CN114395041B - Method for preparing anti-IL-12 and/or IL-23 monoclonal antibody - Google Patents
Method for preparing anti-IL-12 and/or IL-23 monoclonal antibody Download PDFInfo
- Publication number
- CN114395041B CN114395041B CN202210109484.8A CN202210109484A CN114395041B CN 114395041 B CN114395041 B CN 114395041B CN 202210109484 A CN202210109484 A CN 202210109484A CN 114395041 B CN114395041 B CN 114395041B
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- cells
- cell
- ser
- antibody
- 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.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 65
- 108010065637 Interleukin-23 Proteins 0.000 title claims abstract description 54
- 102000013264 Interleukin-23 Human genes 0.000 title claims abstract description 54
- 210000004027 cell Anatomy 0.000 claims description 134
- 108010065805 Interleukin-12 Proteins 0.000 claims description 54
- 102000013462 Interleukin-12 Human genes 0.000 claims description 54
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 50
- 239000013604 expression vector Substances 0.000 claims description 38
- 108090000623 proteins and genes Proteins 0.000 claims description 35
- 239000012634 fragment Substances 0.000 claims description 31
- 239000000427 antigen Substances 0.000 claims description 26
- 108091007433 antigens Proteins 0.000 claims description 26
- 102000036639 antigens Human genes 0.000 claims description 26
- 239000008194 pharmaceutical composition Substances 0.000 claims description 26
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 22
- CMXXUDSWGMGYLZ-XRIGFGBMSA-N (2s)-2-amino-3-(1h-imidazol-5-yl)propanoic acid;hydron;chloride;hydrate Chemical compound O.Cl.OC(=O)[C@@H](N)CC1=CN=CN1 CMXXUDSWGMGYLZ-XRIGFGBMSA-N 0.000 claims description 18
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 18
- 229930006000 Sucrose Natural products 0.000 claims description 18
- 239000005720 sucrose Substances 0.000 claims description 18
- 101150074355 GS gene Proteins 0.000 claims description 16
- 201000004681 Psoriasis Diseases 0.000 claims description 16
- 208000011231 Crohn disease Diseases 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 14
- 238000003259 recombinant expression Methods 0.000 claims description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 13
- 238000010367 cloning Methods 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 239000003550 marker Substances 0.000 claims description 12
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 claims description 12
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 12
- 229920000053 polysorbate 80 Polymers 0.000 claims description 12
- 229940068968 polysorbate 80 Drugs 0.000 claims description 12
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 11
- 238000004113 cell culture Methods 0.000 claims description 11
- 201000010099 disease Diseases 0.000 claims description 11
- 229960002885 histidine Drugs 0.000 claims description 11
- 229960004452 methionine Drugs 0.000 claims description 11
- 108091026890 Coding region Proteins 0.000 claims description 10
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 claims description 10
- 229930195722 L-methionine Natural products 0.000 claims description 10
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 9
- 101150074155 DHFR gene Proteins 0.000 claims description 8
- 201000001263 Psoriatic Arthritis Diseases 0.000 claims description 8
- 208000036824 Psoriatic arthropathy Diseases 0.000 claims description 8
- 150000001413 amino acids Chemical group 0.000 claims description 7
- 229920000136 polysorbate Polymers 0.000 claims description 6
- 229950008882 polysorbate Drugs 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 5
- 238000003306 harvesting Methods 0.000 claims description 5
- 201000006417 multiple sclerosis Diseases 0.000 claims description 5
- 206010002556 Ankylosing Spondylitis Diseases 0.000 claims description 3
- 208000025705 Axial Spondyloarthritis Diseases 0.000 claims description 3
- 206010009900 Colitis ulcerative Diseases 0.000 claims description 3
- 201000002661 Spondylitis Diseases 0.000 claims description 3
- 201000006704 Ulcerative Colitis Diseases 0.000 claims description 3
- 201000000306 sarcoidosis Diseases 0.000 claims description 3
- 201000000596 systemic lupus erythematosus Diseases 0.000 claims description 3
- 208000035475 disorder Diseases 0.000 claims description 2
- 102100024746 Dihydrofolate reductase Human genes 0.000 claims 2
- 108020001096 dihydrofolate reductase Proteins 0.000 claims 2
- 238000012216 screening Methods 0.000 abstract description 18
- 230000004071 biological effect Effects 0.000 abstract description 12
- 229940117681 interleukin-12 Drugs 0.000 description 52
- 229940124829 interleukin-23 Drugs 0.000 description 47
- 239000013612 plasmid Substances 0.000 description 42
- 239000002609 medium Substances 0.000 description 38
- 210000001744 T-lymphocyte Anatomy 0.000 description 23
- 108020002326 glutamine synthetase Proteins 0.000 description 21
- 102000005396 glutamine synthetase Human genes 0.000 description 20
- 230000005764 inhibitory process Effects 0.000 description 20
- 108020004414 DNA Proteins 0.000 description 16
- 230000014509 gene expression Effects 0.000 description 15
- 102000008070 Interferon-gamma Human genes 0.000 description 14
- 108010074328 Interferon-gamma Proteins 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 108010022394 Threonine synthase Proteins 0.000 description 13
- 102000004419 dihydrofolate reductase Human genes 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 10
- 229960003130 interferon gamma Drugs 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 229960000485 methotrexate Drugs 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 8
- 238000007789 sealing Methods 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 8
- 235000010633 broth Nutrition 0.000 description 7
- 239000003480 eluent Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 108091008146 restriction endonucleases Proteins 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 5
- 108010092854 aspartyllysine Proteins 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 235000002639 sodium chloride Nutrition 0.000 description 5
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 4
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 239000012737 fresh medium Substances 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 229940044627 gamma-interferon Drugs 0.000 description 4
- 239000000833 heterodimer Substances 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- OVVUNXXROOFSIM-SDDRHHMPSA-N Arg-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O OVVUNXXROOFSIM-SDDRHHMPSA-N 0.000 description 3
- CUQDCPXNZPDYFQ-ZLUOBGJFSA-N Asp-Ser-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O CUQDCPXNZPDYFQ-ZLUOBGJFSA-N 0.000 description 3
- 208000023275 Autoimmune disease Diseases 0.000 description 3
- 108091033409 CRISPR Proteins 0.000 description 3
- 238000010354 CRISPR gene editing Methods 0.000 description 3
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 3
- GJFYFGOEWLDQGW-GUBZILKMSA-N Ser-Leu-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CO)N GJFYFGOEWLDQGW-GUBZILKMSA-N 0.000 description 3
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 3
- 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 3
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 3
- LMKKMCGTDANZTR-BZSNNMDCSA-N Tyr-Phe-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LMKKMCGTDANZTR-BZSNNMDCSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 108010087924 alanylproline Proteins 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000003209 gene knockout Methods 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 108010031719 prolyl-serine Proteins 0.000 description 3
- 108010029020 prolylglycine Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- ARHJJAAWNWOACN-FXQIFTODSA-N Ala-Ser-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O ARHJJAAWNWOACN-FXQIFTODSA-N 0.000 description 2
- 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 2
- 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 2
- XYBJLTKSGFBLCS-QXEWZRGKSA-N Asp-Arg-Val Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC(O)=O XYBJLTKSGFBLCS-QXEWZRGKSA-N 0.000 description 2
- QNFRBNZGVVKBNJ-PEFMBERDSA-N Asp-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N QNFRBNZGVVKBNJ-PEFMBERDSA-N 0.000 description 2
- 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 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 229940123907 Disease modifying antirheumatic drug Drugs 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- CLPQUWHBWXFJOX-BQBZGAKWSA-N Gln-Gly-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O CLPQUWHBWXFJOX-BQBZGAKWSA-N 0.000 description 2
- LVRKAFPPFJRIOF-GARJFASQSA-N Gln-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N LVRKAFPPFJRIOF-GARJFASQSA-N 0.000 description 2
- OSCLNNWLKKIQJM-WDSKDSINSA-N Gln-Ser-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O OSCLNNWLKKIQJM-WDSKDSINSA-N 0.000 description 2
- YQPFCZVKMUVZIN-AUTRQRHGSA-N Glu-Val-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O YQPFCZVKMUVZIN-AUTRQRHGSA-N 0.000 description 2
- ZYRXTRTUCAVNBQ-GVXVVHGQSA-N Glu-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZYRXTRTUCAVNBQ-GVXVVHGQSA-N 0.000 description 2
- ZOTGXWMKUFSKEU-QXEWZRGKSA-N Gly-Ile-Met Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCSC)C(O)=O ZOTGXWMKUFSKEU-QXEWZRGKSA-N 0.000 description 2
- 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 2
- 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 2
- DNAZKGFYFRGZIH-QWRGUYRKSA-N Gly-Tyr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 DNAZKGFYFRGZIH-QWRGUYRKSA-N 0.000 description 2
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 2
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 2
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 2
- CWJQMCPYXNVMBS-STECZYCISA-N Ile-Arg-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N CWJQMCPYXNVMBS-STECZYCISA-N 0.000 description 2
- FBGXMKUWQFPHFB-JBDRJPRFSA-N Ile-Ser-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N FBGXMKUWQFPHFB-JBDRJPRFSA-N 0.000 description 2
- PXKACEXYLPBMAD-JBDRJPRFSA-N Ile-Ser-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PXKACEXYLPBMAD-JBDRJPRFSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 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 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- 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 2
- GBDMISNMNXVTNV-XIRDDKMYSA-N Leu-Asp-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O GBDMISNMNXVTNV-XIRDDKMYSA-N 0.000 description 2
- YSKSXVKQLLBVEX-SZMVWBNQSA-N Leu-Gln-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 YSKSXVKQLLBVEX-SZMVWBNQSA-N 0.000 description 2
- MVJRBCJCRYGCKV-GVXVVHGQSA-N Leu-Val-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MVJRBCJCRYGCKV-GVXVVHGQSA-N 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- IXHKPDJKKCUKHS-GARJFASQSA-N Lys-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N IXHKPDJKKCUKHS-GARJFASQSA-N 0.000 description 2
- 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 2
- FHIAJWBDZVHLAH-YUMQZZPRSA-N Lys-Gly-Ser Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FHIAJWBDZVHLAH-YUMQZZPRSA-N 0.000 description 2
- CNGOEHJCLVCJHN-SRVKXCTJSA-N Lys-Pro-Glu Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O CNGOEHJCLVCJHN-SRVKXCTJSA-N 0.000 description 2
- 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 2
- JOSAKOKSPXROGQ-BJDJZHNGSA-N Lys-Ser-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JOSAKOKSPXROGQ-BJDJZHNGSA-N 0.000 description 2
- IOQWIOPSKJOEKI-SRVKXCTJSA-N Lys-Ser-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O IOQWIOPSKJOEKI-SRVKXCTJSA-N 0.000 description 2
- SPSSJSICDYYTQN-HJGDQZAQSA-N Met-Thr-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O SPSSJSICDYYTQN-HJGDQZAQSA-N 0.000 description 2
- GHQFLTYXGUETFD-UFYCRDLUSA-N Met-Tyr-Tyr Chemical compound CSCC[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 GHQFLTYXGUETFD-UFYCRDLUSA-N 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 2
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 2
- 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 2
- YFXXRYFWJFQAFW-JHYOHUSXSA-N Phe-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O YFXXRYFWJFQAFW-JHYOHUSXSA-N 0.000 description 2
- ZVJGAXNBBKPYOE-HKUYNNGSSA-N Phe-Trp-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(O)=O)C1=CC=CC=C1 ZVJGAXNBBKPYOE-HKUYNNGSSA-N 0.000 description 2
- IHCXPSYCHXFXKT-DCAQKATOSA-N Pro-Arg-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O IHCXPSYCHXFXKT-DCAQKATOSA-N 0.000 description 2
