分泌PD-1 scFv FLAG的CD19 CAR-T细胞,以及对照组细胞。
实施例2
定量检测分泌PD-1 scFv FLAG的CD19 CAR-T细胞在上清中的PD-1 scFv抗体含量。
(1)将靶细胞按照1×105个/100μl接种于96孔板中,然后分泌PD-1 scFv FLAG的CD19 CAR-T及其对照组细胞按照效靶比10:1、5:1、2.5:1、1.25:1和1:0与RAJI-PD-L1细胞株共培养,收集杀伤4小时后的分泌上清,留作检测PD-1scFv FLAG的样品。
(2)将CHO-PD-1按照1×107cells/ML的密度用PBS重悬,重悬后按照50μl/孔加入到96孔板中,之后将纯化后的PD-1scFv FLAG蛋白标准品按照100μg/ml为起始浓度2倍连续稀释,稀释后按照50μl/孔加入到96孔板中,使蛋白的起始浓度变为50μg/ml。
(3)同时将50微升步骤(1)收集的分泌上清加入对应的孔中,室温染色20分钟,1500rpm 25℃离心5分钟,弃上清,1ML PBS重复清洗三遍,之后按照100μl/孔加入BV421荧光标记的抗FLAG抗体工作液,室温染色20分钟,1500rpm 25℃离心5分钟,弃上清,重复清洗三遍,用流式分析PD-1scFv FLAG蛋白标准品组的PB450平均荧光强度来建立标准曲线,见图6,通过各组分泌上清样品的平均荧光强度来计算PD-1 scFv FLAG的分泌量。
结果见图7,以Nalm6为靶细胞,分泌PD-1 scFv FLAG抗体的GFP-T细胞株在效靶比为10:1、5:1、2.5:1和1.25:1的情况下,PD-1scFv FLAG的分泌量分别为11.9μg/μl、7.02μg/μl、3.9μg/μl和1.9μg/μl;分泌PD-1 scFv FLAG抗体的CART19组在效靶比为10:1、5:1、2.5:1和1.25:1的情况下,PD-1 scFv FLAG的分泌量分别为0.0925μg/μl、0.0785μg/μl、0.0166μg/μl和0.0307μg/μl。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 深圳市菲鹏生物治疗股份有限公司
<120> CAR-T细胞和检测方法
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 57
<212> DNA
<213> 人工序列
<400> 1
atgggatggt cttgtattat tctgtttctg gtggcaactg ctactggggt gcatagt 57
<210> 2
<211> 19
<212> PRT
<213> 人工序列
<400> 2
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser
<210> 3
<211> 339
<212> DNA
<213> 人工序列
<400> 3
caggtgcagc tggtggagag cggcggcgga gtggtgcagc caggcagatc tctgagactg 60
gattgcaagg ccagcggcat caccttcagc aattccggca tgcactgggt gcggcaggcc 120
cccggcaagg gcctggagtg ggtggccgtg atctggtatg acggctctaa gcggtactat 180
gccgactctg tgaagggcag attcaccatc tccagggaca actccaagaa taccctgttc 240
ctgcagatga acagcctgag ggccgaggat accgccgtgt actattgcgc caccaacgac 300
gattactggg gccagggcac actggtgacc gtgtccagc 339
<210> 4
<211> 113
<212> PRT
<213> 人工序列
<400> 4
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser
<210> 5
<211> 45
<212> DNA
<213> 人工序列
<400> 5
ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45
<210> 6
<211> 15
<212> PRT
<213> 人工序列
<400> 6
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 7
<211> 324
<212> DNA
<213> 人工序列
<400> 7
gagatcgtgc tgacccagtc tccagccaca ctgagcctgt ctcctggcga gagagccacc 60
ctgtcttgta gggccagcca gtccgtgagc tcttacctgg cctggtatca gcagaagcca 120
ggccaggccc caagactgct gatctacgac gcctccaaca gagccaccgg catcccagcc 180
agattttctg gctccggctc tggcaccgac ttcacactga ccatcagctc tctggagcca 240
gaggatttcg ccgtgtatta ctgccagcag agctctaact ggccaagaac attcgggcag 300
gggaccaagg tggaaatcaa gagg 324
<210> 8
<211> 108
<212> PRT
