CN1294148C - 环状单链三特异抗体 - Google Patents
环状单链三特异抗体 Download PDFInfo
- Publication number
- CN1294148C CN1294148C CNB011105542A CN01110554A CN1294148C CN 1294148 C CN1294148 C CN 1294148C CN B011105542 A CNB011105542 A CN B011105542A CN 01110554 A CN01110554 A CN 01110554A CN 1294148 C CN1294148 C CN 1294148C
- Authority
- CN
- China
- Prior art keywords
- antibody
- stranded
- cyctic
- fragment
- people
- 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.)
- Expired - Fee Related
Links
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 22
- 108010003723 Single-Domain Antibodies Proteins 0.000 claims abstract description 21
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims abstract description 18
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims abstract description 18
- 239000013604 expression vector Substances 0.000 claims abstract description 16
- 230000000259 anti-tumor effect Effects 0.000 claims abstract description 10
- 239000012634 fragment Substances 0.000 claims description 36
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 21
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 19
- 206010033128 Ovarian cancer Diseases 0.000 claims description 12
- 230000008859 change Effects 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 6
- 230000000968 intestinal effect Effects 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 3
- 125000004122 cyclic group Chemical group 0.000 abstract description 2
- 239000013612 plasmid Substances 0.000 description 32
- 230000029087 digestion Effects 0.000 description 25
- 108020004414 DNA Proteins 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 23
- 108090000623 proteins and genes Proteins 0.000 description 20
- 210000001744 T-lymphocyte Anatomy 0.000 description 19
- 230000000694 effects Effects 0.000 description 17
- 238000000034 method Methods 0.000 description 16
- 239000000427 antigen Substances 0.000 description 15
- 108091007433 antigens Proteins 0.000 description 15
- 102000036639 antigens Human genes 0.000 description 15
- 238000002156 mixing Methods 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 11
- 238000000246 agarose gel electrophoresis Methods 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 238000011160 research Methods 0.000 description 10
- 239000000203 mixture Substances 0.000 description 9
- 102000003960 Ligases Human genes 0.000 description 8
- 108090000364 Ligases Proteins 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 239000003292 glue Substances 0.000 description 8
- 125000000539 amino acid group Chemical group 0.000 description 7
- 230000001186 cumulative effect Effects 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 6
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 230000000139 costimulatory effect Effects 0.000 description 6
- 238000009169 immunotherapy Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 238000001556 precipitation Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 239000013599 cloning vector Substances 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 230000009977 dual effect Effects 0.000 description 5
- 239000013613 expression plasmid Substances 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 206010010254 Concussion Diseases 0.000 description 4
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 4
- 206010025323 Lymphomas Diseases 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 230000000118 anti-neoplastic effect Effects 0.000 description 4
- 230000009514 concussion Effects 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000010353 genetic engineering Methods 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 3
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 3
- 229960000583 acetic acid Drugs 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 239000012362 glacial acetic acid Substances 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 230000037452 priming Effects 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 102100024165 G1/S-specific cyclin-D1 Human genes 0.000 description 2
- 101000980756 Homo sapiens G1/S-specific cyclin-D1 Proteins 0.000 description 2
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 2
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 2
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 102000004856 Lectins Human genes 0.000 description 2
- 108090001090 Lectins Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000011579 SCID mouse model Methods 0.000 description 2
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 230000004940 costimulation Effects 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000002523 lectin Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000002525 ultrasonication Methods 0.000 description 2
- WZZBNLYBHUDSHF-DHLKQENFSA-N 1-[(3s,4s)-4-[8-(2-chloro-4-pyrimidin-2-yloxyphenyl)-7-fluoro-2-methylimidazo[4,5-c]quinolin-1-yl]-3-fluoropiperidin-1-yl]-2-hydroxyethanone Chemical compound CC1=NC2=CN=C3C=C(F)C(C=4C(=CC(OC=5N=CC=CN=5)=CC=4)Cl)=CC3=C2N1[C@H]1CCN(C(=O)CO)C[C@@H]1F WZZBNLYBHUDSHF-DHLKQENFSA-N 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000001382 Experimental Melanoma Diseases 0.000 description 1
- 102000005698 Frizzled receptors Human genes 0.000 description 1
- 108010045438 Frizzled receptors Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 102000012850 Patched-1 Receptor Human genes 0.000 description 1
- 108010065129 Patched-1 Receptor Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 108010083312 T-Cell Antigen Receptor-CD3 Complex Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 230000007503 antigenic stimulation Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 238000005734 heterodimerization reaction Methods 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000000247 postprecipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 102220080600 rs797046116 Human genes 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000005760 tumorsuppression Effects 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
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K16/2809—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 the 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
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明提供了一种抗人肿瘤的环状单链三特异抗体,它是由抗肿瘤单链抗体,改形抗人CD3单链抗体,改形抗人CD28单域抗体由连接肽依次连接而成,并且通过两端引入人抗体分子铰链区片段连接成环状分子。本发明还提供了编码这种抗体的DNA序列,含有这种DNA序列的表达载体和含有这种表达载体的宿主细胞。
Description
本发明涉及一种基因工程环状单链三特异抗体,编码这种抗体的DNA序列,含有所述序列的表达载体和含有这种表达载体的宿主细胞。
三特异抗体的构建基于在同一个分子上引进三个不同的抗原结合部位。构建时所选用的抗体基因不同,从而产生不同的生物学功能。已报道的三特异抗体的构建主要采用化学嵌合方法,杂交瘤细胞进一步杂交或通过融合基因表达而制得;其中各抗体大都采用抗体Fab片段或在三个抗体中只有一个抗体采用单链抗体小片段(Fay TN,et al.,1988;Tutt A,et al.,1991;Jung G,et al.,1991;Schott ME,et al.,1993;French RR,1998;Somasundaram C,et al,1999;Schoonjans R,et al.,2000a;Schoonjans R,etal.,2000b;Wong WM,et al.,2000)。