- QPFJSHSJFIYDJZ-GHCJXIJMSA-N Ser-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CO QPFJSHSJFIYDJZ-GHCJXIJMSA-N 0.000 description 2
- XWCYBVBLJRWOFR-WDSKDSINSA-N Ser-Gln-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O XWCYBVBLJRWOFR-WDSKDSINSA-N 0.000 description 2
- UQFYNFTYDHUIMI-WHFBIAKZSA-N Ser-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CO UQFYNFTYDHUIMI-WHFBIAKZSA-N 0.000 description 2
- 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 2
- UBRMZSHOOIVJPW-SRVKXCTJSA-N Ser-Leu-Lys Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O UBRMZSHOOIVJPW-SRVKXCTJSA-N 0.000 description 2
- CKDXFSPMIDSMGV-GUBZILKMSA-N Ser-Pro-Val Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O CKDXFSPMIDSMGV-GUBZILKMSA-N 0.000 description 2
- UKKROEYWYIHWBD-ZKWXMUAHSA-N Ser-Val-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O UKKROEYWYIHWBD-ZKWXMUAHSA-N 0.000 description 2
- HNDMFDBQXYZSRM-IHRRRGAJSA-N Ser-Val-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HNDMFDBQXYZSRM-IHRRRGAJSA-N 0.000 description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 2
- 210000000447 Th1 cell Anatomy 0.000 description 2
- GXUWHVZYDAHFSV-FLBSBUHZSA-N Thr-Ile-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GXUWHVZYDAHFSV-FLBSBUHZSA-N 0.000 description 2
- KZSYAEWQMJEGRZ-RHYQMDGZSA-N Thr-Leu-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O KZSYAEWQMJEGRZ-RHYQMDGZSA-N 0.000 description 2
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 2
- UJRIVCPPPMYCNA-HOCLYGCPSA-N Trp-Leu-Gly Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N UJRIVCPPPMYCNA-HOCLYGCPSA-N 0.000 description 2
- 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 2
- VCXWRWYFJLXITF-AUTRQRHGSA-N Tyr-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 VCXWRWYFJLXITF-AUTRQRHGSA-N 0.000 description 2
- MTEQZJFSEMXXRK-CFMVVWHZSA-N Tyr-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CC=C(C=C1)O)N MTEQZJFSEMXXRK-CFMVVWHZSA-N 0.000 description 2
- 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 2
- VPEFOFYNHBWFNQ-UFYCRDLUSA-N Tyr-Pro-Tyr Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 VPEFOFYNHBWFNQ-UFYCRDLUSA-N 0.000 description 2
- SSYBNWFXCFNRFN-GUBZILKMSA-N Val-Pro-Ser Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O SSYBNWFXCFNRFN-GUBZILKMSA-N 0.000 description 2
- USXYVSTVPHELAF-RCWTZXSCSA-N Val-Thr-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](C(C)C)N)O USXYVSTVPHELAF-RCWTZXSCSA-N 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 108010008355 arginyl-glutamine Proteins 0.000 description 2
- 230000001363 autoimmune Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- JHRWWRDRBPCWTF-OLQVQODUSA-N captafol Chemical compound C1C=CC[C@H]2C(=O)N(SC(Cl)(Cl)C(Cl)Cl)C(=O)[C@H]21 JHRWWRDRBPCWTF-OLQVQODUSA-N 0.000 description 2
- 238000005277 cation exchange chromatography Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000011210 chromatographic step Methods 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000002988 disease modifying antirheumatic drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012526 feed medium Substances 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 108010078144 glutaminyl-glycine Proteins 0.000 description 2
- 108010010147 glycylglutamine Proteins 0.000 description 2
- 108010084389 glycyltryptophan Proteins 0.000 description 2
- 108010087823 glycyltyrosine Proteins 0.000 description 2
- 108010037850 glycylvaline Proteins 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 2
- 108010034529 leucyl-lysine Proteins 0.000 description 2
- SXTAYKAGBXMACB-UHFFFAOYSA-N methionine sulfoximine Chemical compound CS(=N)(=O)CCC(N)C(O)=O SXTAYKAGBXMACB-UHFFFAOYSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000012261 overproduction Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108010070643 prolylglutamic acid Proteins 0.000 description 2
- ZCCUUQDIBDJBTK-UHFFFAOYSA-N psoralen Chemical compound C1=C2OC(=O)C=CC2=CC2=C1OC=C2 ZCCUUQDIBDJBTK-UHFFFAOYSA-N 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- 238000009121 systemic therapy Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 108010080629 tryptophan-leucine Proteins 0.000 description 2
- 108010044292 tryptophyltyrosine Proteins 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- VXGRJERITKFWPL-UHFFFAOYSA-N 4',5'-Dihydropsoralen Natural products C1=C2OC(=O)C=CC2=CC2=C1OCC2 VXGRJERITKFWPL-UHFFFAOYSA-N 0.000 description 1
- MJZJYWCQPMNPRM-UHFFFAOYSA-N 6,6-dimethyl-1-[3-(2,4,5-trichlorophenoxy)propoxy]-1,6-dihydro-1,3,5-triazine-2,4-diamine Chemical compound CC1(C)N=C(N)N=C(N)N1OCCCOC1=CC(Cl)=C(Cl)C=C1Cl MJZJYWCQPMNPRM-UHFFFAOYSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- WCBVQNZTOKJWJS-ACZMJKKPSA-N Ala-Cys-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(O)=O WCBVQNZTOKJWJS-ACZMJKKPSA-N 0.000 description 1
- DPNZTBKGAUAZQU-DLOVCJGASA-N Ala-Leu-His Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N DPNZTBKGAUAZQU-DLOVCJGASA-N 0.000 description 1
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 1
- OINVDEKBKBCPLX-JXUBOQSCSA-N Ala-Lys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OINVDEKBKBCPLX-JXUBOQSCSA-N 0.000 description 1
- CREYEAPXISDKSB-FQPOAREZSA-N Ala-Thr-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CREYEAPXISDKSB-FQPOAREZSA-N 0.000 description 1
- YJHKTAMKPGFJCT-NRPADANISA-N Ala-Val-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O YJHKTAMKPGFJCT-NRPADANISA-N 0.000 description 1
- GIVATXIGCXFQQA-FXQIFTODSA-N Arg-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N GIVATXIGCXFQQA-FXQIFTODSA-N 0.000 description 1
- ZJBUILVYSXQNSW-YTWAJWBKSA-N Arg-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O ZJBUILVYSXQNSW-YTWAJWBKSA-N 0.000 description 1
- QHUOOCKNNURZSL-IHRRRGAJSA-N Arg-Tyr-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O QHUOOCKNNURZSL-IHRRRGAJSA-N 0.000 description 1
- BVLIJXXSXBUGEC-SRVKXCTJSA-N Asn-Asn-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O BVLIJXXSXBUGEC-SRVKXCTJSA-N 0.000 description 1
- OLVIPTLKNSAYRJ-YUMQZZPRSA-N Asn-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N OLVIPTLKNSAYRJ-YUMQZZPRSA-N 0.000 description 1
- QUAWOKPCAKCHQL-SRVKXCTJSA-N Asn-His-Lys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N QUAWOKPCAKCHQL-SRVKXCTJSA-N 0.000 description 1
- WQLJRNRLHWJIRW-KKUMJFAQSA-N Asn-His-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC(=O)N)N)O WQLJRNRLHWJIRW-KKUMJFAQSA-N 0.000 description 1
- RCFGLXMZDYNRSC-CIUDSAMLSA-N Asn-Lys-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O RCFGLXMZDYNRSC-CIUDSAMLSA-N 0.000 description 1
- RBOBTTLFPRSXKZ-BZSNNMDCSA-N Asn-Phe-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RBOBTTLFPRSXKZ-BZSNNMDCSA-N 0.000 description 1
- HPBNLFLSSQDFQW-WHFBIAKZSA-N Asn-Ser-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O HPBNLFLSSQDFQW-WHFBIAKZSA-N 0.000 description 1
- HNXWVVHIGTZTBO-LKXGYXEUSA-N Asn-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O HNXWVVHIGTZTBO-LKXGYXEUSA-N 0.000 description 1
- KBQOUDLMWYWXNP-YDHLFZDLSA-N Asn-Val-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC(=O)N)N KBQOUDLMWYWXNP-YDHLFZDLSA-N 0.000 description 1
- YNQIDCRRTWGHJD-ZLUOBGJFSA-N Asp-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC(O)=O YNQIDCRRTWGHJD-ZLUOBGJFSA-N 0.000 description 1
- HSWYMWGDMPLTTH-FXQIFTODSA-N Asp-Glu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HSWYMWGDMPLTTH-FXQIFTODSA-N 0.000 description 1
- WSGVTKZFVJSJOG-RCOVLWMOSA-N Asp-Gly-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O WSGVTKZFVJSJOG-RCOVLWMOSA-N 0.000 description 1
- GYWQGGUCMDCUJE-DLOVCJGASA-N Asp-Phe-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(O)=O GYWQGGUCMDCUJE-DLOVCJGASA-N 0.000 description 1
- VNXQRBXEQXLERQ-CIUDSAMLSA-N Asp-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N VNXQRBXEQXLERQ-CIUDSAMLSA-N 0.000 description 1
- YIDFBWRHIYOYAA-LKXGYXEUSA-N Asp-Ser-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O YIDFBWRHIYOYAA-LKXGYXEUSA-N 0.000 description 1
- YUELDQUPTAYEGM-XIRDDKMYSA-N Asp-Trp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC(=O)O)N YUELDQUPTAYEGM-XIRDDKMYSA-N 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- NDUSUIGBMZCOIL-ZKWXMUAHSA-N Cys-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CS)N NDUSUIGBMZCOIL-ZKWXMUAHSA-N 0.000 description 1
- BPHKULHWEIUDOB-FXQIFTODSA-N Cys-Gln-Gln Chemical compound SC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O BPHKULHWEIUDOB-FXQIFTODSA-N 0.000 description 1
- OHLLDUNVMPPUMD-DCAQKATOSA-N Cys-Leu-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N OHLLDUNVMPPUMD-DCAQKATOSA-N 0.000 description 1
- SMEYEQDCCBHTEF-FXQIFTODSA-N Cys-Pro-Ala Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O SMEYEQDCCBHTEF-FXQIFTODSA-N 0.000 description 1
- KSMSFCBQBQPFAD-GUBZILKMSA-N Cys-Pro-Pro Chemical compound SC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 KSMSFCBQBQPFAD-GUBZILKMSA-N 0.000 description 1
- ALTQTAKGRFLRLR-GUBZILKMSA-N Cys-Val-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N ALTQTAKGRFLRLR-GUBZILKMSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241001212789 Dynamis Species 0.000 description 1
- LOJYQMFIIJVETK-WDSKDSINSA-N Gln-Gln Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(O)=O LOJYQMFIIJVETK-WDSKDSINSA-N 0.000 description 1
- NVEASDQHBRZPSU-BQBZGAKWSA-N Gln-Gln-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O NVEASDQHBRZPSU-BQBZGAKWSA-N 0.000 description 1
- GNMQDOGFWYWPNM-LAEOZQHASA-N Gln-Gly-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)CNC(=O)[C@@H](N)CCC(N)=O)C(O)=O GNMQDOGFWYWPNM-LAEOZQHASA-N 0.000 description 1
- FGYPOQPQTUNESW-IUCAKERBSA-N Gln-Gly-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)N)N FGYPOQPQTUNESW-IUCAKERBSA-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
- HMIXCETWRYDVMO-GUBZILKMSA-N Gln-Pro-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O HMIXCETWRYDVMO-GUBZILKMSA-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
- WPJDPEOQUIXXOY-AVGNSLFASA-N Gln-Tyr-Asn Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O WPJDPEOQUIXXOY-AVGNSLFASA-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
- HUFCEIHAFNVSNR-IHRRRGAJSA-N Glu-Gln-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HUFCEIHAFNVSNR-IHRRRGAJSA-N 0.000 description 1
- NJCALAAIGREHDR-WDCWCFNPSA-N Glu-Leu-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NJCALAAIGREHDR-WDCWCFNPSA-N 0.000 description 1
- AAJHGGDRKHYSDH-GUBZILKMSA-N Glu-Pro-Gln Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O AAJHGGDRKHYSDH-GUBZILKMSA-N 0.000 description 1
- BFEZQZKEPRKKHV-SRVKXCTJSA-N Glu-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)O)N)C(=O)N[C@@H](CCCCN)C(=O)O BFEZQZKEPRKKHV-SRVKXCTJSA-N 0.000 description 1
- NNQDRRUXFJYCCJ-NHCYSSNCSA-N Glu-Pro-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O NNQDRRUXFJYCCJ-NHCYSSNCSA-N 0.000 description 1
- DMYACXMQUABZIQ-NRPADANISA-N Glu-Ser-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O DMYACXMQUABZIQ-NRPADANISA-N 0.000 description 1
- ZALGPUWUVHOGAE-GVXVVHGQSA-N Glu-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZALGPUWUVHOGAE-GVXVVHGQSA-N 0.000 description 1
- WGYHAAXZWPEBDQ-IFFSRLJSSA-N Glu-Val-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WGYHAAXZWPEBDQ-IFFSRLJSSA-N 0.000 description 1
- XCLCVBYNGXEVDU-WHFBIAKZSA-N Gly-Asn-Ser Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O XCLCVBYNGXEVDU-WHFBIAKZSA-N 0.000 description 1
- IXKRSKPKSLXIHN-YUMQZZPRSA-N Gly-Cys-Leu Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O IXKRSKPKSLXIHN-YUMQZZPRSA-N 0.000 description 1
- BIRKKBCSAIHDDF-WDSKDSINSA-N Gly-Glu-Cys Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(O)=O BIRKKBCSAIHDDF-WDSKDSINSA-N 0.000 description 1
- AFWYPMDMDYCKMD-KBPBESRZSA-N Gly-Leu-Tyr Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AFWYPMDMDYCKMD-KBPBESRZSA-N 0.000 description 1
- JSLVAHYTAJJEQH-QWRGUYRKSA-N Gly-Ser-Phe Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JSLVAHYTAJJEQH-QWRGUYRKSA-N 0.000 description 1
- CQMFNTVQVLQRLT-JHEQGTHGSA-N Gly-Thr-Gln Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O CQMFNTVQVLQRLT-JHEQGTHGSA-N 0.000 description 1
- XHVONGZZVUUORG-WEDXCCLWSA-N Gly-Thr-Lys Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCCN XHVONGZZVUUORG-WEDXCCLWSA-N 0.000 description 1
- TVMNTHXFRSXZGR-IHRRRGAJSA-N His-Lys-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O TVMNTHXFRSXZGR-IHRRRGAJSA-N 0.000 description 1
- ALPXXNRQBMRCPZ-MEYUZBJRSA-N His-Thr-Phe Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ALPXXNRQBMRCPZ-MEYUZBJRSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-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
- NPAYJTAXWXJKLO-NAKRPEOUSA-N Ile-Met-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)O)N NPAYJTAXWXJKLO-NAKRPEOUSA-N 0.000 description 1