<213> 人工序列
<400> 8
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 9
<211> 24
<212> DNA
<213> 人工序列
<400> 9
gactacaagg acgacgacga caaa 24
<210> 10
<211> 8
<212> PRT
<213> 人工序列
<400> 10
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
Claims (10)
1.一种CAR-T细胞,其特征在于,所述CAR-T细胞能够表达嵌合抗原受体和分泌目标抗体,所述目标抗体的N端或C端融合有检测标签,且所述检测标签能够被检测抗体特异性识别和结合,以对所述目标抗体进行定量检测。
2.根据权利要求1所述的CAR-T细胞,其特征在于,
所述检测标签为多肽;
优选地,所述检测标签选自FLAG、HA、HA1、c-Myc、Poly-His、Poly-Arg、Strep-TagII、AU1、EE、T7、4A6、ε、B、gE以及Ty1中的任意一种。
3.根据权利要求1或2所述的CAR-T细胞,其特征在于,所述目标抗体为能够封闭PD-1/PD-L1信号通路的抗体;
优选地,所述目标抗体为PD-1抗体或其功能性片段;
优选地,所述目标抗体为PD-L1抗体的功能性片段;
优选地,所述功能性片段的结构形式选自F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种;
优选地,所述目标抗体选自PD-1抗体的scFv,或者所述目标抗体选自PD-L1抗体的scFv;
优选地,所述目标抗体的轻链可变区氨基酸序列如SEQ ID NO.8所示,重链可变区氨基酸序列如SEQ ID NO.4所示;
优选地,所述目标抗体的位于轻链可变区与重链之间可变区的铰接区序列的氨基酸序列如SEQ ID NO.6所示。
4.根据权利要求3所述的CAR-T细胞,其特征在于,所述目标抗体的C端融合有所述检测标签;
优选地,所述目标抗体通过如下信号肽实现分泌表达:hIgG SP;
优选地,所述检测抗体标记有信号指示剂;
优选地,所述信号指示剂为荧光基团或者用于催化底物显色的催化酶;
优选地,所述荧光基团选自BV421;
优选地,所述催化酶选自辣根过氧化物酶或碱性磷酸酶。
5.根据权利要求1或2所述的CAR-T细胞,其特征在于,所述嵌合抗原受体上的抗原结合结构域所识别和靶向的靶点为肿瘤;
优选地,所述肿瘤为血液肿瘤或实体瘤;
优选地,所述血液肿瘤选自B细胞淋巴瘤、慢性淋巴细胞白血病、急性髓细胞白血病、急性淋巴白血病和多发性骨髓瘤中的任意一种;
优选地,所述实体瘤选自胃癌、肝癌、肺癌、小肠癌、前列腺癌、宫颈癌、结直肠癌、大肠癌、乳腺癌、卵巢癌、淋巴癌、鼻咽癌、骨癌、肾上腺肿瘤、脑胶质瘤、非小细胞肺癌和膀胱肿瘤中的任意一种;
优选地,所述靶点为B细胞淋巴瘤;
优选地,所述靶点为B细胞淋巴瘤细胞膜上的CD19抗原;
优选地,所述抗原结合结构域选自CD19抗体的scFv;
优选地,所述CD19抗体为FMC63。
6.根据权利要求5所述的CAR-T细胞,其特征在于,所述嵌合抗原受体上的跨膜结构域选自如下蛋白分子中的至少一种的跨膜结构域:CD8、CD3ε、CD4、CD9、CD45、CD37、CD5、CD28、CD33、CD16、CD22、CD154、CD134和CD137;
优选地,所述跨膜结构域为CD8跨膜结构域;
优选地,所述嵌合抗原受体上的共刺激信号传导区包含如下共刺激分子中至少一种的细胞内结构域:4-1BB、CD3ζ、CD3δ、CD134、CD79a、CD137、ICD3ε、CD5、CD79b、CD5、CD66d、OX40、CD22、CD2、CD3γ、CD154和CD28;
优选地,所述共刺激信号传导区包含CD3ζ和4-1BB的胞内结构域。
7.一种定量检测如权利要求1-6任一项所述CAR-T细胞所分泌的抗体的方法,其特征在于,其包括:将所述CAR-T细胞的培养上清液与检测抗体混合,得到混合液。
8.根据权利要求7所述的方法,其特征在于,所述方法还包括:
检测所述混合液的信号强度值;
优选地,将检测得到的所述信号强度值代入标准曲线方程并计算得到所述上清液中的由所述CAR-T细胞分泌的所述目标抗体的含量。
9.根据权利要求8所述的方法,其特征在于,所述方法还包括:所述标准曲线方程是根据不同浓度的目标抗体标准品及其对应的信号强度值建立得到。
10.一种细胞药物,其特征在于,其含有权利要求1-6任一项所述CAR-T细胞。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911181105.0A CN112852740A (zh) | 2019-11-27 | 2019-11-27 | Car-t细胞和检测方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911181105.0A CN112852740A (zh) | 2019-11-27 | 2019-11-27 | Car-t细胞和检测方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112852740A true CN112852740A (zh) | 2021-05-28 |
Family
ID=75985568