本发明的环状单链三特异抗体的构建是基于在肿瘤免疫治疗中起主要作用的T细胞需要双重信号激活而设计的一种新型工程抗体。在肿瘤的免疫治疗中,以T细胞为主的细胞免疫起主要作用。T细胞的激活需要双重信号,第一信号由TCR-CD3复合体提供,与抗原特异性相关;第二信号为APC表面的多个辅助刺激分子提供的共刺激信号。CD3由5种不同的肽链构成,CD3与TCR呈非共价键结合,形成完整的TCR-CD3复合物,共同参与对抗原刺激的免疫应答。如果只有第一信号而没有共刺激信号刺激可导致T细胞的无能甚至凋亡。共刺激信号没有抗原特异性,亦不受MHC限制,但可介导细胞因子分泌,T细胞增殖及效应功能的发挥。CD28是T细胞最主要的一个共刺激信号接受体,在T细胞共刺激信号受体中如CD2,CD4,CD8等,只有CD28可以阻止诱导T细胞产生无能(Slavik et al.1999)。根据这些特点,可以利用抗CD3抗体和抗CD28抗体分别作为它们的配体而起激活T细胞的作用。随着抗体工程技术的不断发展,尤其是基因工程技术在抗体改造上的应用,使得人们可以根据需要对抗体进行改造以更利于应用。目前,已根据抗体的靶向性,构建了既针对肿瘤细胞又可以激活效应细胞的双特异抗体,双特异单链抗体等。
采用基因工程制备的抗体在肿瘤免疫治疗研究报道中,基于抗肿瘤相关抗原抗体(TAA)与抗CD3抗体或抗CD28抗体构成的双特异抗体(BsAb)或单克隆抗体(McAb)的研究报道较多。早期BsAb用于肿瘤免疫治疗的临床试验为单独连接触发分子TCR-CD3和TAA,发现效果不佳,可出现活化的T细胞克隆无能和凋亡,后来采用IL-2或丝裂原植物凝聚素(Lectin)作为共刺激因子在体外予刺激活化T细胞的方法取得一定的效果。随着双信号识别理论的确立以及CD28分子的深入研究,发现抗CD28 McAb可以同B7家族一样传递共刺激信号,协同抗CD3/TAA触发T细胞的充分活化。Demanet et al.(1996)用抗CD3/Id BsAb加抗CD28McAb对BCL1淋巴瘤的Balb/C小鼠模型多次注射,可使负荷105个细胞的淋巴瘤消退,其疗效较单独使用BsAb提高了20倍,而BsAb的用量仅为单独使用的1/10。Bohlen et al.(1997)等用抗CD3/CD19 BsAb和抗CD28 McAb介导自体T细胞治疗人类慢性B淋巴细胞白血病的SCID小鼠模型,得到较好的肿瘤抑制效果,且具有预防复发的价值。进一步的研究是将抗CD3和抗CD28 BsAb共同使用以增强肿瘤的特异性,如Renner et al.(1994)将抗CD3/CD30和抗CD28/CD30两种BsAb联合使用,治疗人何杰金氏淋巴瘤的SCID小鼠取得良好效果。Mazzoni etal.(1996)将抗CD3×抗FBP(卵巢癌TAA)和抗CD28×抗FBP两种双特异抗体同时使用,体外杀伤实验表明,双重信号能有效的激活CD8+T细胞,对带有FBP的卵巢癌细胞特异地杀伤。Manzke et al.(1999)将CD3×CD19双特异抗体和CD28双价抗体联合使用,在治疗B细胞介导的淋巴细胞癌中,比单独使用CD3×CD19双特异抗体效果明显。BsAb在实体瘤的治疗上也取得明显效果,Grosse-Hovest et al.(1999)发现抗CD3/tumorBsAb在与B16黑瘤细胞共育的肺癌细胞小鼠模型中有明显的治疗作用,抗CD28 BsAb的合用可明显地提高对肿瘤细胞的攻击率,且在静脉注射BsAb治疗过的小鼠中用肿瘤细胞再次攻击,长期存活的数量明显提高,表明BsAb可诱导小鼠产生长期的保护免疫。因此,如果将抗肿瘤相关抗原抗体,抗CD3抗体及抗CD28抗体的基因融合表达,可以在更有效激活效应细胞,提高肿瘤的治愈率的同时,大大简化生产工艺流程,提高生产效率,降低生产成本。然而,如果简单地将这三种抗体串联成线状分子,不利于在体内运输,而且不稳定。为了解决这一问题,本发明用抗体铰链区片段将这一分子环化,构建成环状单链三特异抗体。
鼠源抗体在治疗中也存在许多需要解决的问题,特别是鼠原抗体的异源性引起病人产生HAMA(human anti-mouse immunoglobulin antibodies)反应,加快了抗体的清除,封闭其治疗效果,并引起过敏反应。目前很难制备人源单克隆抗体,因此对鼠源抗体进行人源化改造,是充分发挥鼠源抗体在肿瘤治疗中应用的可选择途径。抗体人源化的分子基础是抗体具有清晰的结构与功能域。这个Y-字型的分子具有两条相同的重链和轻链,每一条链由一个可变区(variable region)和一个或多个恒定区(constant region)组成,可变区主要负责与抗原结合,恒定区主要负责结合效应分子。在每一个可变区内有三个在序列和晶体结构上都高度变化的柔性环区(loop),它们主要负责抗原的识别,被称为互补决定区(complementarity-determining regions,CDRs),而可变区的其余部分相对稳定,由较为刚性的β-折叠(β-sheet)组成,被称为框架区(frameworkregions,FRs)。CDRs与FRs间隔排列而形成“三明治”结构。重链和轻链及两条重链之间均以二硫键相连。抗体的这种结构相对保守,有利于应用蛋白质工程对抗体分子加以改造,以达到保持抗原结合位点特异性和有效引发效应功能的同时,最大程度地降低免疫原活性并发挥抗体的临床治疗作用。第一代人源化抗体是嵌合抗体,由鼠源抗体可变区和人源抗体的恒定区组成。有数据表明,嵌合抗体有效地提高了药物动力学系数并明显降低了免疫源性,一些嵌合抗体已成功地进入临床实验。但经反复用药仍有一半以上的病人产生了抗鼠源可变区抗体。第二代人源化抗体被称作CDR-移植抗体(CDR-grafted antibody)或改形抗体,即将鼠源CDRs移植到人源抗体框架内,这就在保留鼠源抗体的抗原结合特异性基础上,较嵌合抗体进一步人源化。实际上,同一个人源框架可植入不同的鼠源CDRs,而生成多种不同序列的改形抗体。然而,简单将鼠源CDRs移植到人源抗体的FRs中,常常导致人源化抗体的免疫学活性下降甚至消失。因此要考虑CDRs和FRs氨基酸残基之间的相互作用,对维持抗体空间结构的鼠源FRs中个别氨基酸残基必须予以保留。例如:从抗人淋巴细胞表面抗原的鼠源单抗出发构建改形抗体时,仅仅将CDRs进行移植并不能得到与原抗原相结合的活性,用计算机模拟VHCDRs与FRs时发现FR1的Phe27与CDR1密切接触,而人源框架中的相应位置为Ser27。当把Ser27诱变为Phe27后,改形抗体获得了原抗原与抗体的结合活性。实际上,一些改形抗体在进行个别氨基酸残基诱变后,亲和力可以提高三倍。CAMPATH-1H是进入临床的第一个改形抗体,在治疗非何杰金(non-Hodgkin)淋巴瘤和类风湿关节炎中取得了良好效果。类似的还有HuRSV-19,D1.3VHFNS/VK。残基替换的一般策略是,选择与鼠源FRs同源性最高的人源序列作为构建改形抗体的框架,参照已知可变区的晶体结构及所属抗体家族的保守序列,在计算机的辅助下建立分子模型,进行取舍。残基替换在提高亲和力的同时,也增加了异源性,因此,在改形抗体的构建中,应权衡二者,优化组合。在环形三特异抗体的构建中,我们采用了由本实验室构建筛选的改形抗CD3的单链抗体和改形抗CD28VH单域抗体,这为环形三特异抗体降低鼠源性及进一步临床应用奠定了基础。
在环形三特异抗体的构建中,连接不同抗体基因的域间连接肽(Interlinker)的选用十分重要,它关系到所构建的抗体是否成功。本发明中分别采用了人免疫球蛋白IgG的Fc片段,人血清白蛋白HSA的片段以及人免疫球蛋白IgG3’CL的铰链区片段;同时在各个连接肽与抗体基因片段之间加入具有柔韧性的短肽Gly4Ser,这为各抗体在空间的正确折叠提供了条件。Interlinker Fc:小分子抗体在应用中的主要问题是,由于缺少Fc,不能引发效应功能。有研究表明,在人IgG四种亚类中,IgG1引发ADCC和CDC的能力最强。它通过CH2C端的一段序列与Clq结合,引发补体的经典激活途径,其中Glu318,Lys320及Lys322在空间构象上靠近成族,位于Fc分子表面,直接与C1q结合。在对Fc引发效应功能的作用方式的研究中发现,尽管糖基化不影响抗原抗体的结合,但却对Fc引发的ADCC及CDC至关重要。IgG的糖基化位点在CH2的Asn297上。因此,本研究选择人IgG1 CH2中297-322长26aa的片段,包含糖基化位点Asn297。Clq结合位点Glu238,Lys320及Lys322,作为scBsAb载体构建中的一种interlinker,使scBsAb具有类似Fc引发CDC效应的功能。Interlinker HSA:小分子抗体在应用中的另一个问题是,在血清中的半衰期短,易被清除。这些虽有利与免疫诊断和中和毒素,却不利于发挥治疗作用。HAS广泛分布于人体的各个部分,在血液中非常丰富,是最主要的血清蛋白。它主要经肝脏缓解清除,体内半衰期可长达几周。尤其重要的是它几乎没有酶学和免疫学活性,与具有生物学活性的目的蛋白偶联不会引起副作用。因此,HAS可以作为稳定的,惰性的天然载体,在体内传输治疗性分子。有研究表明,与HAS偶联的目的蛋白在动物体内的稳定性增加20-40倍,并主要经肝脏清除,降低对肾脏的毒害作用。成熟的HAS长585个氨基酸,含有17个二硫键,由三个几乎相同的结构域组成。有实验表明,结构域III可以起到整个HSA的作用。因此,本研究选取结构域III中的403-427位上的25个氨基酸残基作为构建中的interlinker。人免疫球蛋白铰链区IgG3’CL hinge:人免疫球蛋白铰链区具有半胱氨酸,其天然空间结构形成时易于通过二硫键将抗体的两条重链连接到一起。人IgG3’CL铰链区具有两个二半胱氨酸共17个氨基酸。该序列的长度以及半胱氨酸的数目对构建三特异抗体较为适宜,本研究采用人IgG3’CL中的铰链区17个氨基酸残基序列,将构建的三特异抗体连接成为环形。[参考文献:1.黄华梁,基因工程抗体,单克隆抗体通讯,1991,7(3):1-4;2.刘喜富,黄华梁,基因工程抗体研究进展,生物工程进展,1994,14(1):54;3.黄华梁,人源化抗体,小分子抗体与肿瘤治疗,单克隆抗体通讯,1993,9(3):19;4.Slavik,J.M.,Hutchcroft,J.E.&Bierer,B.E.(1999):CD28/CTLA-4 andCD80/CD86 families,signaling and function.Immunologic Research.19/1:1-24;5.Demanet C,Brissinck J,Leo O et al.:Bispecific antiboddy-mediatedimmunotherapy of the BCL1 lymphoma:increasd efficacy with multipleinjections and CD28-induced costimulation.Blood 1996;87:4390-4398;6.Bohlen H,Manzke O,Titzer S et al.:Prevention of Epstein-Barr virus-induced human B-cell lymphoma in severe combined immunodeficient micetreated with CD3×CD19 bispecific antibodies,CD28 monospecific antibodies,and autologous T cells.Cancer Res.1997;57:1704-1709;7.Renner C,JangW,Sahin U et al.Science 1994;264:833-835;8.Mazzoni A,Mezzanzanica D,Jung G et al.:CD3-CD28 costimulation as a means to avoiding T cellpreactivation in bispecific monoclonal antibody-based treatment of ovariancarcinoma.Cancer Res.1996;56:5443-5449;9.Manzke,O.,Berthold,F,Huebe,K.et al.(1999):CD3×Cd19 bispecific antibodies and CD28 bivalentantibodies enhance T-cell reactivity against autologous leukemic cells inpediatric B-All bone marrow.Int.J.Cancer,80:715-722;10.Grosse-HovestL,Brandl M,Dohlsten M et al.:Int.J.Cancer 1999;80:138-144;11.Boulianne,G.L.,Hozumi,N.&Shulman,M.J.(1984):Production of functionalchimeric mouse/human antibody.Nature.312,643-646;12.Neuberger,M.S.,Williams,G.T.&Fox,R.O.(1984):Recombinant antibodies possessing novelefiector functions.Nature 312,604-608;13.Jones,P.T.,Dear,P.H.,Foote,J.et al.(1986):Replacing the complementarity-determining regions in a humanantibody with those from a mouse.Nature,321,522-525;14.Riechmann,L.,Clark,M.,Waldmann,H.et al.(1988):Reshaping human antibodies fortherapy.Nature,332,323-327;15.Fay TN,Jacobs I,Teisner B.et al.(1988):Two fetal antigens(FA-1 and FA-2)and endometrial proteins(PP12 and PP14)isolated from amniotic fluid;preliminary observations in fetal and maternaltissues.Eur J Obstet Gynecol Reprod Biol,29(1):73-85;16.Tutt A,StevensonGT,Glenie MJ(1991):Trispecific F(ab′)3 derivatives that use cooperativesignaling via the TCR/CD3 complex and CD2 to activate and redirect restingcytotoxic T cells.J Immunol 147(1):60-9;17.Jung G,Freimann U,VonMarschall Z,et al.