- NSPNUMNLZNOPAQ-SJWGOKEGSA-N Ile-Tyr-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N NSPNUMNLZNOPAQ-SJWGOKEGSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- 102000014158 Interleukin-12 Subunit p40 Human genes 0.000 description 1
- 108010011429 Interleukin-12 Subunit p40 Proteins 0.000 description 1
- 102100020790 Interleukin-12 receptor subunit beta-1 Human genes 0.000 description 1
- 101710103841 Interleukin-12 receptor subunit beta-1 Proteins 0.000 description 1
- 102100036672 Interleukin-23 receptor Human genes 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 1
- CQGSYZCULZMEDE-UHFFFAOYSA-N Leu-Gln-Pro Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)N1CCCC1C(O)=O CQGSYZCULZMEDE-UHFFFAOYSA-N 0.000 description 1
- GPICTNQYKHHHTH-GUBZILKMSA-N Leu-Gln-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O GPICTNQYKHHHTH-GUBZILKMSA-N 0.000 description 1
- HPBCTWSUJOGJSH-MNXVOIDGSA-N Leu-Glu-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HPBCTWSUJOGJSH-MNXVOIDGSA-N 0.000 description 1
- VWHGTYCRDRBSFI-ZETCQYMHSA-N Leu-Gly-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 1
- BKTXKJMNTSMJDQ-AVGNSLFASA-N Leu-His-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N BKTXKJMNTSMJDQ-AVGNSLFASA-N 0.000 description 1
- IAJFFZORSWOZPQ-SRVKXCTJSA-N Leu-Leu-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IAJFFZORSWOZPQ-SRVKXCTJSA-N 0.000 description 1
- VCHVSKNMTXWIIP-SRVKXCTJSA-N Leu-Lys-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O VCHVSKNMTXWIIP-SRVKXCTJSA-N 0.000 description 1
- AUNMOHYWTAPQLA-XUXIUFHCSA-N Leu-Met-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AUNMOHYWTAPQLA-XUXIUFHCSA-N 0.000 description 1
- UHNQRAFSEBGZFZ-YESZJQIVSA-N Leu-Phe-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N UHNQRAFSEBGZFZ-YESZJQIVSA-N 0.000 description 1
- WMIOEVKKYIMVKI-DCAQKATOSA-N Leu-Pro-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WMIOEVKKYIMVKI-DCAQKATOSA-N 0.000 description 1
- DPURXCQCHSQPAN-AVGNSLFASA-N Leu-Pro-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DPURXCQCHSQPAN-AVGNSLFASA-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
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 description 1
- MPGHETGWWWUHPY-CIUDSAMLSA-N Lys-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN MPGHETGWWWUHPY-CIUDSAMLSA-N 0.000 description 1
- WSXTWLJHTLRFLW-SRVKXCTJSA-N Lys-Ala-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O WSXTWLJHTLRFLW-SRVKXCTJSA-N 0.000 description 1
- NTSPQIONFJUMJV-AVGNSLFASA-N Lys-Arg-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O NTSPQIONFJUMJV-AVGNSLFASA-N 0.000 description 1
- YKIRNDPUWONXQN-GUBZILKMSA-N Lys-Asn-Gln Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N YKIRNDPUWONXQN-GUBZILKMSA-N 0.000 description 1
- KWUKZRFFKPLUPE-HJGDQZAQSA-N Lys-Asp-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWUKZRFFKPLUPE-HJGDQZAQSA-N 0.000 description 1
- RFQATBGBLDAKGI-VHSXEESVSA-N Lys-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCCN)N)C(=O)O RFQATBGBLDAKGI-VHSXEESVSA-N 0.000 description 1
- SKRGVGLIRUGANF-AVGNSLFASA-N Lys-Leu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SKRGVGLIRUGANF-AVGNSLFASA-N 0.000 description 1
- ODTZHNZPINULEU-KKUMJFAQSA-N Lys-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N ODTZHNZPINULEU-KKUMJFAQSA-N 0.000 description 1
- MIFFFXHMAHFACR-KATARQTJSA-N Lys-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CCCCN MIFFFXHMAHFACR-KATARQTJSA-N 0.000 description 1
- CAVRAQIDHUPECU-UVOCVTCTSA-N Lys-Thr-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAVRAQIDHUPECU-UVOCVTCTSA-N 0.000 description 1
- QLFAPXUXEBAWEK-NHCYSSNCSA-N Lys-Val-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O QLFAPXUXEBAWEK-NHCYSSNCSA-N 0.000 description 1
- XABXVVSWUVCZST-GVXVVHGQSA-N Lys-Val-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN XABXVVSWUVCZST-GVXVVHGQSA-N 0.000 description 1
- GILLQRYAWOMHED-DCAQKATOSA-N Lys-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN GILLQRYAWOMHED-DCAQKATOSA-N 0.000 description 1
- 239000012515 MabSelect SuRe Substances 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- FDJKUWYYUZCUJX-AJKRCSPLSA-N N-glycoloyl-beta-neuraminic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@@H]1O[C@](O)(C(O)=O)C[C@H](O)[C@H]1NC(=O)CO FDJKUWYYUZCUJX-AJKRCSPLSA-N 0.000 description 1
- FDJKUWYYUZCUJX-UHFFFAOYSA-N N-glycolyl-beta-neuraminic acid Natural products OCC(O)C(O)C1OC(O)(C(O)=O)CC(O)C1NC(=O)CO FDJKUWYYUZCUJX-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- QECVIPBZOPUTRD-UHFFFAOYSA-N N=S(=O)=O Chemical compound N=S(=O)=O QECVIPBZOPUTRD-UHFFFAOYSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- HXSUFWQYLPKEHF-IHRRRGAJSA-N Phe-Asn-Arg Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N HXSUFWQYLPKEHF-IHRRRGAJSA-N 0.000 description 1
- YYKZDTVQHTUKDW-RYUDHWBXSA-N Phe-Gly-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)O)N YYKZDTVQHTUKDW-RYUDHWBXSA-N 0.000 description 1
- BWTKUQPNOMMKMA-FIRPJDEBSA-N Phe-Ile-Phe Chemical compound C([C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 BWTKUQPNOMMKMA-FIRPJDEBSA-N 0.000 description 1
- JDMKQHSHKJHAHR-UHFFFAOYSA-N Phe-Phe-Leu-Tyr Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)CC1=CC=CC=C1 JDMKQHSHKJHAHR-UHFFFAOYSA-N 0.000 description 1
- SJRQWEDYTKYHHL-SLFFLAALSA-N Phe-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CC=CC=C3)N)C(=O)O SJRQWEDYTKYHHL-SLFFLAALSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004695 Polyether sulfone Substances 0.000 description 1
- OOLOTUZJUBOMAX-GUBZILKMSA-N Pro-Ala-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O OOLOTUZJUBOMAX-GUBZILKMSA-N 0.000 description 1
- NXEYSLRNNPWCRN-SRVKXCTJSA-N Pro-Glu-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NXEYSLRNNPWCRN-SRVKXCTJSA-N 0.000 description 1
- VPEVBAUSTBWQHN-NHCYSSNCSA-N Pro-Glu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O VPEVBAUSTBWQHN-NHCYSSNCSA-N 0.000 description 1
- KWMUAKQOVYCQJQ-ZPFDUUQYSA-N Pro-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@@H]1CCCN1 KWMUAKQOVYCQJQ-ZPFDUUQYSA-N 0.000 description 1
- FMLRRBDLBJLJIK-DCAQKATOSA-N Pro-Leu-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FMLRRBDLBJLJIK-DCAQKATOSA-N 0.000 description 1
- ULWBBFKQBDNGOY-RWMBFGLXSA-N Pro-Lys-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCCN)C(=O)N2CCC[C@@H]2C(=O)O ULWBBFKQBDNGOY-RWMBFGLXSA-N 0.000 description 1
- FDMKYQQYJKYCLV-GUBZILKMSA-N Pro-Pro-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 FDMKYQQYJKYCLV-GUBZILKMSA-N 0.000 description 1
- KBUAPZAZPWNYSW-SRVKXCTJSA-N Pro-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KBUAPZAZPWNYSW-SRVKXCTJSA-N 0.000 description 1
- QKDIHFHGHBYTKB-IHRRRGAJSA-N Pro-Ser-Phe Chemical compound N([C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C(=O)[C@@H]1CCCN1 QKDIHFHGHBYTKB-IHRRRGAJSA-N 0.000 description 1
- MKGIILKDUGDRRO-FXQIFTODSA-N Pro-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 MKGIILKDUGDRRO-FXQIFTODSA-N 0.000 description 1
- PRKWBYCXBBSLSK-GUBZILKMSA-N Pro-Ser-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O PRKWBYCXBBSLSK-GUBZILKMSA-N 0.000 description 1
- OOZJHTXCLJUODH-QXEWZRGKSA-N Pro-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 OOZJHTXCLJUODH-QXEWZRGKSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- UEJYSALTSUZXFV-SRVKXCTJSA-N Rigin Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O UEJYSALTSUZXFV-SRVKXCTJSA-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
- HZWAHWQZPSXNCB-BPUTZDHNSA-N Ser-Arg-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O HZWAHWQZPSXNCB-BPUTZDHNSA-N 0.000 description 1
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 1
- UGJRQLURDVGULT-LKXGYXEUSA-N Ser-Asn-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UGJRQLURDVGULT-LKXGYXEUSA-N 0.000 description 1
- KNCJWSPMTFFJII-ZLUOBGJFSA-N Ser-Cys-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(O)=O KNCJWSPMTFFJII-ZLUOBGJFSA-N 0.000 description 1
- MOVJSUIKUNCVMG-ZLUOBGJFSA-N Ser-Cys-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O)N)O MOVJSUIKUNCVMG-ZLUOBGJFSA-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
- XXXAXOWMBOKTRN-XPUUQOCRSA-N Ser-Gly-Val Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXXAXOWMBOKTRN-XPUUQOCRSA-N 0.000 description 1
- HMRAQFJFTOLDKW-GUBZILKMSA-N Ser-His-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O HMRAQFJFTOLDKW-GUBZILKMSA-N 0.000 description 1
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 0.000 description 1
- CUXJENOFJXOSOZ-BIIVOSGPSA-N Ser-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CO)N)C(=O)O CUXJENOFJXOSOZ-BIIVOSGPSA-N 0.000 description 1
- PYTKULIABVRXSC-BWBBJGPYSA-N Ser-Ser-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PYTKULIABVRXSC-BWBBJGPYSA-N 0.000 description 1
- DKGRNFUXVTYRAS-UBHSHLNASA-N Ser-Ser-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O DKGRNFUXVTYRAS-UBHSHLNASA-N 0.000 description 1
- NADLKBTYNKUJEP-KATARQTJSA-N Ser-Thr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NADLKBTYNKUJEP-KATARQTJSA-N 0.000 description 1
- HSWXBJCBYSWBPT-GUBZILKMSA-N Ser-Val-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)C(O)=O HSWXBJCBYSWBPT-GUBZILKMSA-N 0.000 description 1
- 230000029662 T-helper 1 type immune response Effects 0.000 description 1
- 210000000068 Th17 cell Anatomy 0.000 description 1
- IGROJMCBGRFRGI-YTLHQDLWSA-N Thr-Ala-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O IGROJMCBGRFRGI-YTLHQDLWSA-N 0.000 description 1
- DWYAUVCQDTZIJI-VZFHVOOUSA-N Thr-Ala-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DWYAUVCQDTZIJI-VZFHVOOUSA-N 0.000 description 1
- LAFLAXHTDVNVEL-WDCWCFNPSA-N Thr-Gln-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O LAFLAXHTDVNVEL-WDCWCFNPSA-N 0.000 description 1
- GKWNLDNXMMLRMC-GLLZPBPUSA-N Thr-Glu-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O GKWNLDNXMMLRMC-GLLZPBPUSA-N 0.000 description 1
- SXAGUVRFGJSFKC-ZEILLAHLSA-N Thr-His-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SXAGUVRFGJSFKC-ZEILLAHLSA-N 0.000 description 1
- 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 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
- RPECVQBNONKZAT-WZLNRYEVSA-N Thr-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H]([C@@H](C)O)N RPECVQBNONKZAT-WZLNRYEVSA-N 0.000 description 1
- SJPDTIQHLBQPFO-VLCNGCBASA-N Thr-Tyr-Trp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O SJPDTIQHLBQPFO-VLCNGCBASA-N 0.000 description 1
- YOPQYBJJNSIQGZ-JNPHEJMOSA-N Thr-Tyr-Tyr Chemical compound C([C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 YOPQYBJJNSIQGZ-JNPHEJMOSA-N 0.000 description 1
- OGOYMQWIWHGTGH-KZVJFYERSA-N Thr-Val-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O OGOYMQWIWHGTGH-KZVJFYERSA-N 0.000 description 1
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-N 0.000 description 1
- QNXZCKMXHPULME-ZNSHCXBVSA-N Thr-Val-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O QNXZCKMXHPULME-ZNSHCXBVSA-N 0.000 description 1
- NLWCSMOXNKBRLC-WDSOQIARSA-N Trp-Lys-Val Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O NLWCSMOXNKBRLC-WDSOQIARSA-N 0.000 description 1
- SSSDKJMQMZTMJP-BVSLBCMMSA-N Trp-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CC=C(O)C=C1 SSSDKJMQMZTMJP-BVSLBCMMSA-N 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- QYSBJAUCUKHSLU-JYJNAYRXSA-N Tyr-Arg-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O QYSBJAUCUKHSLU-JYJNAYRXSA-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
- SLCSPPCQWUHPPO-JYJNAYRXSA-N Tyr-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 SLCSPPCQWUHPPO-JYJNAYRXSA-N 0.000 description 1
- GZUIDWDVMWZSMI-KKUMJFAQSA-N Tyr-Lys-Cys Chemical compound NCCCC[C@@H](C(=O)N[C@@H](CS)C(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 GZUIDWDVMWZSMI-KKUMJFAQSA-N 0.000 description 1
- WURLIFOWSMBUAR-SLFFLAALSA-N Tyr-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC3=CC=C(C=C3)O)N)C(=O)O WURLIFOWSMBUAR-SLFFLAALSA-N 0.000 description 1
- MQGGXGKQSVEQHR-KKUMJFAQSA-N Tyr-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 MQGGXGKQSVEQHR-KKUMJFAQSA-N 0.000 description 1
- SCBITHMBEJNRHC-LSJOCFKGSA-N Val-Asp-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N SCBITHMBEJNRHC-LSJOCFKGSA-N 0.000 description 1
- KTEZUXISLQTDDQ-NHCYSSNCSA-N Val-Lys-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N KTEZUXISLQTDDQ-NHCYSSNCSA-N 0.000 description 1
- SBJCTAZFSZXWSR-AVGNSLFASA-N Val-Met-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N SBJCTAZFSZXWSR-AVGNSLFASA-N 0.000 description 1
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 1
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 1
- TVGWMCTYUFBXAP-QTKMDUPCSA-N Val-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N)O TVGWMCTYUFBXAP-QTKMDUPCSA-N 0.000 description 1