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911181105.0A Pending CN112852740A (zh) | 2019-11-27 | 2019-11-27 | Car-t细胞和检测方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112852740A (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105793287A (zh) * | 2013-09-20 | 2016-07-20 | 百时美施贵宝公司 | 抗lag-3抗体与抗pd-1抗体联合治疗肿瘤 |
US20180127502A1 (en) * | 2015-06-23 | 2018-05-10 | Memorial Sloan-Kettering Cancer Center | Novel pd-1 immune modulating agents |
CN109666651A (zh) * | 2019-01-25 | 2019-04-23 | 苏州茂行生物科技有限公司 | 一种分泌型靶向Lewis-Y的CAR-T细胞及其制备方法和应用 |
CN109735500A (zh) * | 2019-01-25 | 2019-05-10 | 苏州茂行生物科技有限公司 | 一种分泌型靶向cd133的car-t细胞及其制备方法和应用 |
-
2019
- 2019-11-27 CN CN201911181105.0A patent/CN112852740A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105793287A (zh) * | 2013-09-20 | 2016-07-20 | 百时美施贵宝公司 | 抗lag-3抗体与抗pd-1抗体联合治疗肿瘤 |
US20180127502A1 (en) * | 2015-06-23 | 2018-05-10 | Memorial Sloan-Kettering Cancer Center | Novel pd-1 immune modulating agents |
CN109666651A (zh) * | 2019-01-25 | 2019-04-23 | 苏州茂行生物科技有限公司 | 一种分泌型靶向Lewis-Y的CAR-T细胞及其制备方法和应用 |
CN109735500A (zh) * | 2019-01-25 | 2019-05-10 | 苏州茂行生物科技有限公司 | 一种分泌型靶向cd133的car-t细胞及其制备方法和应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108047332B (zh) | 以cd19为靶点的特异性抗体、car-nk细胞及其制备和应用 | |
CN107868791B (zh) | 一种加强型Slit2 CAR-T和CAR-NK细胞制备方法和应用 | |
CN109734813A (zh) | 一种嵌合抗原受体及其应用 | |
AU2017384900A1 (en) | Chimeric antigen receptor and natural killer cells expressing same | |
CN109929039A (zh) | 基于cd276抗体的嵌合抗原受体、慢病毒表达载体及其应用 | |
CN112574311B (zh) | 双mic结合活性的抗体及其应用 | |
CN111748044A (zh) | Cd19和pd-l1双靶点嵌合抗原受体及其应用 | |
CN111234032B (zh) | 用于治疗卵巢癌的双靶点嵌合抗原受体及制备方法与应用 | |
CN105949323A (zh) | 一种EpCAM特异性嵌合抗原受体及其编码基因、应用 | |
CN107298715B (zh) | Slit2D2-嵌合抗原受体及其应用 | |
CN108047333B (zh) | 以cd33为靶点的特异性抗体、car-nk细胞及其制备和应用 | |
CN111848818A (zh) | 一种增强型免疫细胞及其应用 | |
CN108424461A (zh) | Cd47-car-t细胞 | |
CN113717288A (zh) | 逆转肿瘤微环境的融合蛋白及其应用 | |
CN114539411B (zh) | 一种ror1抗体或其抗原结合片段 | |
CN114395047B (zh) | 双特异性抗体及其应用 | |
KR20210143096A (ko) | Cd22에 특이적인 항체 및 이의 용도 | |
CN111875711A (zh) | 一种增强型免疫细胞及其应用 | |
CN101293924A (zh) | 骨桥蛋白的功能表位、与其特异性结合的单克隆抗体及其在制备抗肿瘤转移药物中的用途 | |
CN108276498A (zh) | 一种包含截短cd20分子的嵌合抗原受体、慢病毒载体及应用 | |
CN108285486A (zh) | 以cd20为靶点的特异性抗体、car-nk细胞及其制备和应用 | |
CN113248622B (zh) | 一种靶向cll1和nkg2d配体的双靶点嵌合抗原受体及其应用 | |
CN110655581A (zh) | 一种抗癌胚抗原的抗体及其制备方法和用途 | |
CN113248621B (zh) | Cll1和cd33双靶点嵌合抗原受体及其应用 | |
CN111808200B (zh) | Cd19和cd22双靶点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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210528 |