(1991):Target cell-induced T cell activation with bi-andtrispecific antibody fragments.Eur J Immunol 21(10):2431-5:18.FrenchRR,(1998):Production of bispecific and trispecific F(ab)2 and F(ab)3 antibodyderivatives.Methods Mol Biol,80:121-134;19.Somasundaram C,Sundarapandiyan K,Keler T,et al.,(1999):Development of a trispecificantibody conjugate that directs two distinct tumor-associated antigens to CD64on myeloid efiector cells.Hum Antibodies,9(1):47-54;20.Schoonjans R,Willems A,Schoonooghe S,et al.(2000a):Fab chains As an efficientheterodimerization scaffold for the production of recombinant bispecific andtrispecific antibody derivatives.J Immunol,165(12):7050-7;21.Schoonjans R,Willems A,Grooten J,et al.,(2000b):Efficient heterodimerization ofrecombinant bi-and trispecific antibodies.Bioseparation,9(3):179-83;22.Wong WM,Vakis SA,Ayre KR,et al.,(2000):Rheumatoid arthritis T cellsproduce Th1 cytokines in response to stimulation with a novel trispecificantibody directed against CD2,CD3,and CD28.Scand JRheumatol,29(5):282-7;23.Schott ME,Frazier KA,Pollock DK,et al.,(1993):Preparation,characterization,and in vivo biodistribution properties ofsynthetically cross-linked multivalent antitumor antibody fragments.Bioconjug Chem,4(2):153-65]。
卵巢癌发病率居妇科恶性肿瘤的第二位。由于起病隐匿,临床发现多属晚期,且术后易复发,五年存活率仅为30%。敏感的早期诊断和术后残留病灶的尽早清除,是改善预后的重要环节。因此,该环形三特异抗体在卵巢癌的免疫治疗上的应用有着广阔前景。
本发明的目的是提供一种用基因工程技术构建及表达的具有独特设计,低毒,高效,生产工艺简单等特点的,导向治疗肿瘤的生物制剂---抗人肿瘤×改形抗人CD3×改形抗人CD28VH环状单链三特异抗体。
本发明的另一个目的是提供一种用于构建通用的环状单链三特异抗体的表达载体。
本发明的又一个目的是提供一种含有用于构建通用的环状单链三特异抗体的表达载体的宿主细胞。
本发明的又一个目的是提供一种编码所述的环状单链三特异抗体的核苷酸序列。
抗体分子具有两条相同的重链和轻链,每一条链由一个可变区(variable region)和一个或多个恒定区(constant region)组成,可变区主要负责与抗原结合,恒定区主要负责结合效应分子。在每一个可变区内有三个在序列和晶体结构上都高度变化的柔性环区(loop),它们主要负责抗原的识别,被称为互补决定区(complementarity-determiningregions,CDRs),而可变区的其余部分相对稳定,由较为刚性的β-折叠(β-sheet)组成,支撑着,被称为框架区(framework regions,FRs),CDRs与FRs间隔排列而形成“三明治”结构。在本发明中,所使用的一些术语具有如下的含义:
“Fab抗体”是指由Fd段(重链VH+CH1构成)和完整的轻链组成,二者通过一个链间二硫键连接,形成异二聚体,它是完整抗体分子的三分之一,仅有一个抗原结合位点。
“单链抗体(scFv)”是用基因工程构建的一种抗体片段,由抗体重链可变区(VH)和轻链可变区(VL)通过一连接肽连接而成的重组蛋白,约为完整抗体分子的六分之一。
“单域抗体”是由抗体重链可变区(VH)或轻链可变区(VL)构成,这种抗体只有一个结构域构成,故称为单域抗体,它是完整抗体分子的十二分之一。
“最小识别单位(Minimal recognizing unit,MRU)”是由单个CDR构成,约为完整抗体分子的七十分之一或八十分之一。
“改形抗体(Reshaping antibody)”也叫做CDR移植抗体(CDR-grafted antibody)。用基因合成或定点突变的方法将鼠源CDRs置换人源抗体中的CDRs,从而保留鼠源抗体的抗原结合特异性。但在构建时要考虑人源FRs中某些氨基酸残基会影响鼠源CDRs构成的抗原结合部位的构象,因此需对FRs中个别氨基酸残基进行突变,才能获得既具有高亲和力又最大程度上达到人源化的抗体。
本发明提供了一种抗人肿瘤的环状单链三特异抗体,它是由抗肿瘤的Fab、单域抗体或单链抗体,改形抗人CD3的Fab、单域抗体或单链抗体以及改形抗人CD28的Fab、单域抗体或单链抗体连接而成的。
在本发明的环状单链三特异抗体中,抗肿瘤的Fab、单域抗体或单链抗体可以是抗卵巢癌的Fab、单域抗体或单链抗体。
本发明的环状单链三特异抗体最好是由抗肿瘤的单链抗体,改形抗人CD3的单链抗体,改形抗人CD28的单域抗体连接而成的。
在本发明的环状单链三特异抗体中,所述的改形抗人CD28的单域抗体最好是VH,它最好具有如下两种氨基酸序列中的一种:
Q V Q L Q E S G P G L V K P S Q T
L S L T C T V S G F S L S D Y G
V H W V R Q P P G K G L E W L G V
I W G G G T N Y N S A L M S R R V
T S S D D T S K N Q F S L K L S
S V D T A V Y Y C A R S Y Y Y S M
D Y W G Q G T L V T V S S
和
Q V Q L Q E S G P G L V K P S Q T
L S L T C T V S G F S L S D Y G
V H W V R Q P P G K G L E W L G V
I W A G G G T N Y N S A L M S R R
V T S S D D T S K N Q F S L K L
S L S S V D T A V Y Y C A R D K G
Y S Y Y Y S M D Y W G Q G T L V T
V S S
在本发明的环状单链三特异抗体中,抗肿瘤的Fab、单域抗体或单链抗体,改形抗人CD3的Fab、单域抗体或单链抗体以及改形抗人CD28的Fab、单域抗体或单链抗体之间最好有一种连接肽。所述的连接肽可以具有如下所示的氨基酸序列中的一种:
(1)pelb
M K Y L L P T A A A G L L L L A A Q P A
1 ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCC
TACTTTATGGATAACGGATGCCGTCGGCGACCTAACAATAATGAGCGACGGGTTGGTCGG
M A Q V K L
61 ATGGCCCAGGTGAAACTG
TACCGGGGTCCACTTTGAC
(2)Gly4Ser
G G G G S
1 GGTGGTGGTGGTTCT
CCACCACCACCAAGA
(3)(Gly4Ser)3
G G G G S G G G G S G G G G S
1 GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCT
CCACCACCACCAAGACCACCACCACCAAGACCACCACCACCAAGA
(4)HUMAN-IgG-Fc
N S T Y R V V S V L T V L H Q D W L N G
1 AACAGCACGTACCGGGTTGTAAGCGTCCTCACCGTACTGCACCAGGACTGGCTGAATGGC
TTGTCGTGCATGGCCCAACATTCGCAGGAGTGGCATGACGTGGTCCTGACCGACTTACCG
K E Y K C K
61 AAGGAATACAAATGCAAG
TTCCTTATGTTTACGTTC
(5)HSA
F Q N A L L V R Y T K K V P Q V S T P T
1 TTCCAGAATGCGCTGCTGGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACT
AAGGTCTTACGCGACGACCAAGCAATGTGGTTCTTTCATGGGGTTCACAGTTGAGGTTGA
P V E V S
61 CCTGTAGAGGTCTCA
GGACATCTCCAGAGT
(6)C-myc
E Q K L I S E E D L N
1 GAACAAAAACTCATCTCAGAAGAGGATCTGAAT
CTTGTTTTTGAGTAGAGTCTTCTCCTAGACTTA
本发明的环状三特异抗体最好是通过下述域间连接肽连接成环状分子的:
(1)HINGE(反向):HUMAN-IgG3’CL
P C R P C T H T T D G L P T K L E
(2)HINGE(正向):HUMAN-IgG3’CL
E L K T P L G D T T H T C P R C P
本发明还提供了一种编码本发明的环状单链三特异抗体的核苷酸序列。
本发明还提供了一种含有上述序列的表达载体。这种表达载体可以是pTRI。
本发明还提供了一种含有上述载体的宿主细胞。这种宿主细胞可以是大肠杆菌。
本发明的环状单链三特异抗体的构建是基于双特异抗体在肿瘤免疫治疗中起主要作用的T细胞需要双重信号激活而设计的一种新型工程抗体,它将针对肿瘤细胞的抗体基因与激活T细胞的两个主要刺激信号的改形抗体基因融合表达。不同于其它三特异抗体的构建,该结构具有以下特点:
1.该结构为环状。三特异抗体链状分子的两端引入人抗体分子铰链区片段,它们通过形成二硫键而使单链分子变成环状。环状的形成,使分子更稳定,并使同一分子中的不同抗体与其靶向位点有更大的空间结合而相互不影响,而且有利于抗体药物在人体内的运输;
2.三种抗体都是小分子抗体:单链抗体或单域抗体。尤其是共刺激信号CD28抗体为单域抗体。该结构使整个分子不会很大,分子量约为84kDa,有利于肿瘤的治疗;
3.负责激活T细胞的两种抗体,抗人CD3和抗入CD28抗体,都是经过人源化改造的改形抗体(Reshaping antibody),消除了免疫原性;
4.在每种抗体之间,通过特定的域间连接肽(interlinker),使每种抗体不仅能充分折叠形成正确的空间结构,还可引入其它一些生物学功能;
5.三种抗体连接在一条链上成一个分子,使这一分子具有三种不同的功能;
6.该分子中针对特定肿瘤的抗体可容易地改换成针对其他肿瘤或细胞因子的抗体,从而扩大其使用范围;
7.直接用大肠杆菌发酵生产,产物无需经体外修饰,生产工艺简化,使用方便,大大降低生产成本。
附图简要说明
图1.构建及表达抗肿瘤环状单链三特异抗体的流程图。
图2.抗肿瘤环状单链三特异抗体(anti-tumor scFv×anti-CD3 scFv×anti-CD28VH)各基因及其域间连接肽接头基因的连接结构图。
图3.二种改形抗CD28VH单域抗体基因的核苷酸序列及所编码的氨基酸序列。
图4.各种连接肽接头的核苷酸序列及所编码的氨基酸序列。
图5.重叠PCR过程示意图。
图6.抗肿瘤环状单链三特异抗体的通用表达质粒pTRI的物理图谱。
图7.抗卵巢癌环状单链三特异抗体在pTRI表达的SDS-聚丙烯酰胺凝胶电泳结果。
实施例
设计合成适当大小的核苷酸片段,通过重叠PCR扩增得到部分域间连接肽的基因序列。将该序列插入到质粒pUC19中构成新载体pUHM1。从含有抗卵巢癌的双特异抗体质粒中用XhoI,BamHI双酶切得到双特异抗体基因并且将该基因插入到克隆载体pUHM1中,得到载体pUHM2。将含有改形抗CD28单域抗体基因和含有连接肽的基因插入到表达载体pTMF中,构成表达载体pTCH1。然后将pUHM2中含有抗卵巢癌双特异抗体及部分连接肽的序列插入到pTCH1中,构成表达质粒pTRI。用构建好的表达质粒pTRI转化宿主细胞BL21。挑取鉴定后的阳性克隆菌转接到含卡那霉素50μg/ml的LB中,37℃培养至OD550为0.4-0.5时,向培养物中加入终浓度为0.8mmol/L的IPTG,诱导培养4小时。收集表达产物,超声破碎菌体,12000rpm离心10min,上清及沉淀分别进行8%和12%SDS-PAGE电泳。
具体操作流程(见附图1)如下:
一.克隆载体pUMH1的构建:
本克隆载体由质粒pUC19改建而来,将含有5’-HindIII-pelB-HumanIgG3’CL hinge(反向)-Gly4Ser-XhoI-BamHI-Gly4Ser-HSA-Gly4Ser-NdeI-EcoRI-3’的连接肽片段插入到pUC19的HindIII,EcoRI双酶切位点中而构成。
设计合成6个大小不同的核苷酸片段,
P1:5’-CCCAAgCTTATgAAATACCTATTgCCTACggC-3’32nts
P2:5’-GCCCAGGTGAAACTGCCGTGCCGTCCATGTACTCACACCACTGACGGTCTGCCGACCAAATTGGAA
GGTGGTGGTGGTTC-3’80nts
P3:5’-CTGCTGGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCCTGTAGAGGTCTCAGGTGG
TGGTGGTTCTCAT-3’81nts
RE1:5’-CCggAATTCCATATgAgAACCACCACCACC-3’30nts
RE2:5’-TTCTTGGTGTAACGAACCAGCAGCGCATTCTGGAAAGAACCACCACCACCGGATCCCTCGAGAGAACC
ACCACCACCTTCC-3’81nts
RE3:5’-GGCACGGCAGTTTCACCTGGGCCATGGCTGGTTGGGCAGCGAGTAATAACAATCCAGCGGCTGCCGTA
GGCAATAGGTATT-3’81nts
采用重叠PCR将6个片段扩增得到285bp的核苷酸片段。
1.重叠PCR扩增连接肽核苷酸序列