- GVNLOVJNNDZUHS-RHYQMDGZSA-N Val-Thr-Lys Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(O)=O GVNLOVJNNDZUHS-RHYQMDGZSA-N 0.000 description 1
- BGTDGENDNWGMDQ-KJEVXHAQSA-N Val-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](C(C)C)N)O BGTDGENDNWGMDQ-KJEVXHAQSA-N 0.000 description 1
- ZHWZDZFWBXWPDW-GUBZILKMSA-N Val-Val-Cys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CS)C(O)=O ZHWZDZFWBXWPDW-GUBZILKMSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 108010050025 alpha-glutamyltryptophan Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 1
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012575 bio-layer interferometry Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 238000003320 cell separation method Methods 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 238000011443 conventional therapy Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011026 diafiltration Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 1
- 108010050475 glycyl-leucyl-tyrosine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000019734 interleukin-12 production Effects 0.000 description 1
- 108040003610 interleukin-12 receptor activity proteins Proteins 0.000 description 1
- 108040001844 interleukin-23 receptor activity proteins Proteins 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 108010027338 isoleucylcysteine Proteins 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 108010024654 phenylalanyl-prolyl-alanine Proteins 0.000 description 1
- 108010073101 phenylalanylleucine Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000001126 phototherapy Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920006393 polyether sulfone Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010079317 prolyl-tyrosine Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- 230000009450 sialylation Effects 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 108010071097 threonyl-lysyl-proline Proteins 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 108010071635 tyrosyl-prolyl-arginine Proteins 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229960003824 ustekinumab Drugs 0.000 description 1
- 108010052774 valyl-lysyl-glycyl-phenylalanyl-tyrosine Proteins 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
-
- 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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The present application relates to a method for preparing anti-IL-12 and/or IL-23 monoclonal antibodies, in particular Wu Sinu monoclonal antibodies. Compared with antibodies expressed by SP2/0 cells and CHO-K1 cells, the antibodies obtained by the method have higher yield, simple screening steps and higher biological activity.
Description
Cross Reference to Related Applications
The present application claims priority to patent application No.202110174184.3, entitled "method for preparing anti-IL-12 and/or IL-23 monoclonal antibodies," filed 2 and 9 of 2021 to the national intellectual property agency of china, the contents of which are incorporated herein by reference in their entirety.
Technical Field
The present application relates to a method for preparing anti-IL-12 and/or IL-23 monoclonal antibodies, in particular Wu Sinu monoclonal antibodies.
Background
Interleukins are a class of cytokines that are produced by and act on a variety of cells. Interleukin-12 (IL-12) is a cytokine with broad biological activity, produced mainly by B cells and macrophages, whose molecule is a heterodimer consisting of two subunits, p35 and p 40. Interleukin-23 (IL-23) has a structure homologous to IL-12 and exists as a heterodimer with an alpha chain of p19 containing 189 amino acids and a beta chain identical to p40 of IL-12. Its function is to activate stimulated T cells, promote their proliferation and production of interferon gamma, and induce proliferation of memory T cells. IL-12 and IL-23 play an important role in a series of processes such as maturation, activation, proliferation and immunoregulation of immune cells, and in addition, they are involved in a variety of physiological and pathological responses of the body.
Because IL-12 and IL-23 are involved in a variety of disorders, several therapeutic strategies have been designed to inhibit IL-12 and/or IL-23 activity. Among them, drug Wu Sinu mab (us tekinumab) of qinsheng company was approved by us FDA in 2009 for use in treatment of patients with moderate and severe plaque type psoriasis in adults. Wu Sinu monoclonal antibody is IgG1 antibody targeting common subunit p40 of IL-12 and IL-23, is a fully human monoclonal antibody, and can inhibit the bioactivity of IL-12 and IL-23, thereby preventing the differentiation of initial T cells to Th1 and Th17 cells and further inhibiting the development of inflammatory reaction. Monoclonal antibodies have been approved for the treatment of plaque psoriasis, psoriatic arthritis and crohn's disease in both the united states and europe Wu Sinu, and for the treatment of moderate to severe plaque psoriasis and adult crohn's disease indications in china.
The Wu Sinu monoclonal antibody from Qiangsheng corporation is an antibody expressed by SP2/0 cells, and the antibody expressed by the SP2/0 mouse myeloma cells has low yield and high immunogenicity (due to the sialylation and alpha-1, 3 galactosylation of NGNA). Therefore, there is a need to develop new methods for expressing antibodies, which further increase the yield and reduce the immunogenicity while guaranteeing the biological activity of the antibodies.
Disclosure of Invention
In a first aspect, the invention provides a method for preparing an anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment thereof, comprising expressing said monoclonal antibody or said antigen-binding fragment using a CHO cell with a DHFR or GS gene knockout, said monoclonal antibody or said antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequences of VH-CDR1, VH-CDR2 and VH-CDR3 as shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively; the light chain variable region comprises a VL-CDR1, a VL-CDR2 and a VL-CDR3, the amino acid sequences of which are shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively.
In one embodiment, the anti-IL-12 and/or IL-23 monoclonal antibodies or antigen binding fragments thereof comprise a heavy chain variable region shown in SEQ ID NO. 7 and a light chain variable region shown in SEQ ID NO. 8.
In one embodiment, the monoclonal antibody is a Wu Sinu monoclonal antibody.
The inventors have unexpectedly found, through a number of experiments, that the use of DHFR knock-out CHO cells or GS knock-out CHO cells for expression of the monoclonal antibodies has unexpectedly higher biological activity and at the same time higher yields than antibodies expressed by SP2/0 cells and CHO-K1 cells.
In one embodiment, the invention provides a method of preparing an anti-IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof, the method comprising the steps of:
a) Cloning the anti-IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment coding sequence thereof into an expression vector to obtain a recombinant expression vector, preferably the expression vector contains a selectable marker gene, more preferably the selectable marker gene is a DHFR gene or a GS gene;
b) Transferring the recombinant expression vector obtained in the step a) into DHFR gene knocked-out CHO cells or GS gene knocked-out CHO cells to obtain genetically engineered CHO cells;
c) Culturing the genetically engineered CHO cell obtained in step b), selecting a monoclonal cell strain expressing said antibody;
d) Culturing the monoclonal cell strain expressing the antibody obtained in the step c), harvesting a cell culture solution containing the antibody, and separating and purifying to obtain the monoclonal antibody.
In one embodiment, the method comprises the steps of:
a) Cloning the coding sequence of the anti-IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof into an expression vector to obtain a recombinant expression vector, preferably the expression vector contains a selectable marker gene, more preferably the selectable marker gene is a DHFR gene or a GS gene;
b) Transferring the recombinant expression vector obtained in the step a) into DHFR gene knocked-out CHO cells or GS gene knocked-out CHO cells to obtain genetically engineered CHO cells;
c) Culturing the genetically engineered CHO cell obtained in step b), selecting a monoclonal cell strain highly expressing said antibody;
d) Culturing the monoclonal cell strain which is obtained in the step c) and is used for efficiently expressing the antibody, harvesting a cell culture solution containing the antibody, and separating and purifying to obtain the monoclonal antibody.
In one embodiment, the DHFR knock-out CHO cell is a CHO-DG44 cell.
In one embodiment, the GS gene knocked-out CHO cell is obtained by knocking out the GS gene using Zinc Finger Nuclease (ZFN) technology or using CRISPR methods.
In one embodiment, the GS knock-out CHO cell is-GS cells.
In one embodiment, the method comprises the steps of:
a) Cloning the coding sequence of the anti-IL-12 and/or IL-23 monoclonal antibody or the binding fragment thereof into an expression vector to obtain a recombinant expression vector, wherein the expression vector contains a GS gene;
b) Transferring the recombinant expression vector obtained in the step a)GS cells, genetically engineered -GS cells;
c) Engineering the gene obtained in step b)-GS cells are cultured in glutamine-free medium, selecting a monoclonal cell line expressing the antibody;
d) Culturing the monoclonal cell strain expressing the antibody obtained in the step c), harvesting a cell culture solution containing the antibody, and separating and purifying to obtain the monoclonal antibody.
In one embodiment, the cell culture process described in step d of the method employs a fed-batch culture mode.
In a second aspect, the invention provides an anti-IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof, by the method of the first aspect of the invention.
In a third aspect, the invention provides a pharmaceutical composition comprising a monoclonal antibody produced by the method of the first aspect of the invention, sucrose, L-histidine hydrochloride monohydrate and polysorbate 80.
In one embodiment, the pharmaceutical composition comprises 90+ -9 mg/mL of monoclonal antibody, 76+ -7.6 mg/mL of sucrose, 0.5+ -0.3 mg/mL of L-histidine, 1+ -0.5 mg/mL of L-histidine hydrochloride monohydrate, and 0.04+ -0.004 mg/mL of polysorbate 80 prepared by the method of the first aspect of the present invention.
In one embodiment, the pharmaceutical composition comprises 90mg/mL of the monoclonal antibody, 76mg/mL of sucrose, 0.636mg/mL of L-histidine, 1.237mg/mL of L-histidine hydrochloride monohydrate, and 800.04mg/mL of polysorbate prepared by the method of the first aspect of the present invention.
In a fourth aspect, the invention provides a pharmaceutical composition comprising a monoclonal antibody produced by the method of the first aspect of the invention, L-histidine hydrochloride monohydrate, polysorbate 80, sucrose, EDTA disodium salt dihydrate, and L-methionine.
In one embodiment, the pharmaceutical composition comprises 5.+ -. 0.5mg/mL, 0.77.+ -. 0.077mg/mL L-histidine, 1.03.+ -. 0.10mg/mL L-histidine hydrochloride monohydrate, 80.4.+ -. 0.04mg/mL polysorbate, 85.+ -. 8.5mg/mL sucrose, 0.02.+ -. 0.002mg/mL EDTA disodium salt dihydrate, and 0.4.+ -. 0.04mg/mL L-methionine of monoclonal antibody produced by the method of the first aspect of the invention.
In one embodiment, the pharmaceutical composition comprises 5mg/mL of monoclonal antibody, 0.77mg/mL of L-histidine, 1.03mg/mL of L-histidine hydrochloride monohydrate, 0.4mg/mL of polysorbate, 85mg/mL of sucrose, 0.02mg/mL of EDTA disodium salt dihydrate, and 0.4mg/mL of L-methionine prepared by the method of the first aspect of the present invention.
In a fifth aspect, the invention provides the use of an anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment thereof produced by the method of the first aspect of the invention or a pharmaceutical composition of the third or fourth aspect of the invention in the manufacture of a medicament for the treatment of a disease or condition associated with IL-12 and/or IL-23.
In one embodiment, the disease or condition is selected from plaque psoriasis, psoriatic arthritis, crohn's disease, systemic lupus erythematosus, ulcerative colitis, multiple sclerosis, sarcoidosis, axial spondyloarthritis, and ankylosing spondylitis. Preferably, the disease or condition is selected from plaque psoriasis, psoriatic arthritis and crohn's disease.
The invention has the following beneficial effects:
1. the inventors have unexpectedly found that the monoclonal antibodies produced by the methods of the invention have greater than 20% inhibitory activity against IL-12 and/or IL-23 than the original drug. By adopting the technical means of the invention, the inhibition activity of the monoclonal antibody to IL-12 and/or IL-23 can be obviously improved.
2. The monoclonal antibody produced by the method of the invention can have a yield of about 1.5 g/L.
Detailed Description
Interleukin-12 (also known as IL-12, a cytotoxic lymphocyte maturation factor, or a natural killer cell stimulation factor) is a heterodimer consisting of one p35 and one p40 subunit, and many research results indicate that IL-12 plays a key role in promoting Th1 immune responses and host defense against intracellular pathogens. In addition, IL-12 may promote the development of some autoimmune and inflammatory diseases in driving a Th1 response. Interleukin-23 (IL-23) is a heterodimer consisting of an IL-12p40 subunit and its own p19 subunit, the receptor system of which shares the IL-12Rβ1 subunit with IL-12R (β1 and β2), and the specific receptor subunit of which is IL-23R.
The biological effects of IL-12 and IL-23 are different from each other. IL-12 is secreted by activated inflammatory cells (monocytes, macrophages, neutrophils, microglia, dendritic cells). During inflammation, IL-12 induces production of interferon-gamma (IFN-gamma) by NK cells (natural killer cells) and T cells. IL-12 may then combine with IFN-gamma to induce T cells to differentiate into Th1 cells. This response stimulates the cellular immune system and maximizes the killing effect of macrophages on pathogens and CD8 + Proliferation of T cells (Trinchiier 2003Nat Rev Immunol 3 133-46). Overproduction of IL-12 is associated with increased pro-inflammatory reactivity and autoimmune typical tissue damage (Leonard et al 1997Crit Rev Immunol 17545-53). Dysregulation of IL-12 production involves the following diseases: psoriasis (Yawalmar et al, 1998J Invest Dermatol 111 1053-7), crohn's disease (Neurath et al, 1995J Exp Med 182 1281-90; simpson et al A,1998J Exp Med 187 1225-34; camoglio et al, 2002Eur J Immunol 32 261-9), multiple sclerosis (Fassbender et al, 1998Neurology 51 753-8; raman et al, 1998J Neuroimmunol 86 30-45), rheumatoid arthritis (Kim et al, 2000 Clin Exp Immunol 119 175-81), among other autoimmune diseases.
Because IL-12 and IL-23 contain common subunits, it is difficult to attribute a disease state to overproduction of only one interleukin or the other. However, studies have shown that IL-23 imbalance is involved in the following diseases: psoriasis (Lee et al, 2004J Exp Med 199 125-30), crohn's disease (Neurath 2007Nat Med 13 26-8) and multiple sclerosis (Cua et al, 2003Nature 421 744-8), among other autoimmune diseases.
The term "anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment thereof" as used herein refers to an antibody or antigen-binding fragment thereof that specifically binds to IL-12 and/or IL-23, preferably, an anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment thereof "binds to IL-12 and/or IL-23, resulting in inhibition of the biological activity of IL-12 and/or IL-23; more preferably, the anti-IL-12 and/or IL-23 monoclonal antibodies or antigen binding fragments thereof are anti-IL-12 and IL-23 monoclonal antibodies or antigen binding fragments thereof, causing inhibition of the biological activity of IL-12 and/or IL-23; more preferably, the heavy chain variable region of the monoclonal antibody comprises: the amino acid sequences are shown as VH-CDR1, VH-CDR2 and VH-CDR3 shown in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3 respectively; the light chain variable region of the monoclonal antibody comprises: the amino acid sequences are shown as VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively; more preferably, the monoclonal antibody comprises a heavy chain variable region shown in SEQ ID NO. 7 and a light chain variable region shown in SEQ ID NO. 8; most preferably, the monoclonal antibody is Wu Sinu mab. As used herein, the term "inhibit" refers to a decrease in the biological activity of a cytokine when an antibody specifically binds to the cytokine. Inhibition of IL-12 and/or IL-23 biological activity by antibodies can be assessed by measuring one or more indicators of IL-12 and/or IL-23 biological activity, as is well known in the art.
The term "Wu Sinu mab" (ustekinumab) is a commercial productFully human IgG1 kappa antibodies that bind to the p40 subunits of IL-12 and IL-23, thereby blocking inflammatory responses in patients. The heavy and light chain sequences of Wu Sinu mab are as set forth in SEQ ID NOs: 9 and SEQ ID NO: shown at 10. Monoclonal antibodies have been licensed on the market in europe and america for the treatment of moderate to severe plaque psoriasis, active psoriasis, and moderate to severe active crohn's disease. Wunumumab has been approved in China for the treatment of moderate to severe plaque psoriasis and adult Crohn's diseaseSymptomatic treatment
In the present invention, the term "standard" is used to denote a product that has been approved by the market as compared to a biologically similar product. In the case where the antibody produced is Wu Sinu mab, the standard is approvedAnd (5) a product.
The term "CHO cells" as used herein, is derived from the epithelium of Chinese hamster ovary (Tijo and Puck (1958) J.Exp. Med. 108:259-271); the term "genetically engineered CHO cells" refers to CHO cells modified or altered by any suitable genetic means and methods known to those of skill in the art such that they express a particular protein. Preferably, the genetically engineered CHO cell may express said monoclonal antibody or antigen binding fragment thereof. More preferably, the genetically engineered CHO cell is obtained by transfecting a recombinant expression vector comprising the coding sequence of the monoclonal antibody or antigen binding fragment thereof into a corresponding CHO cell line. Preferably, the CHO cell is a dihydrofolate reductase (DHFR) gene knockout or a Glutamine Synthetase (GS) gene knockout CHO cell; preferably, the DHFR knock-out CHO cells are CHO-DG44 cells. Preferably, the GS gene knocked-out CHO cell is obtained by knocking out the GS gene using Zinc Finger Nuclease (ZFN) technology or using CRISPR method; more preferably, the GS knock-out CHO cell is -GS cells.
As used herein, "CHO-DG44 cells" refers to CHO cells from which the double allelic dihydrofolate reductase (DHFR) gene has been knocked out, and are currently widely used mammalian cell expression systems for recombinant protein expression developed by Thermo-Fisher. Herein, the GS knock-out CHO cells are, for example, CHO cells using Zinc Finger Nuclease (ZFN) technology or using CRISPR method, e.g., merck companyGS lactationAnimal cell expression system using ∈ ->The Zinc Finger Nuclease (ZFN) technology is used for knocking out the endogenous Glutamine Synthetase (GS) gene of the CHO-K1 cell strain, so that the cell growth process is required to depend on the addition of exogenous L-glutamine to form a natural screening mark, and meanwhile, the dependence of a GS screening system on L-Methionine Sulfonimide (MSX) is eliminated. When the plasmid is not transferred, the cell strain can survive in a glutamine-containing culture medium, but after the plasmid is transferred (GS gene is present on the plasmid), the cell strain can survive in a glutamine-free culture medium, so that the screening process is simplified without adding resistant screening substances such as MSX and the like when screening the high-expression cell strain, and the method is more suitable for industrial expansion production.
The inventors have made extensive experiments and have unexpectedly found that CHO cells knocked out with DHFR (dihydrofolate reductase) or GS (glutamine synthetase), in particular CHO-DG44 cells or The GS cells express the monoclonal antibodies, and compared with the monoclonal antibodies expressed by SP2/0 cells and CHO-K1 cells, the monoclonal antibodies, especially Wu Sinu monoclonal antibodies, have higher biological activity, high yield, low immunogenicity, good cell stability and simple screening steps.
The method for preparing the monoclonal antibody comprises the following steps:
a) Cloning the monoclonal antibody coding sequence into an expression vector to obtain a recombinant expression vector, preferably the expression vector contains a selection marker gene, more preferably the selection marker gene is a DHFR gene or a GS gene;
b) Transferring the recombinant expression vector obtained in the step a) into DHFR gene knocked-out CHO cells or GS gene knocked-out CHO cells to obtain genetically engineered CHO cells;
c) Culturing the genetically engineered CHO cell obtained in step b), selecting a monoclonal cell strain expressing said antibody;
d) Culturing the monoclonal cell strain expressing the antibody obtained in the step c), harvesting a cell culture solution containing the antibody, and separating and purifying to obtain the monoclonal antibody.
Methods for cloning antibody coding sequences into expression vectors to construct recombinant expression vectors suitable for expression of antibodies in CHO cells are well known to those skilled in the art, e.g., as described in Makrides et al (1999) Protein expr. Purif.17:183-202 and Kaufman (2000) mol. Biotechnol.16:151-161. The expression vector refers to a vector which is formed by adding an expression element (such as a promoter, a terminator and the like) on the basis of the basic skeleton of a cloning vector so that a target gene can be expressed, such as a recombinant plasmid and the like. Preferably, the expression vector contains a selectable marker gene. The selectable marker gene encodes a protein as follows: (a) conferring resistance to antibiotics or other toxins, (b) supplementing auxotrophs, or (c) providing critical nutrients not available from complex media. More preferably, the selectable marker gene is a dihydrofolate reductase gene or a glutamine synthetase gene. Methods of genetically engineering CHO cells are well known to those skilled in the art and include, inter alia, co-transfecting CHO cells with one expression vector containing genes encoding the heavy and light chains of an antibody or with a first expression vector containing genes encoding the heavy chain of an antibody and a second expression vector containing genes encoding the light chain of an antibody. Methods of transferring recombinant expression vectors into CHO cells, including electroporation, calcium phosphate precipitation, lipofection, DEAE-dextran transfection, and also including the use of commercial transfection kits, such as FreeStyle from Thermo fisher Scientific TM MAX Reagent, etc.
Culturing the obtained genetically engineered CHO cells in a culture medium to obtain a monoclonal cell strain capable of efficiently expressing antibodies. Wherein, for CHO cells from which DHFR (dihydrofolate reductase) gene is knocked out, a corresponding inhibitor, such as methotrexate (Methotrexate, MTX), is added to the target medium according to the selectable marker gene carried on the transfected recombinant expression vector,Etc., thereby obtaining a cell line capable of efficiently expressing an antibody; in the case of a CHO cell having a weakened GS (glutamine synthetase) gene, since it is necessary that the cell survive in a glutamine-containing medium without transferring the cell into a plasmid, and that the cell strain having a high-efficiency antibody expression can be obtained by culturing the cell strain in a glutamine-free medium after transferring the cell strain into a plasmid (having a GS gene on the plasmid), the cell strain is obtained by culturing the cell strain in a glutamine-free medium without adding methionine iminosulfone (methionine sulfoximine, MSX) to the medium>And the like. Then monoclonal screening is carried out to obtain a monoclonal cell strain expressing the antibody; preferably, a monoclonal cell line is obtained which is capable of expressing the antibody with high efficiency.
The obtained genetically engineered CHO cells are cultured in a suitable medium to produce antibodies. The term "medium" as used herein refers to a solution containing nutrients necessary for the growth of mammalian cells. Typically, the medium provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements necessary for the cells.
There are different modes of culture for genetically engineered CHO cells, including batch, continuous and fed-batch cultures. In the present invention, the antibody is preferably produced using a fed-batch culture method. In a fed-batch culture process, the culture is started with a certain amount of medium, and in a subsequent culture process one or more nutrients are supplemented in order to prevent depletion of the nutrients, during which process no product is removed from the cell culture medium.
After production of antibodies by genetically engineered CHO cells, the antibodies are harvested by separating the cell culture broth containing the antibodies from the cells at the end of the culture process, since recombinant proteins expressed from mammalian cells, in particular antibodies, are usually secreted into the cell culture broth during the culture process. Cell separation methods should minimize cell lysis, avoid increased cell debris and release of proteases and other molecules that may affect the quality of immunoglobulin products. Cell culture fluids comprising antibodies are typically obtained by centrifugation and/or filtration, with recombinant proteins present in the supernatant or filtrate, respectively.
After obtaining the cell culture broth containing the antibody, the antibody must be purified from the cell culture broth. Purification of recombinant proteins is typically accomplished by a series of chromatographic steps such as anion exchange chromatography, cation exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and the like. Purification of the recombinant antibody typically begins with protein a affinity chromatography followed by one or more additional chromatographic steps, such as cation exchange chromatography. Further, the purification step may include ultrafiltration, diafiltration, etc.
As described above, the present inventors found that Wu Sinu mab comparison standard produced by the method of the present inventionThe product has higher bioactivity. The biological activity of Wu Sinu monoclonal antibodies is manifested by an ability to bind to IL-23, an ability to bind to IL-12, an ability to bind to FcRn, and/or an inhibition of IL-12 and/or IL-23 induced gene expression, particularly an inhibition of IL-12 and/or IL-23 induced gene expression. The ability of Wu Sinu mab to bind to IL-23, IL-12, or FcRn can be assessed using, but is not limited to, ELISA and biolayer interferometry; the inhibition of IL-12-induced gene expression by monoclonal antibody Wu Sinu can be studied by cells (e.g., T lymphocytes) that respond to IL-12, IL-12 and antibody samples, and then detecting the production of a target molecule, such as interferon-gamma (IFN-gamma). The target molecule interferon-gamma (IFN-gamma) can be detected and/or quantified by any suitable method, such as ELISA.