重叠PCR采取两步扩增获得全长片段,操作见重叠PCR过程示意图图5。第一步获得P1,P2,RE3,RE2的PCR双链产物M1以及P3,RE1的PCR补平双链片段M2;第二步将所得到的两个片段M1,M2等摩尔加入,通过重叠PCR获得全长。1).M1的获得:将P1,P2,RE3,RE2各个片段溶液4ul(约为10pmol)加入到离心管中,分别加入10×PCR Buffer 3ul,4uldNTPs(2.0mmol/l each),1ul pfu DNA聚合酶(3u/ul),用ddH2O补足到总体积到30ul,加入100ul液体石蜡,混匀,进行30轮PCR循环:94℃变性1min.,55℃退火30秒,72℃延伸40秒。PCR产物进行2.5%琼脂糖凝胶电泳,利用华舜琼脂糖凝胶DNA回收Kit,回收所需的DNA片段。
2).M2的获得:将P3,RE1,两个片段溶液4ul(约为10pmol)加入到离心管中,分别加入10×PCR Buffer 3ul,4ul dNTPs(2.0mmol/l each),1ul pfuDNA聚合酶(3u/ul),用ddH2O补足到总体积到30ul,加入100ul液体石蜡,混匀,进行30轮PCR循环:94℃变性1min.,60℃退火30秒,72℃延伸40秒。PCR产物进行2.5%琼脂糖凝胶电泳,利用华舜琼脂糖凝胶DNA回收Kit,回收所需的DNA片段。3).全长PCR产物的获得:将M1,M2各个片段溶液10ul加入到离心管中,分别加入P1,RE1各4ul,10×PCR Buffer 3ul,4ul dNTPs(2.0mmol/l each),1ul pfu DNA聚合酶(3u/ul),用ddH2O补足到总体积到30ul,加入100ul液体石蜡,混匀,进行30轮PCR循环:94℃变性1min.,55℃退火30秒,72℃延伸40秒。PCR产物进行2.5%琼脂糖凝胶电泳,利用华舜琼脂糖凝胶DNA回收Kit,回收所需的全长DNA片段。
2.PCR扩增产物的限制性内切酶消化及纯化
用HindIII和EcoRI对PCR全长DNA进行消化:取约1ug DNA片段在40ul反应体系中(1×Buffer M,30u的HindIII和30u的EcoRI),37℃酶解4小时,1%琼脂糖凝胶电泳检测消化完全后,在长波长紫外灯下切胶回收所需的条带,参照华舜生物技术有限公司产品kit说明,用柱回收和纯化所需的酶切片段。
3.载体质粒pUC19的提取
采用减法小量提取质粒:挑取pUC19/DH5α单菌落,在5ml LB培养液(含Amp 100μg/ml)中37℃培养过夜,12000rpm离心1分钟收集菌体。沉淀悬于100ul SolutionI(50mmol/l Glucose,10mmol/l EDTA,25mmol/l TirspH8.0),充分混匀。加入200ul新配制的SolutionII(0.2mol/l NaOH,1%SDS),盖紧管盖,快速颠倒4-5次,将离心管置于冰浴中放置3分钟。加入150ul予冷的SolutionIII(3mol/l KAc,pH4.8),混匀后与冰浴放置5分钟。4℃,12000rpm离心10分钟,转移上清至另一离心管中,加入2倍体积的予冷无水乙醇,室温放置10分钟,然后4℃,12000rpm离心20分钟。DNA沉淀用70%乙醇洗一遍,待DNA干燥后,将其溶于100ulddH2O中(含RNase 50ug/ml),37℃消化1小时。加入等体积的酚∶氯仿(1∶1),震荡混匀,室温,12000rpm离心5分钟。转移水相至另一离心管中,加入等体积的氯仿/异戊醇(24∶1)震荡混匀,室温,12000rpm离心5分钟。转移上清至另一离心管中,加入1/10体积的NaAc(3mol/l,pH5.2),2倍体积的无水乙醇,4℃放置1小时。4℃,12000rpm离心20分钟,弃上清,DNA沉淀用70%乙醇洗一遍,干燥后沉淀溶于25ul ddH2O。
4.载体质粒pUC19的限制性内切酶消化及纯化
用HindIII和EcoRI对载体DNA进行消化:取1ug质粒pUC19在40ul反应体系中(1×Buffer M,30u的HindIII和30u的EcoRI),37℃酶解4小时,1%琼脂糖凝胶电泳检测消化完全后,在长波长紫外灯下切胶回收所需的条带,参照华舜生物技术有限公司产品kit说明,用柱回收和纯化所需片段。
5.DNA的连接反应
取回收的双酶切pUC19约40ng,双酶切PCR全长片段20ng,加入2ul 10×T4 DNA连接酶缓冲液,1ul T4 DNA连接酶(约20u),加ddH2O至总体积20ul,混匀,16℃连接过夜。
6.感受态细胞的制备
用氯化钙制备大肠杆菌的感受态细胞:挑取LB平板上的野生Top10单菌落,接种于3ml LB液体培养基中,37℃震荡培养过夜。以1%的接种量转接于30ml的LB液体培养基中,37℃震荡培养至OD值为0.3-0.4时,将菌液置于冰浴中10分钟,4℃,4000rpm离心10分钟,弃去上清,沉淀溶于20ml予冷的0.1mol/l CaCl2中,冰浴放置30分钟,4℃,4000rpm离心10分钟,弃去上清,加入2ml予冷的0.1mol/l CaCl2(含20%甘油)重悬细胞,以200ul分装。未及时使用的各管于-70℃保存。
7.重组DNA的转化,阳性克隆的筛选及测序鉴定
10ul连接混合物加入到200ul的感受态细胞中,混匀,冰浴放置30分钟,42℃水浴90秒,立即置于冰浴中5分钟,涂布于LB(含100ug/mlAmp.)的平板上,表面干燥后,37℃倒置培养过夜。挑取平板上生长的单菌落,采用碱法小量提取质粒,分别用HindIII,EcoRI双酶切;以P1,RE1片段为引物的PCR鉴定插入外源片段的质粒。将鉴定后的质粒送生工生物工程公司测序鉴定,鉴定后测序正确的阳性质粒为pUMH1。
二.克隆载体pUMH2的构建:
载体pUMH2是由质粒pUMH1在XhoI,BamHI位点插入以人IgG1 CH2中Fc片段为interlinker的抗卵巢癌scFV×抗CD3 scFv双特异抗体(BsAb)片段构成。PALM-Fc质粒是本实验室构建的含有抗卵巢癌scFV×抗CD3 scFv双特异抗体(BsAb)基因的中间载体,该双特异抗体片段的顺序为:-XhoI-抗Ovarian carcinomaVH-(Gly4Ser)3-抗OvariancarcinomaVL-Fc-抗CD3VL-(Gly4Ser)3-抗CD3VH-BamHI-。
1.双特异抗体片段的制备及纯化
采用碱法小量提取质粒pALM-Fc,用XhoI和BamHI对载体质粒DNA进行消化:取约1ug质粒pALM-Fc在40ul反应体系中(1×BufferM,30u的XhoI和30u的BamHI),37℃酶解4小时,1%琼脂糖凝胶电泳检测消化完全后,在长波长紫外灯下切胶回收所需的条带,参照华舜生物技术有限公司产品kit说明,用柱回收和纯化所需片段。
2.载体质粒pUMH1的限制性内切酶消化及纯化
用XhoI和BamHI对载体DNA进行消化:取1ug质粒pUMH1在40ul反应体系中(1×Buffer M,30u的XhoI和30u的BamHI),37℃酶解4小时,1%琼脂糖凝胶电泳检测消化完全后,在长波长紫外灯下切胶回收所需的条带,参照华舜生物技术有限公司产品kit说明,用柱回收和纯化所需片段。
3.DNA的连接反应,重组DNA的转化,阳性克隆的筛选
取回收的双酶切pUMH1约40ng,双酶切双特异抗体片段20ng,加入2ul10×T4 DNA连接酶缓冲液,1ul T4 DNA连接酶(约20u),加ddH2O至总体积20ul,混匀,16℃连接过夜。10ul连接混合物加入到200ul的感受态细胞中,混匀,冰浴放置30分钟,42℃水浴90秒,立即置于冰浴中5分钟,涂布于LB(含100ug/ml Amp.)的平板上,表面干燥后,37℃倒置培养过夜。挑取平板上生长的单菌落,采用碱法小量提取质粒,分别采用HindIII,EcoRI双酶切;XhoI,BamHI双酶切鉴定插入的外源片段。鉴定后的阳性质粒为pUMH2。
三.环状单链三特异抗体的构建与表达:
环状单链三特异抗体是由表达载体pTCH1和含有域间连接肽的双特异抗体克隆载体pUMH2提供的片段构建而成。pTCH1是由表达载体pTMF插入:-NdeI-(anti-CD28VH)-(c-myc)-Gly4Ser-Human IgG3’CL(17Aa,正向)-BamHI-片段后构成。
1.表达质粒pTCH1的构建
采用碱法小量提取含有-NdeI-(anti-CD28 VH)-(c-myc)-Gly4Ser-HumanIgG3’CL(17Aa,正向)-BamHI-片段的质粒pUC19,取约1ug该质粒加入到40ul反应体系中(1×Buffer M,30u的BamHI和30u的NdeI),37℃酶解4小时,用于制备-NdeI-(anti-CD28VH)-(c-myc)-Gly4Ser-HumanIgG3’CL(17Aa,正向)-BamHI-片段;在同样条件下用NdeI和BamHI水解表达载体pTMF。1%琼脂糖凝胶电泳检测消化完全后,在长波长紫外灯下切胶回收所需的条带,参照华舜生物技术有限公司产品kit说明,用柱回收和纯化所需片段。
取回收的双酶切pTMF约40ng,双酶切的anti-CD28 VH片段20ng,加入2ul 10×T4 DNA连接酶缓冲液,1ul T4 DNA连接酶(约20u),加ddH2O至总体积20ul,混匀,16℃连接过夜。10ul连接混合物加入到200ul的大肠杆菌BL21感受态细胞中,混匀,冰浴放置30分钟,42℃水浴90秒,立即置于冰浴中5分钟,涂布于LB(含50ug/ml Kna.)的平板上,表面干燥后,37℃倒置培养过夜。挑取平板上生长的单菌落,采用减法小量提取质粒,分别采用质粒大小;HindIII,NdeI双酶切鉴定插入的外源片段。鉴定后的阳性质粒为pTCH1。
2.表达质粒pTCH1的限制性内切酶消化及纯化
用HindIII和NdeI对载体DNA进行消化:取1ug质粒pTCH1在40ul反应体系中(1×Buffer M,30u的HindIII和30u的NdeI),37℃酶解4小时,1%琼脂糖凝胶电泳检测消化完全后,在长波长紫外灯下切胶回收所需的条带,参照华舜生物技术有限公司产品kit说明,用柱回收和纯化所需片段。
3.含有域间连接肽双特异抗体片段的制备及纯化
采用碱法小量提取质粒pUMH2,用HindIII和NdeI对质粒DNA进行消化:取约1ug质粒pUMH2在40ul反应体系中(1×Buffer M,30u的HindIII和30u的NdeI),37℃酶解4小时,1%琼脂糖凝胶电泳检测消化完全后,在长波长紫外灯下切胶回收所需的条带,参照华舜生物技术有限公司产品kit说明,用柱回收和纯化所需片段。
4.DNA的连接反应,重组DNA的转化,阳性克隆的筛选
取回收的双酶切pTCH1约40ng,双酶切的含有域间连接肽双特异抗体片段20ng,加入2ul 10×T4 DNA连接酶缓冲液,1ul T4 DNA连接酶(约20u),加ddH2O至总体积20ul,混匀,16℃连接过夜。10ul连接混合物加入到200ul的感受态细胞中,混匀,冰浴放置30分钟,42℃水浴90秒,立即置于冰浴中5分钟,涂布于LB(含50ug/ml Kna.)的平板上,表面干燥后,37℃倒置培养过夜。挑取平板上生长的单菌落,采用碱法小量提取质粒,分别采用HindIII,NdeI双酶切鉴定插入的外源片段。鉴定后的阳性质粒为pTRI。
5.环状单链三特异抗体的表达
挑取鉴定后含有质粒pTRI的阳性克隆单菌落转接到含卡那霉素50μg/ml的LB中,37℃培养至OD550为0.4-0.5时,向培养物中加入终浓度为0.8mmol/l的IPTG,诱导培养4小时。收集表达产物,超声破碎菌体,12000rpm离心10min,上清及沉淀分别进行12%和8%SDS-PAGE电泳。
聚丙烯酰胺凝胶的配制:
浓缩胶5% | 分离胶12% | 分离胶8% | 封口胶 | |
30%Acr/Bis(29∶1)(ml)1.5M Tris-HCl(pH8.8)(ml)1.0M Tris-HCl(pH6.8)(ml)10%SDS(ml)10%AP(ml)TEMED(ml)ddH2O(ml) | 0.5-0.380.030.030.0032.1 | 6.03.8-0.150.150.0064.9 | 4.03.8-0.150.150.0096.9 | 0.53-0.50.020.020.00120.93 |
样品的处理:适量蛋白样品与等体积的2×SDS凝胶加样缓冲液(100mmol/l Tris-HCl pH6.8,200mmol/l DTT,4%SDS,0.2%溴酚蓝,20%甘油)混合,沸水浴加热5分钟,待上样。
电泳:电泳缓冲液25mmol/l Tris-HCl,pH8.3,0.1%SDS。取适量体积的样品上样,恒压60V,待样品进入分离胶,恒压120V。
染色及脱色:采用考马斯亮兰R250染色(0.25g考马斯亮兰R250溶于100ml甲醇-水-冰乙酸溶液中(90ml甲醇∶水(1∶1,v/v)和10ml冰乙酸),室温染色6-12小时。用甲醇-水-冰乙酸溶液脱色,室温脱色至具有明显条带,照相。
Claims (8)
1、一种抗人肿瘤的环状单链三特异抗体,它是由抗肿瘤单链抗体,改形抗人CD3单链抗体,改形抗人CD28单域抗体由连接肽依次连接而成,并且通过两端引入人抗体分子铰链区片段连接成环状分子,其中连接肽选自如下所示的氨基酸序列中的一种:
(1)M K Y L L P T A A A G L L L L A A Q P A
M A Q V K L
(2)G G G G S
(3)G G G G S G G G G S G G G G S
(4)N S T Y R V V S V L T V L H Q D W L N G
K E Y K C K
F Q N A L L V R Y T K K V P Q V S T P T
(5)P V E V S
(6)E Q K L I S E E D L N;
其中人抗体分子铰链区片段为:
P C R P C T H T T D G L P T K L E
或者E L K T P L G D T T H T C P R C P
2、按照权利要求1所述的环状单链三特异抗体,所述的抗肿瘤单链抗体是抗卵巢癌单链抗体。
3、按照权利要求1所述的环状单链三特异抗体,所述的改形抗人CD28的单域抗体选自如下两种氨基酸序列中的一种:
Q V Q L Q E S G P G L V K P S Q T
L S L T C T V S G F S L S D Y G
V H W V R Q P P G K G L E W L G V
I W G G G T N Y N S A L M S R R V
T S S D D T S K N Q F S L K L S
S V D T A V Y Y C A R S Y Y Y S M
D Y W G Q G T L V T V S S
和
Q V Q L Q E S G P G L V K P S Q T
L S L T C T V S G F S L S D Y G
V H W V R Q P P G K G L E W L G V
I W A G G G T N Y N S A L M S R R
V T S S D D T S K N Q F S L K L
S L S S V D T A V Y Y C A R D K G
Y S Y Y Y S M D Y W G Q G T L V T
V S S
4、一种编码权利要求1至3中任何一项所述的环状单链三特异抗体的核苷酸序列。
5、一种含有权利要求4所述序列的表达载体。
6、按照权利要求5所述的表达载体,它是pTRI。
7、一种含有权利要求5或6中任何一项所述的表达载体的宿主细胞。
8、按照权利要求7所述的宿主细胞,它是大肠杆菌。