The inhibition effect of the Wu Sinu monoclonal antibody produced by the method of the invention on IFN-gamma production is greater than that of a standard product, and the inhibition effect of the Wu Sinu monoclonal antibody produced by the method of the invention on IFN-gamma production is more than 20% of that of the standard product.
The invention also relates to a pharmaceutical composition comprising the anti-IL-12 and/or IL-23 monoclonal antibody or antigen binding fragment thereof prepared by the method. The pharmaceutical composition of the invention comprises at least one pharmaceutically acceptable excipient in addition to the monoclonal antibody. Pharmaceutically acceptable excipients refer to substances that are capable of stabilizing the pharmaceutical ingredients and/or increasing the solubility or reducing the viscosity of the pharmaceutical composition without affecting the physiological activity of the active substance, such as Wu Sinu mab. Common pharmaceutical excipients for monoclonal antibodies include buffers, salts, sugars or sugar alcohols, amino acids and surfactants.
In one embodiment, a pharmaceutical composition comprises the monoclonal antibody, sucrose, L-histidine hydrochloride monohydrate, and polysorbate 80; preferably, the pharmaceutical composition comprises 90+ -9 mg/mL of the monoclonal antibody, 76+ -7.6 mg/mL of sucrose, 0.5+ -0.3 mg/mL of L-histidine, 1+ -0.5 mg/mL of L-histidine hydrochloride monohydrate and 0.04+ -0.004 mg/mL of polysorbate 80. More preferably, the pharmaceutical composition comprises 90mg/mL of the monoclonal antibody, 76mg/mL of sucrose, 0.636mg/mL of L-histidine, 1.237mg/mL of L-histidine hydrochloride monohydrate and 0.04mg/mL of polysorbate 80.
In another embodiment, a pharmaceutical composition comprises the monoclonal antibody, sucrose, L-histidine hydrochloride monohydrate, EDTA disodium salt dihydrate, L-methionine, and polysorbate 80. Preferably, the pharmaceutical composition comprises: the monoclonal antibody is 5+/-0.5 mg/mL, L-histidine is 0.77+/-0.077 mg/mL, L-histidine hydrochloride monohydrate is 1.03+/-0.10 mg/mL, polysorbate is 80.4+/-0.04 mg/mL, sucrose is 85+/-8.5 mg/mL, disodium EDTA dihydrate is 0.02+/-0.002 mg/mL and L-methionine is 0.4+/-0.04 mg/mL. More preferably, the pharmaceutical composition comprises: the monoclonal antibody is 5mg/mL, L-histidine is 0.77mg/mL, L-histidine hydrochloride monohydrate is 1.03mg/mL, polysorbate is 0.4mg/mL, sucrose is 85mg/mL, disodium EDTA dihydrate is 0.02mg/mL and L-methionine is 0.4mg/mL.
The anti-IL-12 and/or IL-23 monoclonal antibodies or antigen binding fragments thereof or the pharmaceutical compositions of the invention can be used for preparing medicaments for treating IL-12 and/or IL-23 related diseases or conditions; preferably, the disease or condition is selected from plaque psoriasis, psoriatic arthritis, systemic lupus erythematosus, crohn's disease, ulcerative colitis, multiple sclerosis, sarcoidosis, axial spondyloarthritis and ankylosing spondylitis. More preferably, the disease or condition is selected from plaque psoriasis, psoriatic arthritis and crohn's disease.
In one embodiment, the anti-IL-12 and/or IL-23 monoclonal antibodies or antigen-binding fragments thereof or the pharmaceutical compositions of the invention are useful in the treatment of plaque psoriasis, particularly in adults with moderate to severe plaque psoriasis, who are not responsive to other systemic therapies (including cyclosporine, methotrexate (MTX) or PUVA (psoralen and uv a), or who are contraindicated, or who are intolerant, and in patients who are ill or intolerant in the teenagers of 12 years and older, who are insufficiently controlled or intolerant to other systemic therapies or phototherapy therapies, and in the treatment of psoriatic arthritis, especially in adults with an insufficient therapeutic effect on the traditional non-biological disease modifying antirheumatic drug (DMARD).
In another embodiment, the anti-IL-12 and/or IL-23 monoclonal antibodies or antigen-binding fragments thereof or the pharmaceutical compositions of the invention are useful in treating Crohn's disease, particularly in adult patients with moderate to severe active Crohn's disease, who are hyporesponsive, unresponsive, intolerant, or medically contraindicated to conventional therapies or TNF-a antagonists.
A composition or method described herein as "comprising" or "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are necessary, but that other elements or steps may be added within the scope of the composition or method. Known or disclosed equivalents of any named essential elements or steps may be substituted for those elements or steps in any of the compositions or methods disclosed herein.
Detailed Description
EXAMPLE 1 construction of CHO-DG44 cell line expressing Wu Sinu monoclonal antibody
Vector construction
(1) Preparation of vector for expressing antibody heavy chain protein
Two restriction enzyme sites of BamHI and Xhol are designed at two ends of a coding sequence (SEQ ID No: 11) of a Wu Sinu monoclonal antibody heavy chain and are respectively connected with the BamHI restriction enzyme sites and the Xhol restriction enzyme sites on an expression vector pCDNA3.1 (purchased from Thermo Fisher Scientific company), so that the connection of a target protein coding DNA fragment and the expression vector is realized, and recombinant plasmid DNA is obtained. The recombinant plasmid DNA was introduced into E.coli DH5 a (purchased from Tian Gen Biochemical technologies Co., ltd.) and the single colony plasmid was extracted and sent to sequencing to obtain a plasmid with the correct sequencing result, which was designated as recombinant plasmid pCDNA-HC.
(2) Preparation of antibody light chain protein expression vector
Two restriction sites of HindIII and Xhol are designed at two ends of a coding sequence (SEQ ID No: 12) of a light chain of Wu Sinu monoclonal antibody and are respectively connected with HindIII and Xhol restriction sites on an expression vector pOptivec (purchased from Thermo Fisher Scientific company) to realize connection of a target protein coding DNA fragment and the expression vector, so as to obtain recombinant plasmid DNA, the recombinant plasmid DNA is introduced into escherichia coli DH5 a (purchased from Tiangen Biochemical technologies Co., ltd.), a single colony plasmid is extracted and sent to sequencing, so that a plasmid with a correct sequencing result is obtained, and the plasmid is recorded as recombinant plasmid pOptivec-LC.
Construction of CHO-DG44 cell line expressing Wu Sinu monoclonal antibody
The recombinant plasmids pOptiVec-LC and pCDNA-HC were linearized, and transfected into CHO-DG44 cells (purchased from Thermo Fisher Scientific company) at the same time, and cultured to obtain a cell line with high expression of the target protein. The method comprises the following specific steps:
(1) Transfection of CHO-DG44 cells with recombinant plasmids
The plasmid was linearized using the PvuI cleavage sites on the recombinant plasmids pOptiVec-LC and pCDNA-HC. The digested plasmid DNA was concentrated to a concentration of 1ug/ul by ethanol precipitation.
Taking 1.5X10 7 The CHO-DG44 cells were replaced with 30mL of a medium (available from Thermo Fisher Scientific company) of CD DG44 preheated to 37℃and mixed well; 60ug of plasmid DNA (pcDNA-HC to pOptiVec-LC molar ratio=1:1) and 15uL of FreeStyle were added TM MAX Reagent (available from Thermo Fisher Scientific company) put 1.2mL OptiPRO TM SFM medium (from Thermo Fisher Scienti)fic company) and gently mixed, incubated at room temperature for 10min to obtain a DNA-Reagent complex. 1.2mL of the DNA-Reagent complex was added to 125mL of CHO-DG44 cell shake flask, the shake flask was gently shaken, the shake flask was placed in a shaker at 37℃with 8% CO 2 Culturing at 130-135 rpm. 48 hours after transfection, the cells were passaged to hypoxanthine/thymine (HT) -deficient CD OptiCHO TM Complete medium (from Gibco Life Technologies).
(2) Screening of stably transfected cells
First wheelScreening: counting the cell lines obtained in the previous step; contains 500ug/mL +.>CD OptiCHO reagent (available from Thermo Fisher Scientific Co.) TM The cells were diluted in complete medium (available from Gibco Life Technologies company) to give a final density of 5X 10 cells 5 Individual living cells/mL; placing the flask in incubator at 37deg.C with 8% CO 2 Is cultured under humidified air at a rotation speed of 130-135 rpm. Changing fresh culture medium every 2-3 days, and maintaining for 14-21 days until cell survival rate>90%。
Second round MTX gradient screening: the cells from the first round of screening were counted, centrifuged at 1000rpm for 5 minutes, and the old medium was aspirated off and the cells were inoculated into CD Optifer containing 50nM MTX TM Complete medium (available from Gibco Life Technologies; 500. Mu.g/mL)) Inoculation density of 3X 10 5 cells/mL. Placing the flask in incubator at 37deg.C with 8% CO 2 Is cultured under humidified air at a rotation speed of 130-135 rpm. The fresh medium was changed every 2-3 days, and when the cell viability was greater than 80%, the cells were inoculated to CD OptiCHO containing 100nM MTX TM Complete medium (from Gibco Life Technologies; containing 500. Mu.g/mL +.>) Inoculation density of 3X 10 5 cells/mL, the flask was placed in an incubator at 37℃with 8% CO 2 Is cultured under humidified air at a rotation speed of 130-135 rpm. The fresh medium was changed every 2-3 days, and cells were inoculated to CD OptiCHO containing 500nM MTX at a cell viability of greater than 80% TM Complete medium (from Gibco Life Technologies; containing 500. Mu.g/mL +.>) Inoculation density of 3X 10 5 cell/mL, total incubation time of 20-30 days, up to cell viability>90%。
(3) Monoclonal screening and expanded culture
Cells from the previous step were serially diluted to 1000 viable cells/mL with cloning medium EX-CELL CHO Cloning Medium (purchased from Sigma Aldrich) and seeded into 96-well plates with 0.8 cells per well. The inoculated 96-well plate was placed at 37℃and 5% CO 2 The plates were incubated at rest in an incubator under air conditions for 10-14 days, in this example 11 days, and the growth of the monoclonal colonies in the wells was examined using a microscope.
When cells in the 96-well plate grow to 80% confluence, the yield of each clone is detected by ELISA method, specifically, the content of Wu Sinu monoclonal antibody in the fermentation liquid is detected by double antibody sandwich method, the coated antibody is murine monoclonal anti-human IgG Fc segment antibody (purchased from Sigma, cat# I6260-.5 mL), the secondary antibody is HRP-labeled murine monoclonal anti-human IgG Fab segment antibody (purchased from Kirschner Biotechnology Co., cat# A01855), and the standard substance is original grinding agentThe maximum concentration of dilution was 1ug/mL, and 12 standards were double-graded diluted.
Screening of high yield top 25% clones for gExpansion, sterile collection of the desired monoclonal cells, amplification to 24 well plates. After 3-5 days, the same assay was used to select the first 10% higher yield target clones, which were transferred to 6 well plates and cultured continuously. Using the same assay, the first 10% yield of monoclonal cells in 6-well plates were selected and each cell line was individually transferred into T-25 flasks for expansion when the number of cells in the T-25 flasks reached 1 x 10 7 After that, the cells were transferred to 125mL flasks. At 37℃and with 8% CO 2 Under air condition, shaking speed is 130-135rpm, and fed-batch culture mode is adopted, and feed medium Dynamis is added during culture TM Medium (available from Thermo Fisher Co.) was cultured for 14 days, and then cell fermentation broth of CHO-DG44 cell strain highly expressing Wu Sinu monoclonal antibody was collected.
EXAMPLE 2 expression of Wu Sinu monoclonal antibodyConstruction of GS cell lines
Vector construction
(1) Expression of antibody heavy chain proteins and vector ligation
Two restriction enzyme sites BstB I and Pac I are designed at two ends of a coding sequence of a Wu Sinu monoclonal antibody heavy chain and are respectively connected with BstB I and Pac I restriction enzyme sites on an expression vector pCGS (purchased from Merck KGaA company), so that the connection of a target protein coding DNA fragment and the expression vector is realized, and the recombinant plasmid DNA is obtained. The recombinant plasmid DNA was introduced into E.coli DH5 a (available from Tian Gen Biochemical Co., ltd.) and spread on LB agar medium containing 100ug/ml ampicillin (final concentration) (10 g of tryptone, 5g of yeast extract, 10g of NaCl, and then 1L of deionized water were added to prepare per liter of medium, 950ml of deionized water). The obtained clone was cultured in LB liquid medium containing 100ug/ml ampicillin (final concentration), and plasmids were extracted with a plasmid extraction kit (purchased from Tiangen Biotechnology Co., ltd.) to perform nucleic acid sequencing and identification, and the plasmid with the correct sequencing result was selected and recorded as recombinant plasmid pCGS-HC.