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB011105542A CN1294148C (zh) | 2001-04-11 | 2001-04-11 | 环状单链三特异抗体 |
PCT/CN2002/000252 WO2002083738A1 (fr) | 2001-04-11 | 2002-04-10 | Anticorps trispecifique monocatenaire cyclique |
EP02724089A EP1378520A4 (en) | 2001-04-11 | 2002-04-10 | TRISPICIFIC ANTIBODY WITH CYCLIC SINGLE STRAND |
US10/474,345 US20050175606A1 (en) | 2001-04-11 | 2002-04-10 | Cyclic single-chain trispecific antibody |
JP2002581493A JP2005501517A (ja) | 2001-04-11 | 2002-04-10 | 環状一本鎖三重特異性抗体 |
CA002443705A CA2443705A1 (en) | 2001-04-11 | 2002-04-10 | Cyclic single strand trispecific antibody |
RU2003130072/13A RU2355705C2 (ru) | 2001-04-11 | 2002-04-10 | Одноцепочечное цикличное триспецифическое антитело |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB011105542A CN1294148C (zh) | 2001-04-11 | 2001-04-11 | 环状单链三特异抗体 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1380341A CN1380341A (zh) | 2002-11-20 |
CN1294148C true CN1294148C (zh) | 2007-01-10 |
Family
ID=4658662
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB011105542A Expired - Fee Related CN1294148C (zh) | 2001-04-11 | 2001-04-11 | 环状单链三特异抗体 |
Country Status (7)
Country | Link |
---|---|
US (1) | US20050175606A1 (zh) |
EP (1) | EP1378520A4 (zh) |
JP (1) | JP2005501517A (zh) |
CN (1) | CN1294148C (zh) |
CA (1) | CA2443705A1 (zh) |
RU (1) | RU2355705C2 (zh) |
WO (1) | WO2002083738A1 (zh) |
Families Citing this family (172)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9028822B2 (en) | 2002-06-28 | 2015-05-12 | Domantis Limited | Antagonists against TNFR1 and methods of use therefor |
US9321832B2 (en) | 2002-06-28 | 2016-04-26 | Domantis Limited | Ligand |
US7235641B2 (en) * | 2003-12-22 | 2007-06-26 | Micromet Ag | Bispecific antibodies |
CN100376599C (zh) * | 2004-04-01 | 2008-03-26 | 北京安波特基因工程技术有限公司 | 基因工程重组抗cea抗cd3抗cd28线性单链三特异抗体 |
AU2005250216B2 (en) * | 2004-06-01 | 2009-12-10 | Domantis Limited | Bispecific fusion antibodies with enhanced serum half-life |
CN101133084A (zh) * | 2004-12-02 | 2008-02-27 | 多曼蒂斯有限公司 | 采用白细胞介素-1ⅰ型受体拮抗剂治疗呼吸道疾病的方法 |
JP5185624B2 (ja) * | 2004-12-02 | 2013-04-17 | ドマンティス リミテッド | 血清アルブミンおよびglp−1またはpyyを標的とする二重特異性抗体 |
DK2009101T3 (en) | 2006-03-31 | 2018-01-15 | Chugai Pharmaceutical Co Ltd | Antibody modification method for purification of a bispecific antibody |
EP2014680A1 (en) * | 2007-07-10 | 2009-01-14 | Friedrich-Alexander-Universität Erlangen-Nürnberg | Recombinant, single-chain, trivalent tri-specific or bi-specific antibody derivatives |
WO2009018386A1 (en) | 2007-07-31 | 2009-02-05 | Medimmune, Llc | Multispecific epitope binding proteins and uses thereof |
JP4780134B2 (ja) * | 2008-04-09 | 2011-09-28 | ソニー株式会社 | 画像表示装置及び画像表示装置の駆動方法 |
SG185415A1 (en) | 2010-05-06 | 2012-12-28 | Novartis Ag | Compositions and methods of use for therapeutic low density lipoprotein - related protein 6 (lrp6) multivalent antibodies |
ES2659406T3 (es) | 2010-05-06 | 2018-03-15 | Novartis Ag | Composiciones y procedimientos de uso para anticuerpos terapéuticos contra la proteína 6 relacionada con las lipoproteínas de baja densidad (LRP6) |
TWI638833B (zh) * | 2010-11-30 | 2018-10-21 | 中外製藥股份有限公司 | 細胞傷害誘導治療劑 |
WO2013048243A1 (en) * | 2011-09-29 | 2013-04-04 | Apo-T B.V. | Multi-specific binding molecules targeting aberrant cells |
BR112014010257A2 (pt) | 2011-10-31 | 2017-04-18 | Chugai Pharmaceutical Co Ltd | molécula de ligação ao antígeno tendo conjugação regulada entre cadeias pesadas e cadeias leves |
ES2666856T3 (es) | 2011-11-04 | 2018-05-08 | Novartis Ag | Proteína 6 relacionada con lipoproteínas de baja densidad (LRP6) - constructos extensores de la vida media |
SG11201404007WA (en) | 2012-01-13 | 2014-08-28 | Apo T B V | Aberrant cell-restricted immunoglobulins provided with a toxic moiety |
JP6152120B2 (ja) | 2012-02-15 | 2017-06-21 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Fc受容体に基づくアフィニティークロマトグラフィー |
US9096840B2 (en) * | 2012-10-04 | 2015-08-04 | Research Development Foundation | Serine protease molecules and therapies |
UY35195A (es) | 2012-12-18 | 2014-07-31 | Novartis Ag | Composiciones y metodos para proteinas de accion prolongada |
ES2814962T3 (es) | 2013-02-20 | 2021-03-29 | Novartis Ag | Fijación eficaz como objetivo de la leucemia humana primaria utilizando células T modificadas con receptor de antígeno quimérico anti-CD123 |
DK2958943T3 (da) | 2013-02-20 | 2019-12-09 | Univ Pennsylvania | Behandling af cancer ved anvendelse af humaniseret anti-EGFRvIII kimær antigenreceptor |
EP3623380A1 (en) | 2013-03-15 | 2020-03-18 | Michael C. Milone | Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy |
TWI654206B (zh) | 2013-03-16 | 2019-03-21 | 諾華公司 | 使用人類化抗-cd19嵌合抗原受體治療癌症 |
ES2773306T3 (es) | 2013-09-16 | 2020-07-10 | Helmholtz Zentrum Muenchen Deutsches Forschungszentrum Gesundheit & Umwelt Gmbh | Polipéptidos bi o multiespecíficos de unión a antígenos de superficie de células efectoras inmunitarias y antígenos de VHB para tratar infecciones por VHB y estados asociados |
CA3225453A1 (en) | 2013-12-19 | 2015-06-25 | Novartis Ag | Human mesothelin chimeric antigen receptors and uses thereof |
US10287354B2 (en) | 2013-12-20 | 2019-05-14 | Novartis Ag | Regulatable chimeric antigen receptor |
WO2015112626A1 (en) | 2014-01-21 | 2015-07-30 | June Carl H | Enhanced antigen presenting ability of car t cells by co-introduction of costimulatory molecules |
US20170335281A1 (en) | 2014-03-15 | 2017-11-23 | Novartis Ag | Treatment of cancer using chimeric antigen receptor |
US20170081411A1 (en) | 2014-03-15 | 2017-03-23 | Novartis Ag | Regulatable chimeric antigen receptor |
SG10202109752XA (en) | 2014-04-07 | 2021-10-28 | Novartis Ag | Treatment of cancer using anti-cd19 chimeric antigen receptor |
KR102568808B1 (ko) | 2014-04-07 | 2023-08-18 | 추가이 세이야쿠 가부시키가이샤 | 면역활성화 항원 결합 분자 |
JP6894702B2 (ja) | 2014-05-13 | 2021-06-30 | 中外製薬株式会社 | 免疫抑制機能を有する細胞に対するt細胞リダイレクト抗原結合分子 |
AP2016009586A0 (en) | 2014-05-29 | 2016-11-30 | Macrogenics Inc | Tri-specific binding molecules and methods of use thereof |
US20170290876A1 (en) | 2014-06-25 | 2017-10-12 | Novartis Ag | Compositions and methods for long acting proteins |
EP3160991A2 (en) | 2014-06-25 | 2017-05-03 | Novartis AG | Compositions and methods for long acting proteins |
EP3161001A2 (en) | 2014-06-25 | 2017-05-03 | Novartis AG | Antibodies specific for il-17a fused to hyaluronan binding peptide tags |
EP3172237A2 (en) | 2014-07-21 | 2017-05-31 | Novartis AG | Treatment of cancer using humanized anti-bcma chimeric antigen receptor |
US11542488B2 (en) | 2014-07-21 | 2023-01-03 | Novartis Ag | Sortase synthesized chimeric antigen receptors |
JP2017528433A (ja) | 2014-07-21 | 2017-09-28 | ノバルティス アーゲー | 低い免疫増強用量のmTOR阻害剤とCARの組み合わせ |
AU2015292755B2 (en) | 2014-07-21 | 2020-11-12 | Novartis Ag | Treatment of cancer using a CD33 chimeric antigen receptor |
EP3174546B1 (en) | 2014-07-31 | 2019-10-30 | Novartis AG | Subset-optimized chimeric antigen receptor-containing t-cells |
US10851149B2 (en) | 2014-08-14 | 2020-12-01 | The Trustees Of The University Of Pennsylvania | Treatment of cancer using GFR α-4 chimeric antigen receptor |
MY189028A (en) | 2014-08-19 | 2022-01-20 | Novartis Ag | Anti-cd123 chimeric antigen receptor (car) for use in cancer treatment |
AU2015317608B2 (en) | 2014-09-17 | 2021-03-11 | Novartis Ag | Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy |
MA40764A (fr) | 2014-09-26 | 2017-08-01 | Chugai Pharmaceutical Co Ltd | Agent thérapeutique induisant une cytotoxicité |
SI3215528T1 (sl) | 2014-11-06 | 2019-11-29 | Hoffmann La Roche | Variante regije Fc s spremenjeno vezavo FcRn in postopki uporabe |
US20180334490A1 (en) | 2014-12-03 | 2018-11-22 | Qilong H. Wu | Methods for b cell preconditioning in car therapy |
HRP20220531T1 (hr) * | 2014-12-05 | 2022-06-10 | Xyphos Biosciences Inc. | Varijabilni fragmenti antitijela koji se umeću i modificirane a1-a2 domene nkg2d liganda |
IL303247A (en) | 2014-12-29 | 2023-07-01 | Novartis Ag | Methods for preparing cells expressing a chimeric receptor antigen |
WO2016120217A1 (en) | 2015-01-26 | 2016-08-04 | Cellectis | Anti-hsp70 specific chimeric antigen receptors (cars) for cancer immunotherapy |
US11161907B2 (en) | 2015-02-02 | 2021-11-02 | Novartis Ag | Car-expressing cells against multiple tumor antigens and uses thereof |
JP6726676B2 (ja) * | 2015-03-16 | 2020-07-22 | ヘルムホルツ・ツェントルム・ミュンヒェン・ドイチェス・フォルシュンクスツェントルム・フューア・ゲズントハイト・ウント・ウムベルト(ゲーエムベーハー)Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt (GmbH) | Hbv感染および関連症状を治療するための三重特異的結合分子 |
SG11201708191XA (en) | 2015-04-08 | 2017-11-29 | Novartis Ag | Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car) - expressing cell |
JP7114457B2 (ja) | 2015-04-17 | 2022-08-08 | ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア | キメラ抗原受容体発現細胞の有効性および増殖を改善するための方法 |
EP3286211A1 (en) | 2015-04-23 | 2018-02-28 | Novartis AG | Treatment of cancer using chimeric antigen receptor and protein kinase a blocker |
HUE051661T2 (hu) | 2015-05-18 | 2021-03-29 | Tcr2 Therapeutics Inc | Készítmények és gyógyászati felhasználások a TCR újraprogramozására fúziós fehérjék felhasználásával |
EP3932428A1 (en) * | 2015-05-21 | 2022-01-05 | Harpoon Therapeutics, Inc. | Trispecific binding proteins and methods of use |
TW201708538A (zh) | 2015-07-21 | 2017-03-01 | 諾華公司 | 改良免疫細胞之功效及擴展之方法 |
WO2017027392A1 (en) | 2015-08-07 | 2017-02-16 | Novartis Ag | Treatment of cancer using chimeric cd3 receptor proteins |
AU2016347058B2 (en) | 2015-10-25 | 2023-11-09 | Sanofi | Trispecific and/or trivalent binding proteins for prevention or treatment of HIV infection |
WO2017086419A1 (ja) | 2015-11-18 | 2017-05-26 | 中外製薬株式会社 | 液性免疫応答の増強方法 |
JP6931329B2 (ja) | 2015-11-18 | 2021-09-01 | 中外製薬株式会社 | 免疫抑制機能を有する細胞に対するt細胞リダイレクト抗原結合分子を用いた併用療法 |
EP3380620A1 (en) | 2015-11-23 | 2018-10-03 | Novartis AG | Optimized lentiviral transfer vectors and uses thereof |
EP3398965A4 (en) | 2015-12-28 | 2019-09-18 | Chugai Seiyaku Kabushiki Kaisha | METHOD FOR PROMOTING THE EFFICACY OF PURIFYING A POLYPEPTIDE CONTAINING AN FC REGION |
EP3397756B1 (en) | 2015-12-30 | 2023-03-08 | Novartis AG | Immune effector cell therapies with enhanced efficacy |
EP3851457A1 (en) | 2016-01-21 | 2021-07-21 | Novartis AG | Multispecific molecules targeting cll-1 |
US20200281973A1 (en) | 2016-03-04 | 2020-09-10 | Novartis Ag | Cells expressing multiple chimeric antigen receptor (car) molecules and uses therefore |
EP3431102A4 (en) | 2016-03-14 | 2019-09-25 | Chugai Seiyaku Kabushiki Kaisha | THERAPEUTIC MEDICINE INDUCING CELLULAR INJURY FOR USE IN THE TREATMENT OF CANCER |
US11549099B2 (en) | 2016-03-23 | 2023-01-10 | Novartis Ag | Cell secreted minibodies and uses thereof |
US20190112380A1 (en) | 2016-03-29 | 2019-04-18 | University Of Southern California | Chimeric antigen receptors targeting cancer |
SG11201808911SA (en) * | 2016-04-13 | 2018-11-29 | Sanofi Sa | Trispecific and/or trivalent binding proteins |
DK3443006T5 (da) | 2016-04-13 | 2023-11-06 | Sanofi Sa | Trispecifikke og/eller trivalente bindingsproteiner |
SI3443096T1 (sl) | 2016-04-15 | 2023-07-31 | Novartis Ag | Sestavki in postopki za selektivno izražanje himerni antigenskih receptorjev |
KR102523682B1 (ko) * | 2016-05-02 | 2023-04-19 | 에프. 호프만-라 로슈 아게 | 콘톨스바디 - 단쇄 표적 결합제 |
US10100106B2 (en) | 2016-05-20 | 2018-10-16 | Harpoon Therapeutics, Inc. | Single domain serum albumin binding protein |
KR102365977B1 (ko) | 2016-05-20 | 2022-02-22 | 하푼 테라퓨틱스, 인크. | 단일 쇄 가변 단편 cd3 결합 단백질 |
US11623958B2 (en) | 2016-05-20 | 2023-04-11 | Harpoon Therapeutics, Inc. | Single chain variable fragment CD3 binding proteins |
WO2017210617A2 (en) | 2016-06-02 | 2017-12-07 | Porter, David, L. | Therapeutic regimens for chimeric antigen receptor (car)- expressing cells |
WO2018013918A2 (en) | 2016-07-15 | 2018-01-18 | Novartis Ag | Treatment and prevention of cytokine release syndrome using a chimeric antigen receptor in combination with a kinase inhibitor |
KR20230100748A (ko) | 2016-07-28 | 2023-07-05 | 노파르티스 아게 | 키메라 항원 수용체 및 pd-1 억제제의 조합 요법 |
US20190161542A1 (en) | 2016-08-01 | 2019-05-30 | Novartis Ag | Treatment of cancer using a chimeric antigen receptor in combination with an inhibitor of a pro-m2 macrophage molecule |
JP7109789B2 (ja) | 2016-08-02 | 2022-08-01 | ティーシーアール2 セラピューティクス インク. | 融合タンパク質を使用したtcrの再プログラム化のための組成物及び方法 |
ES2875959T3 (es) | 2016-10-07 | 2021-11-11 | Tcr2 Therapeutics Inc | Composiciones y métodos para reprogramación de receptores de linfocitos T mediante el uso de proteínas de fusión |
CN110225927B (zh) | 2016-10-07 | 2024-01-12 | 诺华股份有限公司 | 用于治疗癌症的嵌合抗原受体 |
WO2018098365A2 (en) | 2016-11-22 | 2018-05-31 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using fusion proteins |
MX2019006045A (es) * | 2016-11-23 | 2019-11-11 | Harpoon Therapeutics Inc | Proteinas triespecificas dirigidas a psma y metodos de uso. |
EP3544997A4 (en) | 2016-11-23 | 2020-07-01 | Harpoon Therapeutics, Inc. | PROSTATE SPECIFIC MEMBRANE ANTIGEN BINDING PROTEIN |
WO2018111340A1 (en) | 2016-12-16 | 2018-06-21 | Novartis Ag | Methods for determining potency and proliferative function of chimeric antigen receptor (car)-t cells |
WO2018120843A1 (zh) * | 2016-12-30 | 2018-07-05 | 上海近岸生物科技有限公司 | 一种三功能分子及其应用 |
CN106589129B (zh) * | 2016-12-30 | 2021-03-05 | 上海近岸生物科技有限公司 | 一种结合cd19、cd3和cd28的三功能分子及其应用 |
ES2912408T3 (es) | 2017-01-26 | 2022-05-25 | Novartis Ag | Composiciones de CD28 y métodos para terapia con receptores quiméricos para antígenos |
WO2018144535A1 (en) | 2017-01-31 | 2018-08-09 | Novartis Ag | Treatment of cancer using chimeric t cell receptor proteins having multiple specificities |
EP3589662A4 (en) | 2017-02-28 | 2020-12-30 | Harpoon Therapeutics, Inc. | INDUCTIBLE MONOVALENT ANTIGBINDING PROTEIN |
AU2018250641A1 (en) | 2017-04-11 | 2019-10-31 | Inhibrx, Inc. | Multispecific polypeptide constructs having constrained CD3 binding and methods of using the same |
EP3615068A1 (en) | 2017-04-28 | 2020-03-04 | Novartis AG | Bcma-targeting agent, and combination therapy with a gamma secretase inhibitor |
US20200055948A1 (en) | 2017-04-28 | 2020-02-20 | Novartis Ag | Cells expressing a bcma-targeting chimeric antigen receptor, and combination therapy with a gamma secretase inhibitor |
AU2018265856B2 (en) | 2017-05-12 | 2023-04-27 | Harpoon Therapeutics, Inc. | Mesothelin binding proteins |
CN115028727A (zh) | 2017-05-12 | 2022-09-09 | 哈普恩治疗公司 | 靶向msln的三特异性蛋白质及使用方法 |
WO2018232020A1 (en) | 2017-06-13 | 2018-12-20 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using fusion proteins |
US11607457B2 (en) | 2017-07-13 | 2023-03-21 | City Of Hope | Anti-cancer phosphorothioate-coupled peptide conjugates and methods of using the same |
CN111788225A (zh) | 2017-10-10 | 2020-10-16 | 赛诺菲 | 抗cd38抗体及与抗cd3和抗cd28抗体的组合 |
AU2018347582A1 (en) | 2017-10-13 | 2020-05-07 | Harpoon Therapeutics, Inc. | Trispecific proteins and methods of use |
US10927180B2 (en) | 2017-10-13 | 2021-02-23 | Harpoon Therapeutics, Inc. | B cell maturation antigen binding proteins |
WO2019079569A1 (en) | 2017-10-18 | 2019-04-25 | Novartis Ag | COMPOSITIONS AND METHODS FOR SELECTIVE DEGRADATION OF A PROTEIN |
MX2020004229A (es) | 2017-10-25 | 2020-07-22 | Novartis Ag | Metodos de produccion de celulas que expresan receptores antigenicos quimericos. |
KR102559706B1 (ko) | 2017-11-01 | 2023-07-25 | 에프. 호프만-라 로슈 아게 | Trifab-콘톨스바디 |
WO2019086394A1 (en) | 2017-11-01 | 2019-05-09 | F. Hoffmann-La Roche Ag | The compbody - a multivalent target binder |
US20210047411A1 (en) * | 2018-03-15 | 2021-02-18 | Biond Biologics Ltd. | Methods and compositions for decreasing soluble immune receptor cd28 |
GB201804701D0 (en) | 2018-03-23 | 2018-05-09 | Gammadelta Therapeutics Ltd | Lymphocytes expressing heterologous targeting constructs |
US20210047405A1 (en) | 2018-04-27 | 2021-02-18 | Novartis Ag | Car t cell therapies with enhanced efficacy |
EP3788369A1 (en) | 2018-05-01 | 2021-03-10 | Novartis Ag | Biomarkers for evaluating car-t cells to predict clinical outcome |
WO2019227003A1 (en) | 2018-05-25 | 2019-11-28 | Novartis Ag | Combination therapy with chimeric antigen receptor (car) therapies |
WO2019237035A1 (en) | 2018-06-08 | 2019-12-12 | Intellia Therapeutics, Inc. | Compositions and methods for immunooncology |
JP7438988B2 (ja) | 2018-06-13 | 2024-02-27 | ノバルティス アーゲー | Bcmaキメラ抗原受容体及びその使用 |
EP3828200A4 (en) | 2018-07-09 | 2022-05-18 | National University Corporation Kumamoto University | CYCLIC SINGLE CHAIN ANTIBODIES |
AR116109A1 (es) | 2018-07-10 | 2021-03-31 | Novartis Ag | Derivados de 3-(5-amino-1-oxoisoindolin-2-il)piperidina-2,6-diona y usos de los mismos |
WO2020047501A1 (en) | 2018-08-30 | 2020-03-05 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using fusion proteins |
JP2021534783A (ja) | 2018-08-31 | 2021-12-16 | ノバルティス アーゲー | キメラ抗原受容体発現細胞を作製する方法 |
WO2020047449A2 (en) | 2018-08-31 | 2020-03-05 | Novartis Ag | Methods of making chimeric antigen receptor-expressing cells |
EP3849565A4 (en) | 2018-09-12 | 2022-12-28 | Fred Hutchinson Cancer Research Center | REDUCING CD33 EXPRESSION FOR SELECTIVE PROTECTION OF THERAPEUTIC CELLS |
KR20210086623A (ko) | 2018-09-25 | 2021-07-08 | 하푼 테라퓨틱스, 인크. | Ddl3 결합 단백질 및 사용 방법 |
WO2020069409A1 (en) | 2018-09-28 | 2020-04-02 | Novartis Ag | Cd19 chimeric antigen receptor (car) and cd22 car combination therapies |
WO2020069405A1 (en) | 2018-09-28 | 2020-04-02 | Novartis Ag | Cd22 chimeric antigen receptor (car) therapies |
SG11202103478RA (en) | 2018-10-09 | 2021-05-28 | Sanofi Sa | Trispecific anti-cd38, anti-cd28, and anti-cd3 binding proteins and methods of use for treating viral infection |
CA3123511A1 (en) | 2018-12-20 | 2020-06-25 | Novartis Ag | Dosing regimen and pharmaceutical combination comprising 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives |
WO2020165833A1 (en) | 2019-02-15 | 2020-08-20 | Novartis Ag | 3-(1-oxo-5-(piperidin-4-yl)isoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof |
CA3123519A1 (en) | 2019-02-15 | 2020-08-20 | Novartis Ag | Substituted 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof |
WO2020172553A1 (en) | 2019-02-22 | 2020-08-27 | Novartis Ag | Combination therapies of egfrviii chimeric antigen receptors and pd-1 inhibitors |
KR20210134339A (ko) | 2019-02-25 | 2021-11-09 | 노파르티스 아게 | 바이러스 전달을 위한 메조다공성 실리카 입자 조성물 |
AU2020241428A1 (en) | 2019-03-15 | 2021-08-12 | Cartesian Therapeutics, Inc. | Anti-BCMA chimeric antigen receptors |
US11613576B2 (en) | 2019-04-09 | 2023-03-28 | Sanofi | Trispecific binding proteins, methods, and uses thereof |
US20220168389A1 (en) | 2019-04-12 | 2022-06-02 | Novartis Ag | Methods of making chimeric antigen receptor-expressing cells |
WO2020219742A1 (en) | 2019-04-24 | 2020-10-29 | Novartis Ag | Compositions and methods for selective protein degradation |
BR112021026832A2 (pt) | 2019-07-02 | 2022-05-10 | Hutchinson Fred Cancer Res | Vetores ad35 recombinantes e aprimoramentos da terapia gênica relacionada |
WO2021035170A1 (en) | 2019-08-21 | 2021-02-25 | Precision Biosciences, Inc. | Compositions and methods for tcr reprogramming using fusion proteins |
WO2021108661A2 (en) | 2019-11-26 | 2021-06-03 | Novartis Ag | Chimeric antigen receptors and uses thereof |
CN115175937A (zh) | 2019-12-20 | 2022-10-11 | 诺华股份有限公司 | 用于治疗骨髓纤维化和骨髓增生异常综合征的抗TIM-3抗体MBG453和抗TGF-β抗体NIS793与或不与地西他滨或抗PD-1抗体斯巴达珠单抗的组合 |
EP4097129A1 (en) * | 2020-01-29 | 2022-12-07 | Inhibrx, Inc. | Cd28 single domain antibodies and multivalent and multispecific constructs thereof |
WO2021163618A1 (en) | 2020-02-14 | 2021-08-19 | Novartis Ag | Method of predicting response to chimeric antigen receptor therapy |
WO2021168303A1 (en) | 2020-02-21 | 2021-08-26 | Harpoon Therapeutics, Inc. | Flt3 binding proteins and methods of use |
TW202146441A (zh) | 2020-02-27 | 2021-12-16 | 瑞士商諾華公司 | 製備表現嵌合抗原受體的細胞之方法 |
WO2021173995A2 (en) | 2020-02-27 | 2021-09-02 | Novartis Ag | Methods of making chimeric antigen receptor-expressing cells |
CN111269326A (zh) | 2020-02-28 | 2020-06-12 | 南京北恒生物科技有限公司 | 新型嵌合抗原受体及其用途 |