(2) Expression antibody light chain protein and vector ligation
Two restriction enzyme sites of HindIII and Xhol are designed at two ends of the coding sequence of the light chain of the Wu Sinu monoclonal antibody and are respectively connected with the HindIII and Xhol restriction enzyme sites on the recombinant plasmid pCGS-HC, so that the connection of the target protein coding DNA fragment and the expression vector is realized, and the novel recombinant plasmid DNA is obtained. The recombinant plasmid DNA was introduced into E.coli DH5 a (available from Tian Gen Biochemical Co., ltd.) and spread on LB agar medium (prepared per liter of medium, 950ml of deionized water was added with 10g of tryptone, 5g of yeast extract, 10g of NaCl, and then deionized water was added to 1L) containing 100ug/ml of ampicillin. The obtained clone was cultured in LB liquid medium containing 100ug/ml ampicillin (final concentration), and plasmids were extracted with a plasmid extraction kit (purchased from Tiangen Biotechnology Co., ltd.) to perform nucleic acid sequencing and identification, and the plasmid with the correct sequencing result was selected and recorded as recombinant plasmid pCGS-HC-LC.
Cell line selection
Transfection of recombinant plasmid pCGS-HC-LC intoGS cells (purchased from Merck KGaA company) were screened for cell lines highly expressing the protein of interest. The method comprises the following specific steps:
(1) Recombinant plasmid transfection-GS cells
a. Cells were passaged to a density of 1X 10 at day before transfection 6 Living cells/mL.
b. Cell count, total number of cells required per sample was 6×10 6 The desired cells were centrifuged at 1000rpm for 10min in a 50mL centrifuge tube.
c. Each sample was resuspended in 600. Mu.LEX-CELL CD CHO medium (from Sigma Aldrich) and 40. Mu.g of plasmid (plasmid concentration > 1. Mu.g/. Mu.L) was added and mixed well before being placed in a refrigerator with an electric rotor for pre-cooling at 4 ℃.
d. The cells with the plasmids added were blown uniformly and then added to an electrorotating cup, electrorotated at 300V, and after completion, added to a medium EX-CELL CD CHO Fusion (purchased from Sigma Aldrich Co.) containing 6mM glutamine preheated at 37℃and placed in a carbon dioxide incubator for resting culture for 48 hours.
(2) Screening of stably transfected cells
After 48h, the cells were passaged into glutamine-free medium EX-CELL CD CHO Fusion (purchased from Sigma Aldrich). Every 2 days of passage, each generation of cells is labeled "P1", "P2". The term "Pn", where n is the number of cell generations. Before each passage, 40. Mu.L of the cell suspension was used for cell counting and cell viability analysis by a Countstar IC 1000 automatic cell counter to determine the viable cell density and viable cell viability of each generation of cells. When the cell viability was >95%, the screening process was completed. The process can also be accomplished by screening cell pools (minipool).
(3) Monoclonal screening and expanded culture
Cells from the previous step were seeded into 96-well plates, 0.5-1 cells per well.
a. 400mL of complete cloning medium EX-CELL CHO Cloning Medium (from Sigma Aldrich) was taken and preheated to 37 ℃;
b. counting the cells to be plated, and calculating the number of cells required, namely, 20 96-well plates, wherein 960 cells are required according to 0.5 cells per well;
c. continuously diluting cells to the final concentration of 1000 living cells/mL by using the complete cloning culture medium, and taking 960uL of cell dilution liquid into the preheated cloning culture medium;
d. gently mixing the cell suspension, and transferring to a sterile sample adding tank; 200 μl of diluted cells were dispensed into 96-well plates under sterile conditions using a multichannel pipette;
e. at 37deg.C, contains 5% CO 2 The plates were incubated at rest in an incubator under air conditions for 10-14 days, in this example 11 days, and the growth of the monoclonal colonies in the wells was examined using a microscope.
When cells in the 96-well plate grew to 80% confluence, the same ELISA method as in example 1 was used to examine the yield of each clone, the first 25% clone with high productivity was screened for clonal expansion, the desired clone was collected aseptically, and the entire contents of each well were gently blown up and down and transferred to each well of the 24-well plate, which contained 1mL of fresh medium EX-CELL CD CHO Fusion (available from Sigma Aldrich Co.) in each well of the 24-well plate.
After 3-5 days, using the same assay, the target clone with the highest yield of 10% before selection was transferred to a 6-well plate, the desired clone was collected aseptically, and fresh medium EX-CELL CD CHO Fusion was added to each well of the 6-well plate, and the culture volume of each well was 3mL.
Using the same assay, the first 10% yield of the monoclonal cell line in 6-well plates was selected and each cell line was individually transferred into T-25 flasks for expansion when the number of cells in the T-25 flasks reached 1 x 10 7 After that, the cells were transferred to 125mL flasks. At 37℃and with 8% CO 2 Under air conditions at a shaking speed of 130-135rpm, using fed-batch mode,
during the culture, a feed medium EX-CELL Advanced CHO Feed 1 (purchased from Sigma Aldrich Co.) was added, and after 14 days of culture, wu Sinu monoclonal antibody was collectedCell fermentation broth of GS cell lines.
EXAMPLE 3 antibody purification procedure
CHO-DG44 cell strain with high expression Wu Sinu monoclonal antibody collected and-cell fermentation broths of GS cell lines, respectively:
1. centrifuging
Setting the rotating speed of a centrifugal machine to 3000rpm, centrifuging for 10min, and removing cells; the centrifuge was then spun at 15000rpm for 20min to remove cell debris. The step can also adopt a deep filtration method to clarify the cell fermentation liquor.
2. Microfiltration of
The samples were microfiltered using a sterilizing grade filter (pore size 0.22 μm, material polyethersulfone) from the company por.
3. Elution
Instrument: AKTA protein purification instrument and Mabselect sure LX Filler (all purchased from GE company)
Reagent: balancing solution: 150mM NaCl, 20mM phosphate buffer pH7.4
Eluent 1:1M NaCl 20mM sodium citrate pH5.5
Eluent 2:20mM sodium citrate pH5.5
Eluent: 20mM sodium citrate pH3.0
Regeneration liquid: 0.1M NaOH
The steps are as follows: 1) Balancing by using the balancing liquid; 2) Loading a sample; 3) Leaching: sequentially eluting with eluent 2, eluent 1 and eluent 2; 4) Eluting with the above eluent; 5) Cleaning In Place (CIP) with 0.5M sodium hydroxide; 6) Rebalancing: the balancing solution was used for balancing.
The CHO-DG44 cell strain is used for producing the Wunumumab, and the Wu Sinu mab product with the purity of 97% is finally obtained, and the yield is about 1.5 g/L.
UsingProduction of Wu Sinu mab by cells of the GS cell line resulted in a final product of Wu Sinu mab with 98% purity, with a yield of about 1.5 g/L.
EXAMPLE 4 IL-12 inhibitory Activity of CHO-DG44 cell-expressed Wu Sinu monoclonal antibody
Isolation and activation of T cells
Peripheral Blood Mononuclear Cells (PBMC) from Lei Debei L were subjected to EasySep Human T Cell Isolation Kit protocol and total CD3 was isolated by removing non-T cells with magnetic bead sorting reagent (STEM cell) + T cells. T cells at a concentration of 1X10 6 The culture medium was either 1640 medium (from Sigma Aldrich) containing 10% FBS (fetal bovine serum) (Gibco Life Technologies) or TexMACX medium (from Miltenyi Biotec) without serum (from Thermo Fisher Scientific) and the stimulator anti-CD3/CD28 antibody (from Thermo Fisher Scientific) was added while IL-2 (30 u/mL) was added to the T cells when culturing them.
Will be 1.0X10 6 ml -1 The T cells were pre-stimulated with magnetic beads to produce gamma-interferon (gamma-IFN) and after 2 days of stimulation, the cell concentration was adjusted to 0.5X10 by centrifugation at day 3 6 ml -1 (change to TexMACX medium).
IL-12 ELISA
The strips required for the assay were removed from the sealed bags equilibrated to room temperature using a human IFN-. Gamma.ELISA kit (available from Shanghai Eisethion Inc.), leaving Bai Kong empty. Regulating the T cells obtained in the previous step to 0.5X10 5 Each well was then filled with IL-12, a Wu Sinu mab sample of the present invention (hereinafter simply referred to as "sample") and a standard in the amounts shown in Table 1 (100 ul/well), and cell activity experiments were conducted in accordance with the design of Table 1. The reaction wells were sealed with sealing plate gummed paper and incubated for 90 minutes at 37 ℃. Plates were washed 5 times and biotinylated antibody working fluid (100 ul/well) was added in addition to blank wells. The reaction wells were sealed with sealing plate gummed paper and incubated for 60 minutes at 37 ℃. The plate was washed 5 times and enzyme conjugate working solution (100 ul/well) was added in addition to the blank wells. Sealing the reaction hole with sealing plate gummed paper, incubating the incubator at 37 ℃ and incubating for 30 minutes in a dark place. The plate was washed 5 times, 100 ul/well of chromogenic substrate (including blank wells) was added and incubated at 37℃for 15 minutes in the incubator protected from light. Stop solution (including blank wells) was added at 100 ul/well and OD450nm was measured immediately after mixing.
Cell activity experiment and results of the monoclonal antibody sample of Table 1 Wu Sinu
Inhibition ratio = (Abs (IL-12 average value) –Abs (antibody) )/(Abs (IL-12 average value) –Abs (average value of cell supernatant) )
According to the data result and the inhibition rate calculation formula in Table 1, the inhibition rate of the CHO-DG44 expressed Wu Sinu monoclonal antibody sample on the IL-12 activity is 43.19%, the inhibition rate of the standard substance on the IL-12 activity is 34.9%, and the inhibition activity of the CHO-DG44 expressed Wu Sinu monoclonal antibody sample on the IL-12 is 23.753% higher than that of the Wu Sinu monoclonal antibody standard substance.
Example 5Wu Sinu monoclonal antibodies expressed by GS cells and inhibiting activity on IL-12
Isolation and activation of T cells
Peripheral Blood Mononuclear Cells (PBMC) from Lei Debei L were subjected to EasySep Human T Cell Isolation Kit protocol and total CD3 was isolated by removing non-T cells with magnetic bead sorting reagent (STEM cell) + T cells. T cells at a concentration of 1X10 6 In culture on a 12-well plate in X-vivo (available from Lonza) medium, the stimulator anti-CD3/CD28 antibody (available from Thermo Fisher Scientific) was added, and IL-2 (30 u/mL) (available from Thermo Fisher Scientific) was added at the same time when T cells were cultured.
Modulating T cell concentration to 1X10 6 And each mL. Will be 1.0X10 6 ml -1 Pre-stimulating T cells with magnetic beads to activate them and produce gamma-interferon (gamma-IFN), and adjusting cell concentration to 0.3X10 by centrifugation 2 days after stimulation 6 ml -1 。
IL-12ELISA
This step was performed using a human IFN-. Gamma.ELISA kit (available from Shanghai Eichhorny Corp.) and a plate 96-well plate required for the test was removed from a sealed bag equilibrated to room temperature, IL-12 was added to the plate 96-well plate (50 uL/well) in the amount shown in Table 2, a sample of Wu Sinu monoclonal antibody of the present invention (hereinafter simply referred to as "sample") and a standard substance were added to the corresponding well (50 uL/well) in the amount shown in Table 2, and after incubation in an incubator for 1 hour, the T cells obtained in the previous step were centrifuged to remove the original medium, and the medium was adjusted to 0.3X10 with X-vio 5 Each well was added to the corresponding well (100 uL/well). The reaction wells were sealed with sealing plate gummed paper and incubated for 90 minutes at 37 ℃. Plates were washed 5 times and biotinylated antibody working solution (100 ul/well) was added. The reaction wells were sealed with sealing plate gummed paper and incubated for 60 minutes at 37 ℃. The plate was washed 5 times and enzyme conjugate working solution (100 ul/well) was added. Sealing the reaction hole with sealing plate gummed paper, incubating the incubator at 37 ℃ and incubating for 30 minutes in a dark place. The plate was washed 5 times, and 100 ul/well of chromogenic substrate was added and incubated at 37℃in an incubator for 15 minutes in the absence of light. 100 ul/well of stop solution is addedOD450nm was measured immediately after mixing.
Table 2, wu Sinu, cell Activity experiments and results of monoclonal antibody samples
From the data in table 2, the inhibition ratios of the respective antibodies shown in table 3 were calculated using the following inhibition ratio calculation formula.
Inhibition ratio = (Abs (IL-12) –Abs (Standard) )/(Abs (IL-12) –Abs (cell supernatant) )
TABLE 3 treatment of experimental results
Antibodies to | 0.5ug standard | 0.5ug sample | 1ug standard | 1ug sample |
Inhibition ratio (%) | 9.52 | 11.67 | 27.64 | 37.98 |
It was calculated that, when the experiment was performed using 0.5ug Wu Sinu mab,the inhibition activity of the GS-expressed Wu Sinu monoclonal antibody sample is 22.58% higher than that of the Wu Sinu monoclonal antibody sample on IL-12, and when 1ug Wu Sinu monoclonal antibody is used for the experiment, the sample is->The GS-expressed Wu Sinu mab sample has 37.41% higher IL-12 inhibition activity than the Wu Sinu mab standard.