AU2021270299A1 (en) | 2020-05-12 | 2022-12-15 | Lyell Immunopharma, Inc. | Chimeric antigen receptor spacers |
CN111849910B (zh) | 2020-05-27 | 2021-06-15 | 南京北恒生物科技有限公司 | 工程化免疫细胞及其用途 |
CN113801238A (zh) | 2020-06-11 | 2021-12-17 | 南京北恒生物科技有限公司 | 表达nk抑制性分子的工程化免疫细胞及其用途 |
JP2023529211A (ja) | 2020-06-11 | 2023-07-07 | ノバルティス アーゲー | Zbtb32阻害剤及びその使用 |
JP2023531676A (ja) | 2020-06-23 | 2023-07-25 | ノバルティス アーゲー | 3-(1-オキソイソインドリン-2-イル)ピぺリジン-2,6-ジオン誘導体を含む投与レジメン |
EP4148125A4 (en) | 2020-07-15 | 2023-11-15 | Bioheng Therapeutics Limited | MODIFIED IMMUNE CELL INTENDED FOR ALLOTRANSPLANTATION |
US11524998B2 (en) | 2020-07-16 | 2022-12-13 | Novartis Ag | Anti-betacellulin antibodies, fragments thereof, and multi-specific binding molecules |
CN114057890A (zh) | 2020-07-31 | 2022-02-18 | 南京北恒生物科技有限公司 | 新型共刺激结构域及其用途 |
WO2022029573A1 (en) | 2020-08-03 | 2022-02-10 | Novartis Ag | Heteroaryl substituted 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof |
CN112063588A (zh) | 2020-08-13 | 2020-12-11 | 南京北恒生物科技有限公司 | 工程化免疫细胞及其用途 |
EP4200332A1 (en) | 2020-08-19 | 2023-06-28 | Xencor, Inc. | Anti-cd28 and/or anti-b7h3 compositions |
CA3188978A1 (en) | 2020-08-21 | 2022-02-24 | Sandeep Tharian Koshy | Compositions and methods for in vivo generation of car expressing cells |
CN114163532A (zh) | 2020-09-10 | 2022-03-11 | 南京北恒生物科技有限公司 | 包含新型共刺激结构域的嵌合抗原受体及其用途 |
CN112300282A (zh) | 2020-11-03 | 2021-02-02 | 南京北恒生物科技有限公司 | 靶向cd7的人源化抗体及其用途 |
CN114525259A (zh) | 2020-11-03 | 2022-05-24 | 南京北恒生物科技有限公司 | 靶向cd7的嵌合抗原受体及其用途 |
CN116635062A (zh) | 2020-11-13 | 2023-08-22 | 诺华股份有限公司 | 使用表达嵌合抗原受体(car)的细胞的组合疗法 |
CN114524876A (zh) | 2020-11-23 | 2022-05-24 | 南京北恒生物科技有限公司 | 靶向nkg2a的抗体及其用途 |
WO2022152168A1 (zh) | 2021-01-12 | 2022-07-21 | 南京北恒生物科技有限公司 | 靶向ror1的抗体及其用途 |
WO2022166365A1 (zh) | 2021-02-03 | 2022-08-11 | 南京北恒生物科技有限公司 | 新型嵌合抗原受体及其用途 |
AU2022227686A1 (en) | 2021-02-25 | 2023-07-27 | Lyell Immunopharma, Inc. | Ror1 targeting chimeric antigen receptor |
TW202304979A (zh) | 2021-04-07 | 2023-02-01 | 瑞士商諾華公司 | 抗TGFβ抗體及其他治療劑用於治療增殖性疾病之用途 |
CN118056008A (zh) | 2021-04-27 | 2024-05-17 | 诺华股份有限公司 | 病毒载体生产系统 |
AU2022330406A1 (en) | 2021-08-20 | 2024-03-07 | Novartis Ag | Methods of making chimeric antigen receptor–expressing cells |
TW202400658A (zh) | 2022-04-26 | 2024-01-01 | 瑞士商諾華公司 | 靶向il—13和il—18的多特異性抗體 |
WO2023214325A1 (en) | 2022-05-05 | 2023-11-09 | Novartis Ag | Pyrazolopyrimidine derivatives and uses thereof as tet2 inhibitors |
WO2024056809A1 (en) | 2022-09-15 | 2024-03-21 | Novartis Ag | Treatment of autoimmune disorders using chimeric antigen receptor therapy |
WO2024089639A1 (en) | 2022-10-26 | 2024-05-02 | Novartis Ag | Lentiviral formulations |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0885614A2 (de) * | 1997-06-17 | 1998-12-23 | GSF-Forschungszentrum für Umwelt und Gesundheit GmbH | Verfahren zur ex vivo-Immunisierung mittels heterologer intakter bispezifischer und/oder trispezifischer Antikörper |
WO2000018806A1 (de) * | 1998-09-25 | 2000-04-06 | Horst Lindhofer | Bispezifische und trispezifische antikörper, die spezifisch mit induzierbaren oberflächenantigenen als operationelle zielstrukturen reagieren |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5601819A (en) * | 1988-08-11 | 1997-02-11 | The General Hospital Corporation | Bispecific antibodies for selective immune regulation and for selective immune cell binding |
WO1991003493A1 (en) * | 1989-08-29 | 1991-03-21 | The University Of Southampton | Bi-or trispecific (fab)3 or (fab)4 conjugates |
ATE218143T1 (de) * | 1996-09-03 | 2002-06-15 | Gsf Forschungszentrum Umwelt | Verwendung bi-und trispezifischer antikörper zur induktion einer tumorimmunität |
US20020064528A1 (en) * | 2000-01-28 | 2002-05-30 | Zhenping Zhu | Antibodies specific to KDR and uses thereof |
-
2001
- 2001-04-11 CN CNB011105542A patent/CN1294148C/zh not_active Expired - Fee Related
-
2002
- 2002-04-10 EP EP02724089A patent/EP1378520A4/en not_active Withdrawn
- 2002-04-10 US US10/474,345 patent/US20050175606A1/en not_active Abandoned
- 2002-04-10 RU RU2003130072/13A patent/RU2355705C2/ru not_active IP Right Cessation
- 2002-04-10 WO PCT/CN2002/000252 patent/WO2002083738A1/zh active Application Filing
- 2002-04-10 CA CA002443705A patent/CA2443705A1/en not_active Abandoned
- 2002-04-10 JP JP2002581493A patent/JP2005501517A/ja active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0885614A2 (de) * | 1997-06-17 | 1998-12-23 | GSF-Forschungszentrum für Umwelt und Gesundheit GmbH | Verfahren zur ex vivo-Immunisierung mittels heterologer intakter bispezifischer und/oder trispezifischer Antikörper |
WO2000018806A1 (de) * | 1998-09-25 | 2000-04-06 | Horst Lindhofer | Bispezifische und trispezifische antikörper, die spezifisch mit induzierbaren oberflächenantigenen als operationelle zielstrukturen reagieren |
Also Published As
Publication number | Publication date |
---|---|
CA2443705A1 (en) | 2002-10-24 |
CN1380341A (zh) | 2002-11-20 |
EP1378520A4 (en) | 2006-08-16 |
RU2355705C2 (ru) | 2009-05-20 |
WO2002083738A1 (fr) | 2002-10-24 |
US20050175606A1 (en) | 2005-08-11 |
JP2005501517A (ja) | 2005-01-20 |
RU2003130072A (ru) | 2005-04-20 |
EP1378520A1 (en) | 2004-01-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1294148C (zh) | 环状单链三特异抗体 | |
US20230303718A1 (en) | Modified Antibody | |
JP7425604B2 (ja) | 抗ctla4-抗pd-1二機能性抗体、その医薬組成物および使用 | |
JP6432121B2 (ja) | Pdl−1抗体、その医薬組成物及びその使用 | |
CN1195779C (zh) | 抗人卵巢癌抗人cd3双特异性抗体 | |
CN110950953B (zh) | 抗b7-h3的单克隆抗体及其在细胞治疗中的应用 | |
JP5774276B2 (ja) | 耐性抗原に対する活性抗体を作製する方法、その方法により得られる抗体およびそれらの使用 | |
CN1186445C (zh) | 抗人肝癌单克隆抗体HAb18轻、重链可变区基因及其应用 | |
CN1603345A (zh) | 一种三价双特异性抗体,其制备方法及用途 | |
JP2010504743A5 (zh) | ||
JP2023106392A (ja) | Cd3抗原結合性断片及びその使用 | |
WO2020199860A1 (zh) | 针对程序性死亡配体的结合物及其应用 | |
CN113354739B (zh) | 一种靶向表达Claudin18.2细胞的嵌合抗原受体及其应用 | |
WO2019195408A1 (en) | Antibody variable domains targeting dll3, and use thereof | |
US20230212317A1 (en) | Heterodimeric proteins | |
CN115850475A (zh) | 人源化抗人ctla4单克隆抗体及其制备方法和用途 | |
AU2369101A (en) | Antibodies against plasma cells | |
CN114805584B (zh) | 抗原结合蛋白及其用途 | |
CN1281753C (zh) | 抗人大肠癌单克隆抗体CAb—1轻、重链可变区基因及其应用 | |
CN114456267B (zh) | 一种抗cd73人源化单克隆抗体及其应用 | |
CN114685682B (zh) | 一种靶向表达cldn 18.2的细胞的嵌合抗原受体 | |
US20230134345A1 (en) | Chimeric antigen receptor comprising third signal receptor and use thereof | |
CN1303105C (zh) | 抗人大肠癌单克隆抗体CAb—2轻、重链可变区基因及其应用 | |
KR20230166120A (ko) | 새로운 tnfr2 결합 분자 | |
CN115073606A (zh) | 一种靶向表达cldn 18.2的细胞的人源化嵌合抗原受体 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
C17 | Cessation of patent right | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070110 Termination date: 20130411 |