From the experimental results of example 4 and example 5, it can be seen that the Wu Sinu monoclonal antibody sample of the invention has an inhibitory activity on IL-12 higher than that of the Wu Sinu monoclonal antibody standard by more than 20%.
Unless otherwise noted, the raw materials and reagents used in the specific examples of the present invention are commercially available.
The present invention has been illustrated by the above-described embodiments, but it should be understood that the above-described embodiments are for purposes of illustration and description only and are not intended to limit the invention to the embodiments described. In addition, it will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that many variations and modifications are possible in light of the teachings of the invention, which variations and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Gan Li pharmaceutical Co., ltd
<120> a method for producing an anti-IL-12 and/or IL-23 monoclonal antibody
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Thr Tyr Trp Leu Gly
1 5
<210> 2
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe Gln
1 5 10 15
Gly
<210> 3
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe
1 5 10
<210> 4
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala
1 5 10
<210> 5
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Ala Ala Ser Ser Leu Gln Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Gln Gln Tyr Asn Ile Tyr Pro Tyr Thr
1 5
<210> 7
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr
20 25 30
Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile
35 40 45
Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr
65 70 75 80
Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 8
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 9
<211> 449
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr
20 25 30
Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile
35 40 45
Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr
65 70 75 80
Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ser Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 10
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 11
<211> 1413
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
gccaccatgg agttcggcct gtcctgggtg ttcctggtgg ccctgttcag gggcgtgcag 60
tgtgaggtgc agctggtgca gtccggcgcc gaggtgaaga agcctggcga gtccctgaag 120
atcagctgca agggctccgg ctactccttc accacatact ggctgggctg ggtgaggcag 180
atgcccggca agggcctgga ttggatcggc atcatgagcc ccgtggatag cgatattagg 240
tacagcccta gcttccaggg ccaggtgaca atgtccgtgg ataagagcat cacaaccgcc 300
tacctgcagt ggaacagcct gaaggccagc gacaccgcca tgtactactg cgccaggaga 360
agacccggcc agggctactt cgacttctgg ggccagggca ccctggtgac agtgagcagc 420
agcagcacca agggccctag cgtgttcccc ctggccccct ctagcaagtc cacctccggc 480
ggcaccgccg ctctgggatg tctggtgaag gactacttcc ccgagcccgt gaccgtgagc 540
tggaactccg gcgccctgac atccggcgtg cacacattcc ccgccgtgct gcagtccagc 600
ggcctgtact ccctgagctc cgtggtgaca gtgccctcca gctccctggg cacccagacc 660
tacatctgca acgtgaacca caagcctagc aacacaaagg tggacaagag ggtggagccc 720
aagagctgcg acaagacaca cacctgccct ccctgtcctg cccctgagct gctgggcgga 780
ccttccgtgt tcctgttccc ccctaagccc aaggacacac tgatgatctc cagaacaccc 840
gaggtgacat gtgtggtggt ggatgtgagc cacgaggacc ccgaggtgaa gttcaactgg 900
tacgtggacg gcgtggaggt gcacaacgcc aagacaaagc ctcgggagga gcagtacaac 960
tccacataca gagtggtgtc cgtgctgacc gtgctgcacc aggattggct gaacggcaag 1020
gagtacaagt gcaaggtgag caacaaggcc ctgcccgccc ctatcgagaa gaccatctcc 1080
aaggccaagg gccagcccag ggagccccag gtttacacac tgccccccag cagagatgag 1140
ctgaccaaga accaggtgtc cctgacctgc ctggtgaagg gcttctaccc ctccgatatt 1200
gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac accccccgtg 1260
ctggatagcg atggcagctt cttcctgtac agcaagctga ccgtggataa gtccagatgg 1320
cagcagggca acgtgttcag ctgttccgtg atgcacgagg ccctgcacaa ccactacaca 1380
cagaagagcc tgtccctgag ccctggcaag tga 1413
<210> 12
<211> 717
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gccaccatgg acatgagggt gcctgcccag ctgctgggcc tgctgctgct gtggctgagc 60
ggcgctagat gcgatattca gatgacacag tcccctagca gcctgagcgc cagcgtgggc 120
gatagagtga ccatcacatg cagggccagc cagggcatct ccagctggct ggcctggtac 180
cagcagaagc ctgagaaggc ccctaagtcc ctgatctacg ccgcctccag cctgcagagc 240
ggcgtgccta gcaggttctc cggcagcggc agcggcacag atttcaccct gacaatcagc 300
tccctgcagc ccgaggactt cgccacctac tactgccagc agtacaacat ctacccctac 360
acattcggcc agggcaccaa gctggagatc aagaggacag tggccgcccc cagcgtgttc 420
atcttccctc ctagcgacga gcagctgaag agcggcacag cctccgtggt gtgtctgctg 480
aacaacttct accctcggga ggccaaggtg cagtggaagg tggacaacgc cctgcagtcc 540
ggcaactccc aggagagcgt gaccgagcag gatagcaagg actccaccta ctccctgagc 600
tccacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg 660
acccaccagg gcctgtccag ccccgtgacc aagagcttca acagaggcga gtgctga 717
Claims (15)
1. A method of making an anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment thereof, comprising expressing the monoclonal antibody or the antigen-binding fragment using DHFR knockout or GS knockout CHO cells, the monoclonal antibody or the antigen-binding fragment comprising a heavy chain variable region comprising VH-CDR1, VH-CDR2 and VH-CDR3 having amino acid sequences set forth in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively; the light chain variable region comprises a VL-CDR1, a VL-CDR2 and a VL-CDR3 which are respectively shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; wherein the GS knock-out CHO cell is A cell; the DHFR knock-out CHO cells are CHO-DG44 cells.
2. The method of claim 1, wherein the anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID No. 7 and a light chain variable region set forth in SEQ ID No. 8.
3. The method of claim 2, wherein the monoclonal antibody is a Wu Sinu monoclonal antibody.
4. A method according to any one of claims 1-3, comprising the steps of:
a) Cloning the monoclonal antibody coding sequence into an expression vector to obtain a recombinant expression vector;
b) Transferring the recombinant expression vector obtained in the step a) into DHFR gene knocked-out CHO cells or GS gene knocked-out CHO cells to obtain genetically engineered CHO cells;
c) Culturing the genetically engineered CHO cell obtained in step b), selecting a monoclonal cell strain expressing said antibody;
d) Culturing the monoclonal cell strain expressing the antibody obtained in the step c), harvesting a cell culture solution containing the antibody, and separating and purifying to obtain the monoclonal antibody.
5. The method of claim 4, wherein the expression vector in step a) comprises a selectable marker gene.
6. The method according to claim 5, wherein the selectable marker gene in step a) is a DHFR gene or a GS gene.
7. An anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment thereof, prepared by the method of any one of claims 1-6.
8. A pharmaceutical composition comprising the monoclonal antibody produced by the method of any one of claims 1-6, sucrose, L-histidine hydrochloride monohydrate, and polysorbate 80.
9. The pharmaceutical composition of claim 8, comprising 90±9mg/mL of the monoclonal antibody, 76±7.6mg/mL of sucrose, 0.5±0.3mg/mL of L-histidine, 1±0.5mg/mL of L-histidine hydrochloride monohydrate, and 800.04 ±0.004mg/mL of polysorbate.
10. The pharmaceutical composition of claim 9, comprising 90mg/mL of the monoclonal antibody, 76mg/mL of sucrose, 0.636mg/mL of L-histidine, 1.237mg/mL of L-histidine hydrochloride monohydrate, and 0.04mg/mL of polysorbate 80.
11. A pharmaceutical composition comprising the monoclonal antibody produced by the method of any one of claims 1-6, L-histidine hydrochloride monohydrate, polysorbate 80, sucrose, EDTA disodium salt dihydrate, and L-methionine.
12. The pharmaceutical composition of claim 11, comprising 5±0.5mg/mL of the monoclonal antibody, 0.77±0.077mg/mL of L-histidine hydrochloride monohydrate 1.03±0.10mg/mL, 0.4±0.04mg/mL of polysorbate 80, 85±8.5mg/mL of sucrose, 0.02±0.002mg/mL of EDTA disodium salt dihydrate, and 0.4±0.04mg/mL of L-methionine.
13. The pharmaceutical composition of claim 12, comprising 5mg/mL of the monoclonal antibody, 0.77mg/mL of L-histidine, 1.03mg/mL of L-histidine hydrochloride monohydrate, 0.4mg/mL of polysorbate 80, 85mg/mL of sucrose, 0.02mg/mL of EDTA disodium salt dihydrate, and 0.4mg/mL of L-methionine.
14. Use of an anti-IL-12 and/or IL-23 monoclonal antibody or antigen-binding fragment prepared by the method of any one of claims 1-6, or a pharmaceutical composition of any one of claims 8-13, in the manufacture of a medicament for treating an IL-12 and/or IL-23 related disease or disorder selected from plaque psoriasis, psoriatic arthritis, crohn's disease, systemic lupus erythematosus, ulcerative colitis, multiple sclerosis, sarcoidosis, central axial spondyloarthritis, and ankylosing spondylitis.
15. The use according to claim 14, the disease or condition being selected from plaque psoriasis, psoriatic arthritis and crohn's disease.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2021101741843 | 2021-02-09 | ||
CN202110174184 | 2021-02-09 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114395041A CN114395041A (en) | 2022-04-26 |
CN114395041B true CN114395041B (en) | 2023-12-01 |
Family
ID=81232608
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210109484.8A Active CN114395041B (en) | 2021-02-09 | 2022-01-29 | Method for preparing anti-IL-12 and/or IL-23 monoclonal antibody |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114395041B (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100021911A1 (en) * | 2008-07-23 | 2010-01-28 | Boehringer Ingelheim Pharma Gmbh & Co Kg | Production host cell lines |
CN102876684A (en) * | 2012-09-17 | 2013-01-16 | 青岛康立泰药业有限公司 | Coding gene, eukaryotic host cell and expression method of human interleukin-12 |
WO2020183418A1 (en) * | 2019-03-14 | 2020-09-17 | Janssen Biotech, Inc. | Manufacturing methods for producing anti-il12/il23 antibody compositions |
-
2022
- 2022-01-29 CN CN202210109484.8A patent/CN114395041B/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100021911A1 (en) * | 2008-07-23 | 2010-01-28 | Boehringer Ingelheim Pharma Gmbh & Co Kg | Production host cell lines |
CN102876684A (en) * | 2012-09-17 | 2013-01-16 | 青岛康立泰药业有限公司 | Coding gene, eukaryotic host cell and expression method of human interleukin-12 |
WO2020183418A1 (en) * | 2019-03-14 | 2020-09-17 | Janssen Biotech, Inc. | Manufacturing methods for producing anti-il12/il23 antibody compositions |
Non-Patent Citations (2)
Title |
---|
Jianwei Zhu等.Mammalian cell protein expression for biopharmaceutical production.Biotechnology Advances.2011,第30卷(第5期),1158-1170. * |
杨林等.细胞因子对类风湿性关节炎骨平衡的调节作用.生命科学.2019,第31卷(第1期),35-43. * |
Also Published As
Publication number | Publication date |
---|---|
CN114395041A (en) | 2022-04-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
TWI791894B (en) | Novel interleukin 2 and use thereof | |
TW516962B (en) | A human TNFR1-IgG1 preparation | |
TW202017576A (en) | Biologically relevant orthogonal cytokine/receptor pairs | |
WO2021185362A1 (en) | Interleukin-2 mutant and use thereof | |
TW202045547A (en) | Dll3 targeting chimeric antigen receptors and binding agents | |
MXPA04005190A (en) | Cell culture process. | |
CN102007146A (en) | Methods for manufacturing a polyclonal protein | |
SA94150015A (en) | lymphokine production and purification | |
CN102994441A (en) | Cell culture medium, and preparation method and use thereof | |
KR20210102194A (en) | Implantable devices and related methods for cell therapy | |
US20220064610A1 (en) | Mutated piggybac transposase | |
CN103012590A (en) | Anti-CD20 monoclonal antibody, preparation method and application thereof | |
JP2012525826A (en) | CHO / CERT cell line | |
EP3101035B1 (en) | Bifunctional fusion protein, preparation method therefor, and use thereof | |
CN114395041B (en) | Method for preparing anti-IL-12 and/or IL-23 monoclonal antibody | |
CN111378046A (en) | Immune effector cell conversion receptor | |
CN112458149B (en) | Method for detecting activation degree of antibody drug on T cells by taking CD3 as target | |
KR20230160823A (en) | Compositions and methods for therapeutic delivery | |
KR101452286B1 (en) | Method for culturing cell expressing gdf5 as single clone in serum free medium with mtx and zeocin | |
TWI834031B (en) | Interleukin 2 mutein and the use thereof | |
WO1992021763A1 (en) | Enhancement of expression by gene targeting in endogenous retrovirus-like sequences | |
WO2022228429A1 (en) | Bcma-targeting chimeric antigen receptor and use thereof | |
US20110189734A1 (en) | Novel methods and cell lines | |
JPH0763363B2 (en) | Method for obtaining fused cells | |
JP2024517711A (en) | Hypersialylated Cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |