CA2908331A1 - Human-mouse chimeric anti-cd147 antibody with non-fucosylated glycosylation - Google Patents
Human-mouse chimeric anti-cd147 antibody with non-fucosylated glycosylation Download PDFInfo
- Publication number
- CA2908331A1 CA2908331A1 CA2908331A CA2908331A CA2908331A1 CA 2908331 A1 CA2908331 A1 CA 2908331A1 CA 2908331 A CA2908331 A CA 2908331A CA 2908331 A CA2908331 A CA 2908331A CA 2908331 A1 CA2908331 A1 CA 2908331A1
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- seq
- cell line
- metuzumab
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000013595 glycosylation Effects 0.000 title claims description 24
- 238000006206 glycosylation reaction Methods 0.000 title claims description 24
- 239000013598 vector Substances 0.000 claims abstract description 51
- 239000012634 fragment Substances 0.000 claims abstract description 49
- 239000002773 nucleotide Substances 0.000 claims abstract description 47
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 47
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 46
- 241000282414 Homo sapiens Species 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 30
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical group C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims abstract description 18
- 230000014509 gene expression Effects 0.000 claims abstract description 18
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 claims abstract description 17
- 201000010099 disease Diseases 0.000 claims abstract description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 14
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 13
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims abstract description 9
- 101000798441 Homo sapiens Basigin Proteins 0.000 claims abstract description 7
- 102100032412 Basigin Human genes 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 134
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 claims description 24
- 230000000694 effects Effects 0.000 claims description 23
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 18
- 239000002246 antineoplastic agent Substances 0.000 claims description 17
- 229940127089 cytotoxic agent Drugs 0.000 claims description 17
- 238000012258 culturing Methods 0.000 claims description 15
- 229950006780 n-acetylglucosamine Drugs 0.000 claims description 14
- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 claims description 13
- 230000002950 deficient Effects 0.000 claims description 13
- 229920001542 oligosaccharide Polymers 0.000 claims description 13
- 150000002482 oligosaccharides Chemical class 0.000 claims description 13
- 102000004357 Transferases Human genes 0.000 claims description 12
- 108090000992 Transferases Proteins 0.000 claims description 12
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 12
- 229960004316 cisplatin Drugs 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 12
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 claims description 12
- 229960005277 gemcitabine Drugs 0.000 claims description 12
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 9
- 201000005202 lung cancer Diseases 0.000 claims description 9
- 208000020816 lung neoplasm Diseases 0.000 claims description 9
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 8
- 229930012538 Paclitaxel Natural products 0.000 claims description 5
- 229940086322 navelbine Drugs 0.000 claims description 5
- 229960001592 paclitaxel Drugs 0.000 claims description 5
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 claims description 5
- CILBMBUYJCWATM-PYGJLNRPSA-N vinorelbine ditartrate Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O.OC(=O)[C@H](O)[C@@H](O)C(O)=O.C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC CILBMBUYJCWATM-PYGJLNRPSA-N 0.000 claims description 5
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000005571 anion exchange chromatography Methods 0.000 claims description 3
- 238000011282 treatment Methods 0.000 abstract description 13
- 206010028980 Neoplasm Diseases 0.000 description 30
- 108020004705 Codon Proteins 0.000 description 17
- 201000011510 cancer Diseases 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 11
- 239000012636 effector Substances 0.000 description 11
- 150000002772 monosaccharides Chemical class 0.000 description 11
- 230000005764 inhibitory process Effects 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 8
- 108700010070 Codon Usage Proteins 0.000 description 8
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 8
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 8
- 239000000427 antigen Substances 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 239000013604 expression vector Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 239000003981 vehicle Substances 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 6
- 230000009089 cytolysis Effects 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 201000007270 liver cancer Diseases 0.000 description 6
- 208000014018 liver neoplasm Diseases 0.000 description 6
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 6
- 230000005012 migration Effects 0.000 description 6
- 238000013508 migration Methods 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 210000004408 hybridoma Anatomy 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- 239000011534 wash buffer Substances 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 206010006187 Breast cancer Diseases 0.000 description 4
- 208000026310 Breast neoplasm Diseases 0.000 description 4
- 206010008342 Cervix carcinoma Diseases 0.000 description 4
- 206010009944 Colon cancer Diseases 0.000 description 4
- 241000880493 Leptailurus serval Species 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 206010033128 Ovarian cancer Diseases 0.000 description 4
- 206010061535 Ovarian neoplasm Diseases 0.000 description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 description 4
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 description 4
- 201000010881 cervical cancer Diseases 0.000 description 4
- 208000029742 colonic neoplasm Diseases 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 206010017758 gastric cancer Diseases 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 4
- 201000011549 stomach cancer Diseases 0.000 description 4
- 230000004614 tumor growth Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 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 3
- 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 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108010087819 Fc receptors Proteins 0.000 description 3
- 102000009109 Fc receptors Human genes 0.000 description 3
- 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 3
- 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 3
- 241001529936 Murinae Species 0.000 description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 3
- FGWUALWGCZJQDJ-URLPEUOOSA-N Phe-Thr-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FGWUALWGCZJQDJ-URLPEUOOSA-N 0.000 description 3
- 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 3
- 206010052428 Wound Diseases 0.000 description 3
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 201000004101 esophageal cancer Diseases 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 3
- 108010073969 valyllysine Proteins 0.000 description 3
- 230000003442 weekly effect Effects 0.000 description 3
- LBJYAILUMSUTAM-ZLUOBGJFSA-N Ala-Asn-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O LBJYAILUMSUTAM-ZLUOBGJFSA-N 0.000 description 2
- GGNHBHYDMUDXQB-KBIXCLLPSA-N Ala-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)N GGNHBHYDMUDXQB-KBIXCLLPSA-N 0.000 description 2
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 2
- AAWLEICNDUHIJM-MBLNEYKQSA-N Ala-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)N)O AAWLEICNDUHIJM-MBLNEYKQSA-N 0.000 description 2
- CWRBRVZBMVJENN-UVBJJODRSA-N Ala-Trp-Met Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCSC)C(=O)O)N CWRBRVZBMVJENN-UVBJJODRSA-N 0.000 description 2
- QDGMZAOSMNGBLP-MRFFXTKBSA-N Ala-Trp-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)N QDGMZAOSMNGBLP-MRFFXTKBSA-N 0.000 description 2
- ZDILXFDENZVOTL-BPNCWPANSA-N Ala-Val-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZDILXFDENZVOTL-BPNCWPANSA-N 0.000 description 2
- VKKYFICVTYKFIO-CIUDSAMLSA-N Arg-Ala-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N VKKYFICVTYKFIO-CIUDSAMLSA-N 0.000 description 2
- PRLPSDIHSRITSF-UNQGMJICSA-N Arg-Phe-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PRLPSDIHSRITSF-UNQGMJICSA-N 0.000 description 2
- JOTRDIXZHNQYGP-DCAQKATOSA-N Arg-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N JOTRDIXZHNQYGP-DCAQKATOSA-N 0.000 description 2
- IYVSIZAXNLOKFQ-BYULHYEWSA-N Asn-Asp-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O IYVSIZAXNLOKFQ-BYULHYEWSA-N 0.000 description 2
- FMNBYVSGRCXWEK-FOHZUACHSA-N Asn-Thr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O FMNBYVSGRCXWEK-FOHZUACHSA-N 0.000 description 2
- JJQGZGOEDSSHTE-FOHZUACHSA-N Asp-Thr-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JJQGZGOEDSSHTE-FOHZUACHSA-N 0.000 description 2
- KNOGLZBISUBTFW-QRTARXTBSA-N Asp-Trp-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C(C)C)C(O)=O KNOGLZBISUBTFW-QRTARXTBSA-N 0.000 description 2
- CZIVKMOEXPILDK-SRVKXCTJSA-N Asp-Tyr-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O CZIVKMOEXPILDK-SRVKXCTJSA-N 0.000 description 2
- YWEHYKGJWHPGPY-XGEHTFHBSA-N Cys-Thr-Arg Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CS)N)O YWEHYKGJWHPGPY-XGEHTFHBSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- MIWJDJAMMKHUAR-ZVZYQTTQSA-N Glu-Trp-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N MIWJDJAMMKHUAR-ZVZYQTTQSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 2
- MIIVFRCYJABHTQ-ONGXEEELSA-N Gly-Leu-Val Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O MIIVFRCYJABHTQ-ONGXEEELSA-N 0.000 description 2
- WNZOCXUOGVYYBJ-CDMKHQONSA-N Gly-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)CN)O WNZOCXUOGVYYBJ-CDMKHQONSA-N 0.000 description 2
- HTZKFIYQMHJWSQ-INTQDDNPSA-N His-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N HTZKFIYQMHJWSQ-INTQDDNPSA-N 0.000 description 2
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 2
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 2
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 2
- 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 2
- NXRNRBOKDBIVKQ-CXTHYWKRSA-N Ile-Tyr-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N NXRNRBOKDBIVKQ-CXTHYWKRSA-N 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- WIDZHJTYKYBLSR-DCAQKATOSA-N Leu-Glu-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WIDZHJTYKYBLSR-DCAQKATOSA-N 0.000 description 2
- FAELBUXXFQLUAX-AJNGGQMLSA-N Leu-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C FAELBUXXFQLUAX-AJNGGQMLSA-N 0.000 description 2
- KIZIOFNVSOSKJI-CIUDSAMLSA-N Leu-Ser-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N KIZIOFNVSOSKJI-CIUDSAMLSA-N 0.000 description 2
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 2
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 description 2
- GQFDWEDHOQRNLC-QWRGUYRKSA-N Lys-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN GQFDWEDHOQRNLC-QWRGUYRKSA-N 0.000 description 2
- 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 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
- FRWZTWWOORIIBA-FXQIFTODSA-N Met-Asn-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N FRWZTWWOORIIBA-FXQIFTODSA-N 0.000 description 2
- MHQXIBRPDKXDGZ-ZFWWWQNUSA-N Met-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)[C@@H](N)CCSC)C(O)=O)=CNC2=C1 MHQXIBRPDKXDGZ-ZFWWWQNUSA-N 0.000 description 2
- AUEJLPRZGVVDNU-UHFFFAOYSA-N N-L-tyrosyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 AUEJLPRZGVVDNU-UHFFFAOYSA-N 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- INHMISZWLJZQGH-ULQDDVLXSA-N Phe-Leu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 INHMISZWLJZQGH-ULQDDVLXSA-N 0.000 description 2
- XDMMOISUAHXXFD-SRVKXCTJSA-N Phe-Ser-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O XDMMOISUAHXXFD-SRVKXCTJSA-N 0.000 description 2
- QTDBZORPVYTRJU-KKXDTOCCSA-N Phe-Tyr-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O QTDBZORPVYTRJU-KKXDTOCCSA-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
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 2
- NAIPAPCKKRCMBL-JYJNAYRXSA-N Pro-Pro-Phe Chemical compound C([C@@H](C(=O)O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H]1NCCC1)C1=CC=CC=C1 NAIPAPCKKRCMBL-JYJNAYRXSA-N 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- OBXVZEAMXFSGPU-FXQIFTODSA-N Ser-Asn-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)CN=C(N)N OBXVZEAMXFSGPU-FXQIFTODSA-N 0.000 description 2
- INCNPLPRPOYTJI-JBDRJPRFSA-N Ser-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CO)N INCNPLPRPOYTJI-JBDRJPRFSA-N 0.000 description 2
- 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 2
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 2
- HBTCFCHYALPXME-HTFCKZLJSA-N Ser-Ile-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HBTCFCHYALPXME-HTFCKZLJSA-N 0.000 description 2
- DOSZISJPMCYEHT-NAKRPEOUSA-N Ser-Ile-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O DOSZISJPMCYEHT-NAKRPEOUSA-N 0.000 description 2
- IFLVBVIYADZIQO-DCAQKATOSA-N Ser-Met-Lys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N IFLVBVIYADZIQO-DCAQKATOSA-N 0.000 description 2
- BSXKBOUZDAZXHE-CIUDSAMLSA-N Ser-Pro-Glu Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O BSXKBOUZDAZXHE-CIUDSAMLSA-N 0.000 description 2
- NMZXJDSKEGFDLJ-DCAQKATOSA-N Ser-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CCCCN)C(=O)O NMZXJDSKEGFDLJ-DCAQKATOSA-N 0.000 description 2
- MFQMZDPAZRZAPV-NAKRPEOUSA-N Ser-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CO)N MFQMZDPAZRZAPV-NAKRPEOUSA-N 0.000 description 2
- YAAPRMFURSENOZ-KATARQTJSA-N Thr-Cys-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N)O YAAPRMFURSENOZ-KATARQTJSA-N 0.000 description 2
- 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 2
- GVMXJJAJLIEASL-ZJDVBMNYSA-N Thr-Pro-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O GVMXJJAJLIEASL-ZJDVBMNYSA-N 0.000 description 2
- KSGKJSFPWSMJHK-JNPHEJMOSA-N Tyr-Tyr-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KSGKJSFPWSMJHK-JNPHEJMOSA-N 0.000 description 2
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 2
- SLLKXDSRVAOREO-KZVJFYERSA-N Val-Ala-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N)O SLLKXDSRVAOREO-KZVJFYERSA-N 0.000 description 2
- ZXYPHBKIZLAQTL-QXEWZRGKSA-N Val-Pro-Asp Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N ZXYPHBKIZLAQTL-QXEWZRGKSA-N 0.000 description 2
- DEGUERSKQBRZMZ-FXQIFTODSA-N Val-Ser-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O DEGUERSKQBRZMZ-FXQIFTODSA-N 0.000 description 2
- WUFHZIRMAZZWRS-OSUNSFLBSA-N Val-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C(C)C)N WUFHZIRMAZZWRS-OSUNSFLBSA-N 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- 238000003491 array Methods 0.000 description 2
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 2
- 108010038633 aspartylglutamate Proteins 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000004709 cell invasion Effects 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011284 combination treatment Methods 0.000 description 2
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 108010060199 cysteinylproline Proteins 0.000 description 2
- 230000006334 disulfide bridging Effects 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 210000003238 esophagus Anatomy 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 108010045126 glycyl-tyrosyl-glycine Proteins 0.000 description 2
- 102000044491 human BSG Human genes 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 108010064486 phenylalanyl-leucyl-valine Proteins 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 108010020755 prolyl-glycyl-glycine Proteins 0.000 description 2
- 108010077112 prolyl-proline Proteins 0.000 description 2
- 108010079317 prolyl-tyrosine Proteins 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 108010038745 tryptophylglycine Proteins 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- DIBLBAURNYJYBF-XLXZRNDBSA-N (2s)-2-[[(2s)-2-[[2-[[(2s)-6-amino-2-[[(2s)-2-amino-3-methylbutanoyl]amino]hexanoyl]amino]acetyl]amino]-3-phenylpropanoyl]amino]-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@H](NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 DIBLBAURNYJYBF-XLXZRNDBSA-N 0.000 description 1
- PNDPGZBMCMUPRI-HVTJNCQCSA-N 10043-66-0 Chemical compound [131I][131I] PNDPGZBMCMUPRI-HVTJNCQCSA-N 0.000 description 1
- PMUNIMVZCACZBB-UHFFFAOYSA-N 2-hydroxyethylazanium;chloride Chemical compound Cl.NCCO PMUNIMVZCACZBB-UHFFFAOYSA-N 0.000 description 1
- KXEVYGKATAMXJJ-ACZMJKKPSA-N Ala-Glu-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O KXEVYGKATAMXJJ-ACZMJKKPSA-N 0.000 description 1
- MNZHHDPWDWQJCQ-YUMQZZPRSA-N Ala-Leu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O MNZHHDPWDWQJCQ-YUMQZZPRSA-N 0.000 description 1
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 1
- SUHLZMHFRALVSY-YUMQZZPRSA-N Ala-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)NCC(O)=O SUHLZMHFRALVSY-YUMQZZPRSA-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
- MDNAVFBZPROEHO-UHFFFAOYSA-N Ala-Lys-Val Natural products CC(C)C(C(O)=O)NC(=O)C(NC(=O)C(C)N)CCCCN MDNAVFBZPROEHO-UHFFFAOYSA-N 0.000 description 1
- WQLDNOCHHRISMS-NAKRPEOUSA-N Ala-Pro-Ile Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WQLDNOCHHRISMS-NAKRPEOUSA-N 0.000 description 1
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 1
- NVGWESORMHFISY-SRVKXCTJSA-N Asn-Asn-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O NVGWESORMHFISY-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
- QNNBHTFDFFFHGC-KKUMJFAQSA-N Asn-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O QNNBHTFDFFFHGC-KKUMJFAQSA-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
- UZFHNLYQWMGUHU-DCAQKATOSA-N Asp-Lys-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UZFHNLYQWMGUHU-DCAQKATOSA-N 0.000 description 1
- DONWIPDSZZJHHK-HJGDQZAQSA-N Asp-Lys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)O)N)O DONWIPDSZZJHHK-HJGDQZAQSA-N 0.000 description 1
- USNJAPJZSGTTPX-XVSYOHENSA-N Asp-Phe-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O USNJAPJZSGTTPX-XVSYOHENSA-N 0.000 description 1
- 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 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
- KNDCWFXCFKSEBM-AVGNSLFASA-N Asp-Tyr-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O KNDCWFXCFKSEBM-AVGNSLFASA-N 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 102000015279 Basigin Human genes 0.000 description 1
- 108010064528 Basigin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000699802 Cricetulus griseus Species 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
- VIRYODQIWJNWNU-NRPADANISA-N Cys-Glu-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CS)N VIRYODQIWJNWNU-NRPADANISA-N 0.000 description 1
- WVLZTXGTNGHPBO-SRVKXCTJSA-N Cys-Leu-Leu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O WVLZTXGTNGHPBO-SRVKXCTJSA-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
- ZXCAQANTQWBICD-DCAQKATOSA-N Cys-Lys-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CS)N ZXCAQANTQWBICD-DCAQKATOSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 241001524679 Escherichia virus M13 Species 0.000 description 1
- 108091006020 Fc-tagged proteins Proteins 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- JJKKWYQVHRUSDG-GUBZILKMSA-N Glu-Ala-Lys Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O JJKKWYQVHRUSDG-GUBZILKMSA-N 0.000 description 1
- JVSBYEDSSRZQGV-GUBZILKMSA-N Glu-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O JVSBYEDSSRZQGV-GUBZILKMSA-N 0.000 description 1
- CKOFNWCLWRYUHK-XHNCKOQMSA-N Glu-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)N)C(=O)O CKOFNWCLWRYUHK-XHNCKOQMSA-N 0.000 description 1
- MWMJCGBSIORNCD-AVGNSLFASA-N Glu-Leu-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O MWMJCGBSIORNCD-AVGNSLFASA-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
- QDMVXRNLOPTPIE-WDCWCFNPSA-N Glu-Lys-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QDMVXRNLOPTPIE-WDCWCFNPSA-N 0.000 description 1
- BPCLDCNZBUYGOD-BPUTZDHNSA-N Glu-Trp-Glu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O)=CNC2=C1 BPCLDCNZBUYGOD-BPUTZDHNSA-N 0.000 description 1
- MFYLRRCYBBJYPI-JYJNAYRXSA-N Glu-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O MFYLRRCYBBJYPI-JYJNAYRXSA-N 0.000 description 1
- VSVZIEVNUYDAFR-YUMQZZPRSA-N Gly-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN VSVZIEVNUYDAFR-YUMQZZPRSA-N 0.000 description 1
- GRIRDMVMJJDZKV-RCOVLWMOSA-N Gly-Asn-Val Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O GRIRDMVMJJDZKV-RCOVLWMOSA-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
- BUEFQXUHTUZXHR-LURJTMIESA-N Gly-Gly-Pro zwitterion Chemical compound NCC(=O)NCC(=O)N1CCC[C@H]1C(O)=O BUEFQXUHTUZXHR-LURJTMIESA-N 0.000 description 1
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 1
- NNCSJUBVFBDDLC-YUMQZZPRSA-N Gly-Leu-Ser Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O NNCSJUBVFBDDLC-YUMQZZPRSA-N 0.000 description 1
- HAOUOFNNJJLVNS-BQBZGAKWSA-N Gly-Pro-Ser Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O HAOUOFNNJJLVNS-BQBZGAKWSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-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
- OLIFSFOFKGKIRH-WUJLRWPWSA-N Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CN OLIFSFOFKGKIRH-WUJLRWPWSA-N 0.000 description 1
- FFJQHWKSGAWSTJ-BFHQHQDPSA-N Gly-Thr-Ala Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O FFJQHWKSGAWSTJ-BFHQHQDPSA-N 0.000 description 1
- NVTPVQLIZCOJFK-FOHZUACHSA-N Gly-Thr-Asp Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O NVTPVQLIZCOJFK-FOHZUACHSA-N 0.000 description 1
- ZZWUYQXMIFTIIY-WEDXCCLWSA-N Gly-Thr-Leu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O ZZWUYQXMIFTIIY-WEDXCCLWSA-N 0.000 description 1
- SYOJVRNQCXYEOV-XVKPBYJWSA-N Gly-Val-Glu Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SYOJVRNQCXYEOV-XVKPBYJWSA-N 0.000 description 1
- 102000001398 Granzyme Human genes 0.000 description 1
- 108060005986 Granzyme Proteins 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- HIAHVKLTHNOENC-HGNGGELXSA-N His-Glu-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O HIAHVKLTHNOENC-HGNGGELXSA-N 0.000 description 1
- HZWWOGWOBQBETJ-CUJWVEQBSA-N His-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N)O HZWWOGWOBQBETJ-CUJWVEQBSA-N 0.000 description 1
- CSTDQOOBZBAJKE-BWAGICSOSA-N His-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](CC2=CN=CN2)N)O CSTDQOOBZBAJKE-BWAGICSOSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 description 1
- MKWSZEHGHSLNPF-NAKRPEOUSA-N Ile-Ala-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O)N MKWSZEHGHSLNPF-NAKRPEOUSA-N 0.000 description 1
- VGSPNSSCMOHRRR-BJDJZHNGSA-N Ile-Ser-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N VGSPNSSCMOHRRR-BJDJZHNGSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- SXTAYKAGBXMACB-DPVSGNNYSA-N L-methionine sulfoximine Chemical compound CS(=N)(=O)CC[C@H](N)C(O)=O SXTAYKAGBXMACB-DPVSGNNYSA-N 0.000 description 1
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- WSGXUIQTEZDVHJ-GARJFASQSA-N Leu-Ala-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O WSGXUIQTEZDVHJ-GARJFASQSA-N 0.000 description 1
- BTNXKBVLWJBTNR-SRVKXCTJSA-N Leu-His-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(N)=O)C(O)=O BTNXKBVLWJBTNR-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
- 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-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
- BRTVHXHCUSXYRI-CIUDSAMLSA-N Leu-Ser-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O BRTVHXHCUSXYRI-CIUDSAMLSA-N 0.000 description 1
- QWWPYKKLXWOITQ-VOAKCMCISA-N Leu-Thr-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QWWPYKKLXWOITQ-VOAKCMCISA-N 0.000 description 1
- VUBIPAHVHMZHCM-KKUMJFAQSA-N Leu-Tyr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 VUBIPAHVHMZHCM-KKUMJFAQSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-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
- NTBFKPBULZGXQL-KKUMJFAQSA-N Lys-Asp-Tyr Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NTBFKPBULZGXQL-KKUMJFAQSA-N 0.000 description 1
- FGMHXLULNHTPID-KKUMJFAQSA-N Lys-His-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CN=CN1 FGMHXLULNHTPID-KKUMJFAQSA-N 0.000 description 1
- WQDKIVRHTQYJSN-DCAQKATOSA-N Lys-Ser-Arg Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N WQDKIVRHTQYJSN-DCAQKATOSA-N 0.000 description 1
- CTJUSALVKAWFFU-CIUDSAMLSA-N Lys-Ser-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N CTJUSALVKAWFFU-CIUDSAMLSA-N 0.000 description 1
- 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 1
- 239000004472 Lysine Substances 0.000 description 1
- 239000012515 MabSelect SuRe Substances 0.000 description 1
- 206010027476 Metastases Diseases 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
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 102000004503 Perforin Human genes 0.000 description 1
- 108010056995 Perforin Proteins 0.000 description 1
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 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
- JOXIIFVCSATTDH-IHPCNDPISA-N Phe-Asn-Trp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N JOXIIFVCSATTDH-IHPCNDPISA-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
- JLLJTMHNXQTMCK-UBHSHLNASA-N Phe-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=CC=C1 JLLJTMHNXQTMCK-UBHSHLNASA-N 0.000 description 1
- QARPMYDMYVLFMW-KKUMJFAQSA-N Phe-Pro-Glu Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=CC=C1 QARPMYDMYVLFMW-KKUMJFAQSA-N 0.000 description 1
- IIEOLPMQYRBZCN-SRVKXCTJSA-N Phe-Ser-Cys Chemical compound N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O IIEOLPMQYRBZCN-SRVKXCTJSA-N 0.000 description 1
- BPIMVBKDLSBKIJ-FCLVOEFKSA-N Phe-Thr-Phe Chemical compound C([C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 BPIMVBKDLSBKIJ-FCLVOEFKSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000010806 PrimeScriptTM RT Reagent kit Methods 0.000 description 1
- CGBYDGAJHSOGFQ-LPEHRKFASA-N Pro-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 CGBYDGAJHSOGFQ-LPEHRKFASA-N 0.000 description 1
- KIPIKSXPPLABPN-CIUDSAMLSA-N Pro-Glu-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1 KIPIKSXPPLABPN-CIUDSAMLSA-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
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-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
- HWLKHNDRXWTFTN-GUBZILKMSA-N Pro-Pro-Cys Chemical compound C1C[C@H](NC1)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CS)C(=O)O HWLKHNDRXWTFTN-GUBZILKMSA-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
- OWQXAJQZLWHPBH-FXQIFTODSA-N Pro-Ser-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O OWQXAJQZLWHPBH-FXQIFTODSA-N 0.000 description 1
- GMJDSFYVTAMIBF-FXQIFTODSA-N Pro-Ser-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O GMJDSFYVTAMIBF-FXQIFTODSA-N 0.000 description 1
- KHRLUIPIMIQFGT-AVGNSLFASA-N Pro-Val-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O KHRLUIPIMIQFGT-AVGNSLFASA-N 0.000 description 1
- YDTUEBLEAVANFH-RCWTZXSCSA-N Pro-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 YDTUEBLEAVANFH-RCWTZXSCSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 238000011579 SCID mouse model Methods 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- OYEDZGNMSBZCIM-XGEHTFHBSA-N Ser-Arg-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OYEDZGNMSBZCIM-XGEHTFHBSA-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
- VGNYHOBZJKWRGI-CIUDSAMLSA-N Ser-Asn-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO VGNYHOBZJKWRGI-CIUDSAMLSA-N 0.000 description 1
- BPMRXBZYPGYPJN-WHFBIAKZSA-N Ser-Gly-Asn Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O BPMRXBZYPGYPJN-WHFBIAKZSA-N 0.000 description 1
- OQPNSDWGAMFJNU-QWRGUYRKSA-N Ser-Gly-Tyr Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 OQPNSDWGAMFJNU-QWRGUYRKSA-N 0.000 description 1
- 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 1
- GZSZPKSBVAOGIE-CIUDSAMLSA-N Ser-Lys-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O GZSZPKSBVAOGIE-CIUDSAMLSA-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
- 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 1
- SRSPTFBENMJHMR-WHFBIAKZSA-N Ser-Ser-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SRSPTFBENMJHMR-WHFBIAKZSA-N 0.000 description 1
- 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 1
- OZPDGESCTGGNAD-CIUDSAMLSA-N Ser-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CO OZPDGESCTGGNAD-CIUDSAMLSA-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
- PCJLFYBAQZQOFE-KATARQTJSA-N Ser-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N)O PCJLFYBAQZQOFE-KATARQTJSA-N 0.000 description 1
- SNXUIBACCONSOH-BWBBJGPYSA-N Ser-Thr-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CO)C(O)=O SNXUIBACCONSOH-BWBBJGPYSA-N 0.000 description 1
- BDMWLJLPPUCLNV-XGEHTFHBSA-N Ser-Thr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O BDMWLJLPPUCLNV-XGEHTFHBSA-N 0.000 description 1
- RCOUFINCYASMDN-GUBZILKMSA-N Ser-Val-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCSC)C(O)=O RCOUFINCYASMDN-GUBZILKMSA-N 0.000 description 1
- 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 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
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- ASJDFGOPDCVXTG-KATARQTJSA-N Thr-Cys-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O ASJDFGOPDCVXTG-KATARQTJSA-N 0.000 description 1
- DSLHSTIUAPKERR-XGEHTFHBSA-N Thr-Cys-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O DSLHSTIUAPKERR-XGEHTFHBSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- XSEPSRUDSPHMPX-KATARQTJSA-N Thr-Lys-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O XSEPSRUDSPHMPX-KATARQTJSA-N 0.000 description 1
- DXPURPNJDFCKKO-RHYQMDGZSA-N Thr-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O DXPURPNJDFCKKO-RHYQMDGZSA-N 0.000 description 1
- SGAOHNPSEPVAFP-ZDLURKLDSA-N Thr-Ser-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SGAOHNPSEPVAFP-ZDLURKLDSA-N 0.000 description 1
- ZESGVALRVJIVLZ-VFCFLDTKSA-N Thr-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@@H]1C(=O)O)N)O ZESGVALRVJIVLZ-VFCFLDTKSA-N 0.000 description 1
- BEZTUFWTPVOROW-KJEVXHAQSA-N Thr-Tyr-Arg Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N)O BEZTUFWTPVOROW-KJEVXHAQSA-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
- 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
- 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
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 description 1
- SCCKSNREWHMKOJ-SRVKXCTJSA-N Tyr-Asn-Ser Chemical compound N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O SCCKSNREWHMKOJ-SRVKXCTJSA-N 0.000 description 1
- QAYSODICXVZUIA-WLTAIBSBSA-N Tyr-Gly-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O QAYSODICXVZUIA-WLTAIBSBSA-N 0.000 description 1
- AUZADXNWQMBZOO-JYJNAYRXSA-N Tyr-Pro-Arg Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C1=CC=C(O)C=C1 AUZADXNWQMBZOO-JYJNAYRXSA-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
- SQUMHUZLJDUROQ-YDHLFZDLSA-N Tyr-Val-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O SQUMHUZLJDUROQ-YDHLFZDLSA-N 0.000 description 1
- WDIGUPHXPBMODF-UMNHJUIQSA-N Val-Glu-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N WDIGUPHXPBMODF-UMNHJUIQSA-N 0.000 description 1
- OACSGBOREVRSME-NHCYSSNCSA-N Val-His-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(N)=O)C(O)=O OACSGBOREVRSME-NHCYSSNCSA-N 0.000 description 1
- ZIGZPYJXIWLQFC-QTKMDUPCSA-N Val-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C(C)C)N)O ZIGZPYJXIWLQFC-QTKMDUPCSA-N 0.000 description 1
- XTDDIVQWDXMRJL-IHRRRGAJSA-N Val-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N XTDDIVQWDXMRJL-IHRRRGAJSA-N 0.000 description 1
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 1
- FMQGYTMERWBMSI-HJWJTTGWSA-N Val-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C(C)C)N FMQGYTMERWBMSI-HJWJTTGWSA-N 0.000 description 1
- VCIYTVOBLZHFSC-XHSDSOJGSA-N Val-Phe-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N VCIYTVOBLZHFSC-XHSDSOJGSA-N 0.000 description 1
- 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 1
- KRAHMIJVUPUOTQ-DCAQKATOSA-N Val-Ser-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N KRAHMIJVUPUOTQ-DCAQKATOSA-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
- SDHZOOIGIUEPDY-JYJNAYRXSA-N Val-Ser-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CO)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 SDHZOOIGIUEPDY-JYJNAYRXSA-N 0.000 description 1
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 1
- QPJSIBAOZBVELU-BPNCWPANSA-N Val-Tyr-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](C(C)C)N QPJSIBAOZBVELU-BPNCWPANSA-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
- ZLNYBMWGPOKSLW-LSJOCFKGSA-N Val-Val-Asp Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLNYBMWGPOKSLW-LSJOCFKGSA-N 0.000 description 1
- LLJLBRRXKZTTRD-GUBZILKMSA-N Val-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N LLJLBRRXKZTTRD-GUBZILKMSA-N 0.000 description 1
- JVGDAEKKZKKZFO-RCWTZXSCSA-N Val-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)N)O JVGDAEKKZKKZFO-RCWTZXSCSA-N 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 1
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000013377 clone selection method Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- -1 coatings Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 229940073579 ethanolamine hydrochloride Drugs 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 239000005350 fused silica glass Substances 0.000 description 1
- 238000009650 gentamicin protection assay Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 102000005396 glutamine synthetase Human genes 0.000 description 1
- 108020002326 glutamine synthetase Proteins 0.000 description 1
- 108010057083 glutamyl-aspartyl-leucine Proteins 0.000 description 1
- 108010013768 glutamyl-aspartyl-proline Proteins 0.000 description 1
- 230000002414 glycolytic effect Effects 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
- 108010037850 glycylvaline Proteins 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 230000003118 histopathologic effect Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 description 1
- 229940097277 hygromycin b Drugs 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000013115 immunohistochemical detection Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 108010027338 isoleucylcysteine Proteins 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000012737 microarray-based gene expression Methods 0.000 description 1
- HZAXFHJVJLSVMW-UHFFFAOYSA-N monoethanolamine hydrochloride Natural products NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 238000012243 multiplex automated genomic engineering Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 108010024654 phenylalanyl-prolyl-alanine Proteins 0.000 description 1
- 108010073101 phenylalanylleucine Proteins 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108010067988 prolactin-binding protein Proteins 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011363 radioimmunotherapy Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 108010071097 threonyl-lysyl-proline Proteins 0.000 description 1
- 230000009258 tissue cross reactivity Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000010877 transwell invasion assay Methods 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 108010079202 tyrosyl-alanyl-cysteine Proteins 0.000 description 1
- 108010071635 tyrosyl-prolyl-arginine Proteins 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 108010052774 valyl-lysyl-glycyl-phenylalanyl-tyrosine Proteins 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 108010027345 wheylin-1 peptide Proteins 0.000 description 1
- 238000012447 xenograft mouse model Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
-
- 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
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
-
- 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
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
- C07K2317/41—Glycosylation, sialylation, or fucosylation
-
- 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
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Biomedical Technology (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
- Biophysics (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present disclosure relates to a nucleotide sequence comprising the sequence of SEQ ID NO:
5, and/or sequence of SEQ ID NO: 6, vector and host cell line comprising the nucleotide. The present disclosure also relates to an antibody that binds to extracellular region of human CD147, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2, and/or a light chain variable region having the amino acid sequence of SEQ ID
NO: 1, and the antibody contains a glycoform lacking both fucose residues and xylose residues, pharmaceutical composition comprising the antibody or fragment thereof, the method of producing the antibody or fragment thereof, and use of the pharmaceutical composition in treatment of CD147 expression-related diseases.
5, and/or sequence of SEQ ID NO: 6, vector and host cell line comprising the nucleotide. The present disclosure also relates to an antibody that binds to extracellular region of human CD147, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2, and/or a light chain variable region having the amino acid sequence of SEQ ID
NO: 1, and the antibody contains a glycoform lacking both fucose residues and xylose residues, pharmaceutical composition comprising the antibody or fragment thereof, the method of producing the antibody or fragment thereof, and use of the pharmaceutical composition in treatment of CD147 expression-related diseases.
Description
GLYCOSYLATION
FIELD OF THE INVENTION
[0001] The present disclosure relates to a human-mouse chimeric anti-CD147 antibody with specific glycosylation profile in Fc region.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
[0001] The present disclosure relates to a human-mouse chimeric anti-CD147 antibody with specific glycosylation profile in Fc region.
BACKGROUND OF THE INVENTION
[0002] CD147 is a highly glycosylated transmembrane protein that belongs to the immunoglobulin superfamily. CD147 is commonly over-expressed in many tumors (Li et al., 2009. HAb18G (CD147), a cancer-associated biomarker and its role in cancer detection.
Histopathology 54, 677-687.), including carcinomas of liver, lung, breast, pancreas, prostate, and bladder. CD147 over expression level is correlated with tumor histopathologic type and clinical stage of disease. Importantly, CD147 surface expression is closely associated with tumorigenesis, tumor progression and reduced patient survival in various cancers, and these facts validate CD147 as a therapeutic target for cancer treatment.
Histopathology 54, 677-687.), including carcinomas of liver, lung, breast, pancreas, prostate, and bladder. CD147 over expression level is correlated with tumor histopathologic type and clinical stage of disease. Importantly, CD147 surface expression is closely associated with tumorigenesis, tumor progression and reduced patient survival in various cancers, and these facts validate CD147 as a therapeutic target for cancer treatment.
[0003] Antibody Dependent Cell-mediated Cytotoxicity (ADCC) is a major clinical mechanism of action for therapeutic antibodies. Generally, when the antibody binds to a tumor target on the surface of a cell in a tumor tissue of a cancer patient, the Fc region of the antibody attracts effector cells, such as NK cells, by binding to the surface Fc receptor (e.g. CD16) of the NK cells. Binding of the Fc region to CD16 of the effector cells induces secretion of perforin and granzyme that lead to apoptosis of the target cells. Macrophages, neutrophils and eosinophils can also be activated by the Fc effector function.
[0004] ADCC efficacy of IgG antibodies is significantly dependent on Fc glycosylation patterns (Jefferis, 2009). We previously generated an anti-CD147 murine monoclonal antibody, HAb18 (U.S. 7,638,619, PCT/CNO3/00188 and China patent No. ZL02114471.0), and developed a 131I-labeled HAb18 F(ab')2 (named Licartin) to treat liver cancer (Xu et al., 2007a; A
randomized controlled trial of Licartin for preventing hepatoma recurrence after liver transplantation. Hepatology 45, 269-276). Licartin has been shown to be effective in the treatment of liver cancer, demonstrating anti-CD147 antibodies as molecular targeted therapeutics. Although efficient, safe and tolerated in therapy, Licartin suffered from immunogenicity which triggers generation of human¨anti-mouse antibodies (HAMA) in 4 out of 130 treated patients without affecting therapeutic results (Chen et al., 2006;
Targeting radioimmunotherapy of hepatocellular carcinoma with iodine (131I) metuximab injection:
clinical phase I/II trials. International journal of radiation oncology, biology, physics 65, 435-444.). Such limitations have prompted efforts to improve the efficacy and tolerability of HAb 18 through antibody engineering, as well as the inconvenience of administration of a non-radioactively labelled therapeutic antibody. The present disclosure addresses these and other needs and provides several additional benefits for efficient and safe treatment of cancer patients, which will be described in the remainder of this document.
BRIEF SUMMARY OF THE INVENTION
randomized controlled trial of Licartin for preventing hepatoma recurrence after liver transplantation. Hepatology 45, 269-276). Licartin has been shown to be effective in the treatment of liver cancer, demonstrating anti-CD147 antibodies as molecular targeted therapeutics. Although efficient, safe and tolerated in therapy, Licartin suffered from immunogenicity which triggers generation of human¨anti-mouse antibodies (HAMA) in 4 out of 130 treated patients without affecting therapeutic results (Chen et al., 2006;
Targeting radioimmunotherapy of hepatocellular carcinoma with iodine (131I) metuximab injection:
clinical phase I/II trials. International journal of radiation oncology, biology, physics 65, 435-444.). Such limitations have prompted efforts to improve the efficacy and tolerability of HAb 18 through antibody engineering, as well as the inconvenience of administration of a non-radioactively labelled therapeutic antibody. The present disclosure addresses these and other needs and provides several additional benefits for efficient and safe treatment of cancer patients, which will be described in the remainder of this document.
BRIEF SUMMARY OF THE INVENTION
[0005] In one aspect, the present disclosure provides nucleotide sequences comprising the sequence of SEQ ID NO: 5, and/or sequence of SEQ ID NO: 6. In one aspect, the present disclosure provides nucleotide sequences comprising the sequence of SEQ ID NO:
7, and/or sequence of SEQ ID NO: 8.
7, and/or sequence of SEQ ID NO: 8.
[0006] In another aspect, the present disclosure provides vectors comprising the nucleotide sequence comprising the sequence of SEQ ID NO: 5, and/or sequence of SEQ ID
NO: 6. In another aspect, the present disclosure provides vectors comprising the nucleotide sequence comprising the sequence of SEQ ID NO: 7, and/or sequence of SEQ ID NO: 8.
NO: 6. In another aspect, the present disclosure provides vectors comprising the nucleotide sequence comprising the sequence of SEQ ID NO: 7, and/or sequence of SEQ ID NO: 8.
[0007] In another aspect, the present disclosure provides host cells comprising a vector, wherein the vector comprises the nucleotide sequence comprising the sequence of SEQ ID NO: 5, and/or sequence of SEQ ID NO: 6. In another aspect, the present disclosure provides host cells comprising a vector, wherein the vector comprises the nucleotide sequence comprising the sequence of SEQ ID NO: 7, and/or sequence of SEQ ID NO: 8.
[0008] In another aspect, the present disclosure provides antibodies that bind to an extracellular region of human CD147, wherein the antibodies comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2, and the antibodies contain a glycoform lacking both fucose residues and xylose residues. In some embodiments, the antibodies further comprise a light chain variable region having the amino acid sequence of SEQ
ID NO: 1. In some embodiments, the antibodies comprise a light chain having the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibodies comprise a heavy chain having the amino acid sequence of SEQ ID NO: 4.
ID NO: 1. In some embodiments, the antibodies comprise a light chain having the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibodies comprise a heavy chain having the amino acid sequence of SEQ ID NO: 4.
[0009]
In some embodiments, the antibodies or fragments thereof comprises a predominant portion of glycoforms comprising N-linked oligosaccharides comprising five mannose residues (also called Mannose-5),In some embodiments, the antibodies or fragments thereof comprise solely a glycoform comprising N-linked Mannose-5. In some embodiments, the antibody has a glycosylation profile as analyzed by High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) that is substantially equivalent to that of Metuzumab as shown in Figure 6.
In some embodiments, the antibodies or fragments thereof comprises a predominant portion of glycoforms comprising N-linked oligosaccharides comprising five mannose residues (also called Mannose-5),In some embodiments, the antibodies or fragments thereof comprise solely a glycoform comprising N-linked Mannose-5. In some embodiments, the antibody has a glycosylation profile as analyzed by High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) that is substantially equivalent to that of Metuzumab as shown in Figure 6.
[0010]
In some embodiments, the antibodies are obtained from an acetyl-glucosamine transferase deficient cell line. In some embodiments, the cell line is CHO
cell line.
In some embodiments, the antibodies are obtained from an acetyl-glucosamine transferase deficient cell line. In some embodiments, the cell line is CHO
cell line.
[0011] In some embodiments, the ADCC activity of the antibodies containing a glycoform that lacks both fucose residues and xylose residues is at least 2 times, 5 times, 10 times or 20 times higher than the ADCC activity of an antibody that has a glycoform comprising fucose residues, xylose residues or both.
[0012]
In another aspect, the present disclosure provides pharmaceutical compositions comprising an antibody that binds to an extracellular region of human CD147, or a fragment of such antibody, and a pharmaceutically acceptable vehicle or excipient, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ
ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 1, and the antibody contains a glycoform lacking both fucose residues and xylose residues. In some embodiments, the antibodies comprise a light chain having the amino acid sequence of SEQ ID
NO: 3. In some embodiments, the antibodies comprise a heavy chain having the amino acid sequence of SEQ ID
NO: 4.
In some embodiments, the pharmaceutical compositions further comprise a chemotherapeutic agent. In some embodiment, the chemotherapeutic agent is selected from gemcitabine, cisplatin, paclitaxel, and navelbine.
In another aspect, the present disclosure provides pharmaceutical compositions comprising an antibody that binds to an extracellular region of human CD147, or a fragment of such antibody, and a pharmaceutically acceptable vehicle or excipient, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ
ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 1, and the antibody contains a glycoform lacking both fucose residues and xylose residues. In some embodiments, the antibodies comprise a light chain having the amino acid sequence of SEQ ID
NO: 3. In some embodiments, the antibodies comprise a heavy chain having the amino acid sequence of SEQ ID
NO: 4.
In some embodiments, the pharmaceutical compositions further comprise a chemotherapeutic agent. In some embodiment, the chemotherapeutic agent is selected from gemcitabine, cisplatin, paclitaxel, and navelbine.
[0013] In another aspect, the present disclosure provides methods for producing an antibody that binds to an extracellular region of human CD147, or a fragment of such antibody, comprising: obtaining a nucleotide sequence encoding for a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and nucleotide sequence encoding for a light chain variable region having the amino acid sequence of SEQ ID NO: 1; constructing an vector comprising the nucleotide sequence or fragment of the nucleotide sequence;
transfecting the vector into an acetyl-glucosamine transferase deficient cell line; culturing the transfected cell line in media; and obtaining the antibody or the fragment of the antibody from the culture.
transfecting the vector into an acetyl-glucosamine transferase deficient cell line; culturing the transfected cell line in media; and obtaining the antibody or the fragment of the antibody from the culture.
[0014] In another aspect, the present disclosure provides methods for producing an antibody that binds to an extracellular region of human CD147, or fragment thereof, comprising: culturing a host cell comprising a vector comprising a first nucleotide sequence encoding for a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a second nucleotide sequence encoding for a light chain variable region having the amino acid sequence of SEQ ID
NO: 1 under a condition to allow expression of the antibody or fragment thereof comprising the heavy chain and the light chain; and obtaining the antibody or the fragment thereof from the culture.
NO: 1 under a condition to allow expression of the antibody or fragment thereof comprising the heavy chain and the light chain; and obtaining the antibody or the fragment thereof from the culture.
[0015] In some embodiments, the step of culturing the transfected cell line in media comprises culturing the transfected cell line at 36-38 C for a first period and then at 30-32 C for a second period. In some embodiments, the first period is 4-10 days. In some embodiments, the second period is 11-21 days.
[0016] In some embodiments, the expression level of the antibody by the cell line is at least 100 mg/L, at least 200 mg/L, or at least 300 mg/L. In some embodiments, the percentage of non-glycosylated heavy chain calculated based on the total amount of antibody obtained is less than 10%, 7%, 5% or 3%. In some embodiments, the cell line is an acetyl-glucosamine transferase deficient CHO cell line.
[0017] In another aspect, the present disclosure provides methods for treating a disease associated with human CD147 in a subject in need thereof, comprising administering an effective amount of an antibody that binds to an extracellular region of human CD147, or fragment thereof, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO:
1, and the antibody contains a glycoform lacking both fucose residues and xylose residues. In some embodiments, the antibody comprises a light chain having the amino acid sequence of SEQ
ID NO: 3. In some embodiments, the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 4. In some embodiments, the methods further comprise administering a chemotherapeutic agent to the subject. In some embodiments, the chemotherapeutic agent is selected from gemcitabine, cisplatin, paclitaxel, and navelbine. In some embodiments, the disease is cancer. In some embodiments, the cancer is lung cancer, liver cancer, esophagus i cancer, ovarian cancer, stomach cancer, breast cancer, cervical cancer, or colon cancer. In some embodiments, the disease is lung cancer. In some embodiments, the disease is non-small cell lung cancer.
BRIEF DESCFRIPTION OF FIGURES
1, and the antibody contains a glycoform lacking both fucose residues and xylose residues. In some embodiments, the antibody comprises a light chain having the amino acid sequence of SEQ
ID NO: 3. In some embodiments, the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 4. In some embodiments, the methods further comprise administering a chemotherapeutic agent to the subject. In some embodiments, the chemotherapeutic agent is selected from gemcitabine, cisplatin, paclitaxel, and navelbine. In some embodiments, the disease is cancer. In some embodiments, the cancer is lung cancer, liver cancer, esophagus i cancer, ovarian cancer, stomach cancer, breast cancer, cervical cancer, or colon cancer. In some embodiments, the disease is lung cancer. In some embodiments, the disease is non-small cell lung cancer.
BRIEF DESCFRIPTION OF FIGURES
[0018] Figure 1 shows the construction diagram of the pQD-Hyg-GSeu-cHAb 1 8 expression vector.
[0019] Figure 2 shows the antibody production levels of Metuzumab in CHO MAGE1.5 cells and cHAbl8 mAb produced in CHO K1 cells.
[0020] Figure 3A shows the antigen binding kinetics of Metuzumab and cHAbl8 mAb.
[0021] Figure 3B shows the antigen binding kinetics of Metuzumab and cHAbl 8 mAb.
[0022] Figure 4 shows the results of Metuzumab and cHAbl8 mAbs bound to A549 cells, which are human CD147 expression cells. The antibody concentration is 0.001 vtg/mL, 0.01p.g/mL, 0.1 g/mL, 1 vtg/m1 and 10 g/mL, respectively.
[0023] Figure 5 shows the results of CD147-binding specificity of Metuzumab in human cancer tissue array by immunohistochemistry.
[0024] Figure 6 shows the oligosaccharide and monosaccharide profiles of Metuzumab and cHAbl8.
[0025] Figure 7 shows an ADCC activity of Metuzumab on non-small cell lung cancer cells.
NCI-H520, A549 and NCI-H446 cells (1x104 per well) were incubated with Metuzumab or cHAb 18 mAb for 30 min at 37 C. Effector cells (Spleen cells) were added at the ratio of effector cells to T cells (E/T) 50:1 for 24 hours at 37 C in CO2 incubator. After centrifugation, supernatants were harvested and lactate dehydrogenase (LDH) activity were measured. Data represent mean SD of five independent experiments.
NCI-H520, A549 and NCI-H446 cells (1x104 per well) were incubated with Metuzumab or cHAb 18 mAb for 30 min at 37 C. Effector cells (Spleen cells) were added at the ratio of effector cells to T cells (E/T) 50:1 for 24 hours at 37 C in CO2 incubator. After centrifugation, supernatants were harvested and lactate dehydrogenase (LDH) activity were measured. Data represent mean SD of five independent experiments.
[0026] Figure 8A shows inhibition of cancer migration by Metuzumab.
[0027] Figure 8B shows inhibition of cancer invasion by Metuzumab.
[0028] Figure 9A shows inhibition of tumor growth in vivo by the Metuzumab. Mice injected with NCI-H520 cells subcutaneously in the right dorsal flank were treated by intravenous saline;
2mg/kg Cisplatin and 100mg/kg Gemcitabine; Metuzumab infusion at 2 mg/kg, 10 mg/kg and 30 mg/kg; combination of 10 mg/kg Metuzumab with 2mg/kg Cisplatin and 100mg/kg Gemcitabine;
wherein the antibodies were injected twice weekly for 3 week consecutively, while the I
chemotherapeutic agents were injected once a week for 3 weeks consecutively.
The tumors were measured twice a week.
2mg/kg Cisplatin and 100mg/kg Gemcitabine; Metuzumab infusion at 2 mg/kg, 10 mg/kg and 30 mg/kg; combination of 10 mg/kg Metuzumab with 2mg/kg Cisplatin and 100mg/kg Gemcitabine;
wherein the antibodies were injected twice weekly for 3 week consecutively, while the I
chemotherapeutic agents were injected once a week for 3 weeks consecutively.
The tumors were measured twice a week.
[0029] Figure 9B shows inhibition of tumor growth in vivo by the Metuzumab. Mice injected with A549 cells subcutaneously in the right dorsal flank were treated by intravenous saline;
2mg/kg Cisplatin and 100mg/kg Gemcitabine; Metuzumab infusion at 2 mg/kg, 10 mg/kg and 30 mg/kg; combination of 10 mg/kg Metuzumab with 2mg/kg Cisplatin and 100mg/kg Gemcitabine;
wherein the antibodies were injected twice weekly for 3 week consecutively, while the chemotherapeutic agents were injected once a week for 3 weeks consecutively.
The tumors were measured twice a week.
DETAILED DESCRIPTION OF THE INVENTION
2mg/kg Cisplatin and 100mg/kg Gemcitabine; Metuzumab infusion at 2 mg/kg, 10 mg/kg and 30 mg/kg; combination of 10 mg/kg Metuzumab with 2mg/kg Cisplatin and 100mg/kg Gemcitabine;
wherein the antibodies were injected twice weekly for 3 week consecutively, while the chemotherapeutic agents were injected once a week for 3 weeks consecutively.
The tumors were measured twice a week.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.
Nucleotide Sequence
Nucleotide Sequence
[0031] In one aspect, the present disclosure provides nucleotide sequences encoding an antibody or a fragment thereof, comprising the sequence of SEQ ID NO: 5 or a degenerate variant thereof, and/or sequence of SEQ ID NO: 6 or a degenerate variant thereof
[0032] In some embodiments, the nucleotide sequence comprises the sequence of SEQ ID
NO: 5. In some embodiments, the nucleotide sequence comprises the sequence of SEQ ID NO: 6.
In some embodiments, the nucleotide sequence comprises both the sequence of SEQ ID NO: 5 and the sequence of SEQ ID NO: 6.
NO: 5. In some embodiments, the nucleotide sequence comprises the sequence of SEQ ID NO: 6.
In some embodiments, the nucleotide sequence comprises both the sequence of SEQ ID NO: 5 and the sequence of SEQ ID NO: 6.
[0033] In some embodiments, the nucleotide sequence comprises the sequence of SEQ ID
NO: 7. In some embodiments, the nucleotide sequence comprises the sequence of SEQ ID NO: 8.
In some embodiments, the nucleotide sequence comprises both the sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 8.
!
NO: 7. In some embodiments, the nucleotide sequence comprises the sequence of SEQ ID NO: 8.
In some embodiments, the nucleotide sequence comprises both the sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 8.
!
[0034] The term "degenerate variant" as used herein refers to a nucleic acid molecule which includes one or more alternative genetic codons to those present in the parent sequence, yet encodes the same amino acid sequence due to the degeneracy of the genetic codons.
[0035] Without wishing to be bound by theory, but it is believed that SEQ ID NO: 5 and SEQ
ID NO: 6 are particularly advantageous in expression in certain types of host cells such as CHO
cells, partly because of the optimization in the genetic codon usage based on the host cells or other types of cells.
ID NO: 6 are particularly advantageous in expression in certain types of host cells such as CHO
cells, partly because of the optimization in the genetic codon usage based on the host cells or other types of cells.
[0036] The nucleotide sequence used in the present disclosure can be prepared by any methods known in the art, including but not limited to, chemical synthesis, molecular cloning and etc. (see, e.g. for details, Sambrook, Fritsch & Maniatis, Molecular Cloning: A laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, 1989).
[0037] The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, and bispecific (bivalent) antibody that binds to a specific antigen. A native intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region (VH) and a first, second, and third constant region (CHI, CH2, CH3), while each light chain consists of a variable region (VI) and a constant region (CO. The antibody has a "Y" shape, with the stem of the Y
consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding.
Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The Fc portion of an antibody refers to that portion of the antibody consisting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding. The Fc portion of the antibody is responsible for various effector functions such as ADCC, and Complement Dependent Cytotoxicity (CDC), but does not function in antigen binding.
consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding.
Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The Fc portion of an antibody refers to that portion of the antibody consisting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding. The Fc portion of the antibody is responsible for various effector functions such as ADCC, and Complement Dependent Cytotoxicity (CDC), but does not function in antigen binding.
[0038] A "fragment" of an antibody as used herein refers to an antigen-binding portion or full length of a heavy chain or a light chain of an antibody, or an antigen-binding portion of both the heavy chain and the light chain of an antibody.
Vector I
Vector I
[0039] In another aspect, the nucleotide sequence described above may be inserted into one or more vectors. The term "vector" as used herein, refers to a polynucleotide vehicle capable of transforming or transfecting cells and allowing gene expression in the cells.
A vector can be an expression vector and a cloning vector. The present disclosure provides vectors (e.g. expression vectors) containing the nucleotide sequence provided herein encoding the antibody or a fragment thereof, at least one promoter operably linked to the nucleotide sequence, and at least one selection marker. The expression vectors of the present disclosure can be viral vectors, plasmids, phages and cosmids. Examples include, such as, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40), lambda phage, and M13 phage, plasmid PCR 2.1, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, and etc.
A vector can be an expression vector and a cloning vector. The present disclosure provides vectors (e.g. expression vectors) containing the nucleotide sequence provided herein encoding the antibody or a fragment thereof, at least one promoter operably linked to the nucleotide sequence, and at least one selection marker. The expression vectors of the present disclosure can be viral vectors, plasmids, phages and cosmids. Examples include, such as, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40), lambda phage, and M13 phage, plasmid PCR 2.1, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, and etc.
[0040] In some embodiments, the present disclosure provides vectors capable of expressing the antibody provided herein or a fragment thereof, comprising the nucleotide sequence having the sequence of SEQ ID NO: 5, and/or sequence of SEQ ID NO: 6.
[0041] In some embodiments, the vectors comprise the nucleotide sequence comprising the sequence of SEQ ID NO: 5. In some embodiments, the vectors comprise the nucleotide sequence comprising the sequence of SEQ ID NO: 6. In some embodiments, the vectors comprise the nucleotide sequence comprising both the sequence of SEQ ID NO: 5 and the sequence of SEQ
ID NO: 6. In some embodiments, the vectors comprise the nucleotide sequence having the sequence of SEQ ID NO: 7, and/or sequence of SEQ ID NO: 8.
Host Cell
ID NO: 6. In some embodiments, the vectors comprise the nucleotide sequence having the sequence of SEQ ID NO: 7, and/or sequence of SEQ ID NO: 8.
Host Cell
[0042] In another aspect, the present disclosure provides host cells comprising a vector provided herein, wherein the vector comprises the nucleotide sequence having the sequence of SEQ ID NO: 5, and/or sequence of SEQ ID NO: 6. In some embodiments, the host cells comprise a vector provided comprising the nucleotide sequence having the sequence of SEQ ID
NO: 7, and/or sequence of SEQ ID NO: 8.
NO: 7, and/or sequence of SEQ ID NO: 8.
[0043] In some embodiments, the host cells comprise a vector provided herein, wherein the vector comprises the nucleotide sequence having the sequence of SEQ ID NO: 5.
In some embodiments, the host cells comprise a vector provided herein, wherein the vector comprises the nucleotide sequence having the sequence of SEQ ID NO: 6. In some embodiments, the host cells i I
comprise a vector provided herein, wherein the vector comprises the nucleotide sequence having both the sequence of SEQ ID NO: 5 and the sequence of SEQ ID NO: 6.
In some embodiments, the host cells comprise a vector provided herein, wherein the vector comprises the nucleotide sequence having the sequence of SEQ ID NO: 6. In some embodiments, the host cells i I
comprise a vector provided herein, wherein the vector comprises the nucleotide sequence having both the sequence of SEQ ID NO: 5 and the sequence of SEQ ID NO: 6.
[0044] In some embodiments, the nucleotide sequence included in the vector contains genetic codons which are selected for accommodating host cell codon usage bias. The term "codon usage bias" as used herein, refers to the extent to which one codon is preferentially used by the host cell to code for a particular amino acid over all other possible codons for the same amino acid. Codon usage bias is due to the differences in the codon usage in the genome of different species. The genetic codons contained in the vector can be accommodated based on the host cell or other types of cells.
[0045] The term "host cell" as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced. A host cell can be transfected with a vector of the present disclosure. Host cells may be eukaryotic cells that are capable of producing antibody with glycosylation. Examples of eukaryotic cells include animal cells such as mammalian cells. For example, the host cell may be cell lines, such as but are not limited to HeLa, HEK-293, NIH3T3, COS, Chinese Hamster Ovary (CHO) cells, NSO, PER.C6, K562, L1.2, JY, BHK, K562, 293F, 3T3, and Jurkat. The CHO cells may include, but not be limited to, CHO/DHFR-(dihyrofolate reductase deficiency) or CHO/GS- (glutamine synthetase deficientcy) cells. Examples of CHO cells include those described in documents such as Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. ScL USA, 60 , 1275 (1968);
Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996);
Radiation Research, 148, 260 (1997); Proc. Natl. Acad. ScL USA, 77, 4216 (1980); Proc. Natl.
Acad. ScL, 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp.
883-900); and the like. In addition, CHO-K1 (ATCC CCL-61), DUXB11 (ATCC CCL-9096), CHO DG44 and Pro-5 (ATCC CCL-1781) registered in ATCC (The American Type Culture Collection) and a commercially available CHO-S (Life Technologies, Cat #
11619) or sub-cell lines obtained by adapting the cell lines using various media can also be exemplified. In some embodiments, the CHO cells are adapted to suspension cell culture. In some embodiments, the CHO cells are adapted to serum free culture.
Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996);
Radiation Research, 148, 260 (1997); Proc. Natl. Acad. ScL USA, 77, 4216 (1980); Proc. Natl.
Acad. ScL, 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp.
883-900); and the like. In addition, CHO-K1 (ATCC CCL-61), DUXB11 (ATCC CCL-9096), CHO DG44 and Pro-5 (ATCC CCL-1781) registered in ATCC (The American Type Culture Collection) and a commercially available CHO-S (Life Technologies, Cat #
11619) or sub-cell lines obtained by adapting the cell lines using various media can also be exemplified. In some embodiments, the CHO cells are adapted to suspension cell culture. In some embodiments, the CHO cells are adapted to serum free culture.
[0046] In some embodiments, the CHO cells are modified to provide for a variant glycosylation pattern as compared to an unmodified parental host cell. For example, the CHO
cells can be modified to over-express or under-express or knock-out one or more enzymes responsible for glycosylation. In some embodiments, the CHO cells are acetyl-glucosamine transferase deficient.
Antibody
cells can be modified to over-express or under-express or knock-out one or more enzymes responsible for glycosylation. In some embodiments, the CHO cells are acetyl-glucosamine transferase deficient.
Antibody
[0047] The present disclosure provides antibodies that bind to an extracellular region of human CD147, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 4, and the antibody contains a glycoform lacking fucose residues, xylose residues, or both. In some embodiments, the antibodies further comprise a light chain having the amino acid sequence of SEQ ID NO: 3.
[0048] In some embodiments, the antibody is a human-mouse chimeric antibody. A "human-mouse chimeric antibody" refers to an antibody in which the heavy/light chain variable regions are derived from mouse, and the heavy/light chain constant regions are derived from human. In some embodiments, the human-mouse chimeric antibodies comprise a constant region derived from human antibody IgG1 , IgG2, or IgG4. In some embodiments, the antibody comprises a constant region of human antibody IgGl.
[0049] In some embodiments, the antibodies provided herein are recombinant antibodies and fragments thereof that recognize an extracellular region of CD147 and have cytotoxicity on CD147 expressing cells. Exemplary antibodies of the present disclosure include, HAb18, cHAb18, and variants thereof having certain glycoforms.
[0050] The HAbl 8 antibody is a monoclonal murine antibody produced by a hybridoma cell line generated using a BALB/c mice immunized with a cell suspension extracted from fresh human HCC tissues. The hybridoma cell line has been deposited under the deposit number of CGMCC NO. 0426 with China General Microbiological Culture Collection Center having the address of NO.1 West Beichen Road, Chaoyang District, Beijing 100101, P. R.
China.
China.
[0051] The cHAbl8 antibody is a human-mouse chimeric anti-CD147 antibody which comprises a heavy chain variable domain and a light chain variable domain derived from HAb18, and constant domains derived from a human antibody IgGl. The cHAbl8 comprises a heavy chain amino acid sequence of SEQ ID NO: 4, and light chain amino acid sequence of SEQ ID
NO: 3, in which the heavy chain variable domain has an amino acid sequence of SEQ ID NO: 2, and light chain variable domain has an amino acid sequence of SEQ ID NO: 1.
NO: 3, in which the heavy chain variable domain has an amino acid sequence of SEQ ID NO: 2, and light chain variable domain has an amino acid sequence of SEQ ID NO: 1.
[0052] In some embodiments, the antibodies and fragments thereof comprise a particular glycoform. As used herein, the term "glycoform" with respect to an antibody or fragment thereof, refers to a particular glycosylated form of the antibody or fragment thereof. When there is at least one amino acid residue in the antibody or fragment thereof that has the potential to be glycosylated (i.e., to be linked to a glycan or different glycans or different sets of glycans), each different version of the antibody or antibody fragment linked to a particular glycan or glycans is referred to as a glycoform. In other words, different glycoforms share the same amino acid sequence but have different glycans or oligosaccharides attached to the glycosylation site(s).
Glycosylated antibodies and fragments thereof can be recombinantly produced in host cells where the cellular glycosylation machinery transfers one or more glycans to the amino acid sequence of the antibodies and fragments thereof, thereby producing a population of glycosylated antibodies containing certain glycoforms.
Glycosylated antibodies and fragments thereof can be recombinantly produced in host cells where the cellular glycosylation machinery transfers one or more glycans to the amino acid sequence of the antibodies and fragments thereof, thereby producing a population of glycosylated antibodies containing certain glycoforms.
[0053] In some embodiments, the antibodies and fragments thereof provided herein are glycosylated in the Fc region. In some embodiments, the antibodies and fragments thereof provided herein carry N-linked oligosaccharides in its Fc region. For example, the antibodies and fragments thereof can carry two N-linked oligosaccharides in its Fc region, one on each heavy chain.
[0054] In some embodiments, the antibodies and fragment thereof provided herein contain a glycoform which specifically lacks fucose residues, xylose residues, or both.
In some embodiments, the antibodies and fragment thereof provided herein consist of one or more glycoforms which specifically lack(s) fucose residues, xylose residues, or both. The term "lack"
or "lacking" with respect to certain monosaccharide residues as used herein is intended to mean such monosaccharide residues are absent or are at a level that is too low to be detected using a detection method known in the art, for example, High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). Other detection methods may also be used to detect the oligosaccharides, for example, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI/TOF-MS).
In some embodiments, the antibodies and fragment thereof provided herein consist of one or more glycoforms which specifically lack(s) fucose residues, xylose residues, or both. The term "lack"
or "lacking" with respect to certain monosaccharide residues as used herein is intended to mean such monosaccharide residues are absent or are at a level that is too low to be detected using a detection method known in the art, for example, High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). Other detection methods may also be used to detect the oligosaccharides, for example, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI/TOF-MS).
[0055] In some embodiments, the ADCC activity of the antibodies that contain a glycoform lacking both fucose residues and xylose residues are at least 2 times, 5 times, 10 times, 20 times, 40 times, 60 times, or 100 times higher than the ADCC activity of an antibody that contains a glycoform comprising fucose residues, xylose residues, or both.
[0056] ADCC is a cell-mediated reaction in which antigen-nonspecific cytotoxic cells that express Fc Receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages which are included in PBMC or spleen) recognize antibody bound to the surface of a target cell and subsequently cause lysis of (i.e., "kill") the target cell. The term "ADCC
activity" refers to the ability of an antibody or Fc fusion protein to elicit an ADCC reaction. ADCC
activity can be assessed directly using an in vitro assay, e.g., a 51Cr release assay using peripheral blood mononuclear cells (PBMC) and/or spleen effector cells as described in the Examples and Shields et al. (2001) J. Biol. Chem., 276:6591-6604, or any other suitable method.
ADCC activity may be expressed as a concentration of antibody at which the lysis of target cells is half-maximal. The ADCC activity of an antibody generally depends on the binding affinity of the antibody to target cell, which could be affected by the glycoform of N-linked oligosaccharide of the antibody.
activity" refers to the ability of an antibody or Fc fusion protein to elicit an ADCC reaction. ADCC
activity can be assessed directly using an in vitro assay, e.g., a 51Cr release assay using peripheral blood mononuclear cells (PBMC) and/or spleen effector cells as described in the Examples and Shields et al. (2001) J. Biol. Chem., 276:6591-6604, or any other suitable method.
ADCC activity may be expressed as a concentration of antibody at which the lysis of target cells is half-maximal. The ADCC activity of an antibody generally depends on the binding affinity of the antibody to target cell, which could be affected by the glycoform of N-linked oligosaccharide of the antibody.
[0057] In some embodiments, the antibodies and fragment thereof provided herein contain a glycoform comprising Mannose-5 glycans. In some embodiments, the antibodies and fragments thereof provided herein contain N-linked Mannose-5 glycans. The term "Mannose-5" as used herein refers to a chain of five mannose residues which are added sequentially to the core monosaccharide(s). Core monosaccharide(s) are the monosaccharide(s) which are synthesized at the initial stage of glycosylation and form the starting point for addition of more monosaccharides. For example, in certain glycosylation process, two N-acetylglucosamine (G1cNAc) monosaccharides form the core monosaccharides upon which the additional sugar residues, for example, mannose residues are further added. Mannose-5 can be a linear sugar chain or a branched sugar chain. In some embodiments, Mannose-5 is a two-armed branched structure in which one arm consists of one mannose residue and the other arm consists of three mannose residues, with a joint mannose residue which links both arms to the core monosaccharides of the antibody peptide at the glycosylation site. More details can be found at, for example, Varki A et al, Essentials of glycobiology, Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1999, Chapter 7.
[0058] In some embodiments, the antibodies or fragments thereof in the present disclosure comprise a predominant portion of glycoforms containing N-linked Mannose-5 glycans. "A
predominant portion" as used herein refers to a portion that is above 70%, above 80%, above 90%, above 95%, above 96%, above 97%, above 98%, above 99%, or 100% of the whole population of glycosylated antibodies containing N-linked glycoforms. The amount of glycoform contained on the antibodies or fragments thereof may be measured by a conventional detection method known in the art, for example, HPAEC-PAD.
predominant portion" as used herein refers to a portion that is above 70%, above 80%, above 90%, above 95%, above 96%, above 97%, above 98%, above 99%, or 100% of the whole population of glycosylated antibodies containing N-linked glycoforms. The amount of glycoform contained on the antibodies or fragments thereof may be measured by a conventional detection method known in the art, for example, HPAEC-PAD.
[0059] In some embodiments, the antibody is Metuzumab or a fragment thereof.
Metuzumab is an antibody whose heavy chain amino acid sequence is SEQ ID NO: 4 and light chain amino acid sequence is SEQ ID NO: 3, and which further comprises one or more glycoform(s) lacking both fucose residues and xylose residues. In some embodiments, Metuzumab contains N-linked Mannose-5 glycans.
Metuzumab is an antibody whose heavy chain amino acid sequence is SEQ ID NO: 4 and light chain amino acid sequence is SEQ ID NO: 3, and which further comprises one or more glycoform(s) lacking both fucose residues and xylose residues. In some embodiments, Metuzumab contains N-linked Mannose-5 glycans.
[0060] A glycosylation profile of Metuzumab is shown in Figure 6. In some embodiments, the antibodies or fragments thereof provided herein have a glycosylation profile as analyzed by HPAEC-PAD that is substantially equivalent to that of Metuzumab as shown in Figure 6.
"Substantially equivalent" as used herein with respect to glycosylation profile is intended to mean the glycosylation profile is in general consistency to that shown in Figure 6. For example, the glycosylation profile has the same number of peak(s), similar peak shape, and/or similar peak position.
"Substantially equivalent" as used herein with respect to glycosylation profile is intended to mean the glycosylation profile is in general consistency to that shown in Figure 6. For example, the glycosylation profile has the same number of peak(s), similar peak shape, and/or similar peak position.
[0061] In some embodiments, the antibody is obtained from an acetyl-glucosamine transferase deficient cell line. In some embodiments, the cell line is acetyl-glucosamine transferase deficient CHO cell line.
[0062] In some embodiments, the heavy chain and light chain of Metuzumab are encoded by a nucleic acid of SEQ ID NO: 8 and a nucleic acid of SEQ ID NO: 7, respectively. Metuzumab has a heavy chain and/or a light chain encoded by nucleic acids containing altered genetic codon usage based on the codon usage bias in CHO cells to increase the expression level of the heavy chain and/or the light chain in the host cell. For example, the nucleic acid encoding the light chain variant region of Metuzumab comprises an altered codon for Serine (Ser, S) which is AGC, and the nucleic acid encoding the heavy chain variant region comprises an altered codon for Leucine (Leu, L) which is CTG, and an altered codon for Lysine (Lys, L) which is AAG. In some embodiments, the host cell for production of Metuzumab is acetyl-glucosamine transferase deficient CHO-Kl MAGE 1.5 cell (obtained from Eureka therapeutics, U.S.
8,025,879, U.S.
8,080,415, U.S. 8,084,222, PCT/1JS2009/051325, CN200980145664.4). Although the amino acid sequences of Metuzumab are identical to those of cHAb18, the glycosylation profile of Metuzumab is different from that of cHAb18. In some embodiments, the glycosylation profile of Metuzumab exhibits homogeneous glycoform and comprises solely Mannose-5 N-linked oligosaccharide and lacks fucose and xylose.
Pharmaceutical Composition
8,025,879, U.S.
8,080,415, U.S. 8,084,222, PCT/1JS2009/051325, CN200980145664.4). Although the amino acid sequences of Metuzumab are identical to those of cHAb18, the glycosylation profile of Metuzumab is different from that of cHAb18. In some embodiments, the glycosylation profile of Metuzumab exhibits homogeneous glycoform and comprises solely Mannose-5 N-linked oligosaccharide and lacks fucose and xylose.
Pharmaceutical Composition
[0063] The present disclosure further provides pharmaceutical compositions comprising an antibody that binds to an extracellular region of human CD147, or a fragment thereof, and a pharmaceutically acceptable vehicle or excipient, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 4 and a light chain having the amino acid sequence of SEQ ID NO: 3, and the antibody contains a glycoform lacking both fucose residues and xylose residues.
[0064] In some embodiments, the pharmaceutical compositions comprise an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 4, or a fragment thereof, and a pharmaceutically acceptable vehicle or excipient. In some embodiments, the pharmaceutical composition comprises an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 3, or a fragment thereof, and a pharmaceutically acceptable vehicle or excipient. In some embodiments, the pharmaceutical composition comprise an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO:
4 and a light chain having the amino acid sequence of SEQ ID NO:3, or a fragment thereof, and a pharmaceutically acceptable vehicle or excipient.
4 and a light chain having the amino acid sequence of SEQ ID NO:3, or a fragment thereof, and a pharmaceutically acceptable vehicle or excipient.
[0065] As used herein, the phrase "pharmaceutically acceptable"
means that compounds, materials, compositions, and/or dosage forms are, within the scope of sound medical judgment, suitable for use in vivo in human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments, compounds, materials, compositions, and/or dosage forms that are pharmaceutically acceptable refer to those approved by a regulatory agency (such as U.S. Food and Drug Administration, China Food and Drug Administration or European Medicines Agency) or listed in generally recognized pharmacopoeia (such as U.S. Pharmacopoeia, China Pharmacopoeia or European Pharmacopoeia) for use in animals, and more particularly in humans.
means that compounds, materials, compositions, and/or dosage forms are, within the scope of sound medical judgment, suitable for use in vivo in human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments, compounds, materials, compositions, and/or dosage forms that are pharmaceutically acceptable refer to those approved by a regulatory agency (such as U.S. Food and Drug Administration, China Food and Drug Administration or European Medicines Agency) or listed in generally recognized pharmacopoeia (such as U.S. Pharmacopoeia, China Pharmacopoeia or European Pharmacopoeia) for use in animals, and more particularly in humans.
[0066] As used herein, the term "vehicle or excipient" refers to any and all carriers, fillers, solvents, coatings, antibacterial and antifungal agents, flavoring agents, isotonic and absorption delaying agents, and the like that are pharmaceutically acceptable and can facilitate storage and administration of the compounds of the present application to a subject.
Pharmaceutically acceptable vehicle or excipient that can be employed in present disclosure includes those generally known in the art, such as those described in "Remington Pharmaceutical Sciences"
Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference.
!
Pharmaceutically acceptable vehicle or excipient that can be employed in present disclosure includes those generally known in the art, such as those described in "Remington Pharmaceutical Sciences"
Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference.
!
[0067] In some embodiments, the pharmaceutical composition of the present disclosure further comprises one or more chemotherapeutic agents. In some embodiments, the chemotherapeutic agent is selected from gemcitabine, cisplatin, paclitaxel, navelbine, or a combination thereof. In some embodiments, the chemotherapeutic agent is gemcitabine and cisplatin.
Method for producing an antibody
Method for producing an antibody
[0068] In another aspect, the present disclosure provides methods for producing an antibody that binds to an extracellular region of human CD147, or a fragment thereof, comprising:
culturing a host cell comprising a vector comprising a first nucleotide sequence encoding a heavy chain having the amino acid sequence of SEQ ID NO: 4 and a second nucleotide sequence encoding a light chain having the amino acid sequence of SEQ ID NO: 3 under a condition to allow expression of the antibody comprising the heavy chain and the light chain or a fragment thereof; and obtaining the antibody or the fragment thereof from the culture.
culturing a host cell comprising a vector comprising a first nucleotide sequence encoding a heavy chain having the amino acid sequence of SEQ ID NO: 4 and a second nucleotide sequence encoding a light chain having the amino acid sequence of SEQ ID NO: 3 under a condition to allow expression of the antibody comprising the heavy chain and the light chain or a fragment thereof; and obtaining the antibody or the fragment thereof from the culture.
[0069] In yet another aspect, the present disclosure provides methods for producing an antibody that binds to an extracellular region of human CD147, or fragment thereof, comprising:
obtaining nucleotide sequence encoding a heavy chain having the amino acid sequence of SEQ
ID NO: 4 and nucleotide sequence encoding a light chain having the amino acid sequence of SEQ ID NO: 3; constructing an vector comprising the nucleotide sequence or a fragment of the nucleotide sequence; transfecting the vector into acetyl-glucosamine transferase deficient cell line; culturing the transfected cell line in media; and obtaining the antibody or the fragment of the antibody from the culture.
obtaining nucleotide sequence encoding a heavy chain having the amino acid sequence of SEQ
ID NO: 4 and nucleotide sequence encoding a light chain having the amino acid sequence of SEQ ID NO: 3; constructing an vector comprising the nucleotide sequence or a fragment of the nucleotide sequence; transfecting the vector into acetyl-glucosamine transferase deficient cell line; culturing the transfected cell line in media; and obtaining the antibody or the fragment of the antibody from the culture.
[0070] In some embodiments, the step of obtaining the nucleotide sequence comprises altering the nucleotide sequence to accommodate codon usage bias to host acetyl-glucosamine transferase deficient cell line.
[0071] The step of transfecting the vector into the acetyl-glucosamine transferase deficient cell line can be performed via any method known in the art, including but are not limited to, electro-transfection, liposome transfection, viral transfection.
[0072] The culture media used in present disclosure may be any culture media suitable for culturing the host cell line, including but are not limited to, RPMI 1640, DMEM, CDM4 CHO
(Hyclone), CD CHO Opti (Gibco), CHO Efficient Feed A or B (Gibco). In some embodiments, !
i the culture media is serum free media. In some embodiments, the culture media is animal component free media. In some embodiments, the culture media is CD CHO Opti (Gibco).
(Hyclone), CD CHO Opti (Gibco), CHO Efficient Feed A or B (Gibco). In some embodiments, !
i the culture media is serum free media. In some embodiments, the culture media is animal component free media. In some embodiments, the culture media is CD CHO Opti (Gibco).
[0073] In some embodiments, the step of culturing the transfected cell line in media includes a fed-batch process. In some embodiments, the step of culturing the transfected cell line in media comprises culturing the transfected cell line at 36-38 C for a first period and then at 30-32 C for a second period. In some embodiments, the first period is 4-10 days.
In some embodiments, the second period is 11-21 days.
In some embodiments, the second period is 11-21 days.
[0074] In some embodiments, the expression level of the antibody in the cell line is at least 100 mg/L, at least 200 mg/L, or at least 300 mg/L. In some embodiments, the expression level of the antibody in the cell line increases by at least 20 times, or at least 50 times, or at least 100 times. In some embodiments, the percentage of non-glycosylated heavy chain calculated based on the total amount of the antibody obtained is less than 10%, 7%, 5% or 3%.
Method for treating human CD147 expression-related diseases
Method for treating human CD147 expression-related diseases
[0075] In another aspect, the present disclosure provides methods for treating human CD147 expression-related diseases in a subject in need thereof, comprising administering an effective amount of an antibody that binds to an extracellular region of human CD147, or a fragment thereof, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ
ID NO: 4 and a light chain having the amino acid sequence of SEQ ID NO: 3, and the antibody contains a glycoform lacking both fucose residues and xylose residues. In some embodiments, the methods further comprise administering a chemotherapeutic agent to the subject. In some embodiments, the chemotherapeutic agent is selected from gemcitabine, cisplatin, paclitaxel, and navelbine. In some embodiments, the disease is cancer. In some embodiments, the cancer is lung cancer, liver cancer, esophagus cancer, ovarian cancer, stomach cancer, breast cancer, cervical cancer, or colon cancer. In some embodiments, the disease is lung cancer. In some embodiments, the disease is non-small cell lung cancer.
ID NO: 4 and a light chain having the amino acid sequence of SEQ ID NO: 3, and the antibody contains a glycoform lacking both fucose residues and xylose residues. In some embodiments, the methods further comprise administering a chemotherapeutic agent to the subject. In some embodiments, the chemotherapeutic agent is selected from gemcitabine, cisplatin, paclitaxel, and navelbine. In some embodiments, the disease is cancer. In some embodiments, the cancer is lung cancer, liver cancer, esophagus cancer, ovarian cancer, stomach cancer, breast cancer, cervical cancer, or colon cancer. In some embodiments, the disease is lung cancer. In some embodiments, the disease is non-small cell lung cancer.
[0076] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[0077] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood !
that the terms "comprises", "comprising", "includes" , "including", "have"
and/or "having" if used herein, specify the presence of stated features, steps, operations, elements and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components and/or groups thereof. The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited.
Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "3000 mm2" is intended to mean "about 3000 mm2". As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%.
that the terms "comprises", "comprising", "includes" , "including", "have"
and/or "having" if used herein, specify the presence of stated features, steps, operations, elements and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components and/or groups thereof. The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited.
Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "3000 mm2" is intended to mean "about 3000 mm2". As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%.
[0078] Every document cited herein, including any cross referenced or related patent or application is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.
[0079] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are now described.
EXAMPLES
EXAMPLES
[0080] The following examples are presented to illustrate the present disclosure. They are not intended to limiting in any manner. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
EXAMPLE 1: Production of chimeric antibody against CD147
EXAMPLE 1: Production of chimeric antibody against CD147
[0081] The hybridoma cell line deposited in China General Microbiological Culture Collection Center (CGMCC NO. 0426) is used to produce a mouse monoclonal antibody against human CD147, named HAb18. DNA encoding the variable region of HAbl 8 was cloned as described below. Then, the mouse heavy/light chain variable region sequences were ligated to sequences of the constant region of human IgGl, and then inserted into a vector for use of producing a chimeric antibody, cHAb18.
[0082] Cloning Variable Region of HAbl8
[0083] (i) Extraction of total RNA from hybridoma cell culture:
[0084] Total RNA of hybridoma cell culture producing HAbl 8 antibody was isolated with TRIzol reagent (Invitrogen) according to manufacturer's instruction. The quality and quantity of the RNA were determined by ultraviolet spectroscopy (260/280 nm, 1.8<ratio<2.0) and 1%
denaturing polyacrylamide gel electrophoresis.
denaturing polyacrylamide gel electrophoresis.
[0085] (ii) Preparation of cDNA library from the extracted total RNA:
[0086] First strand cDNA was synthesized by PrimeScript RT reagent Kit (from TaKaRa) according to the manufacturer's instruction.
[0087] (iii) Amplification of variable regions of HAb18 by PCR
[0088] The DNA encoding the variable regions of heavy chain and light chain (VH and VI) of murine anti-human monoclonal antibody HAb18 were amplified by PCR with specific primers.
PCR was performed with the Phusion High-Fidelity DNA Polymerase from NEB.
PCR was performed with the Phusion High-Fidelity DNA Polymerase from NEB.
[0089] Primers for cloning the variable region of the light Chain are:
[0090] VL-5': 5'-AGCATTGTGATGACCCAGACTCCCACATT-3' (SEQ ID NO: 9)
[0091] VL-3': 5'-CCGCTTGATTTCCAACTTTGTCCCCGAGCC-3' (SEQ ID NO: 10).
[0092] Primers for cloning the variable region of the heavy chain are:
[0093] VH-S': 5'-GAAGTGAAGCTGGAGGAGTCTGGAGGAGGCT-3' (SEQ ID NO: 11)
[0094] VH-3': 5'-TGCAGAGACAGTGACCAGAGTCCCTTGGCCC-3' (SEQ ID NO: 12)
[0095] The PCR reaction was carried out under the following conditions:
40 cycles of 94 C, 1 min; 55 C, 1 min; and 72 C, 1 mm; for the extension at 72 C in the final cycle, 10min.
40 cycles of 94 C, 1 min; 55 C, 1 min; and 72 C, 1 mm; for the extension at 72 C in the final cycle, 10min.
[0096] (iv) PCR Product Purification and ligation into pMD18 T vector
[0097] The PCR products (about 360bp and about 410bp, respectively) was separated on a 1% agarose gel, excised, purified on E.Z.N.A.TM Gel Extraction Kit (Omega bio-tek) according to manufactures' instruction and eluted in DNAse free water.
i
i
[0098] PCR Products were ligated into pMD18 T vector (TaKaRa, Otsu, Japan) according to manufactures' instruction, transformed into E. coli cells, screened for positive clones and sequenced.
[0099] Construction of expression vector for Metuzumab
[00100] To enhance the antibody productivity in CHO-Kl cells, we optimized the codon of some amino acid codon in light/heavy chain variable region to increase the expression levels of the peptides in CHO cells. In heavy chain variable region, the codon for Leu4 was changed from CTT to CTG, the codon for Lys78 was changed from AAA to AAG. In light chain variable region, the codon for Serl was modified from AGT to AGC, the codon for Leu103 was changed from CTG to TTG, the codon for Lys106 was changed from AAA to AAG, the codon for Arg107 was changed from CGC to CGG. Optimized nucleotide sequences of light/heavy chain variable region are shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively.
[00101] The pQD-Hyg-GSeu vector was previously constructed in-house based on a commercial vector pcDNA3.1(-) (Invitrogen). Briefly, pcDNA3.1(-) was modified to give rise to pQD vector, followed by further engineering to incorporate hygromycin gene and the GS region.
Light/heavy chain variable region sequences were synthesized with the signal peptide sequence (SEQ ID NO 13) on the 5' terminal and inserted into Nhe I + Barn HI (VH) and Hind III + Xba I
(VL) restriction site of pQD-Hyg-GSeu vector by T4-ligase (NEB, U.S.) so that the light chain variable region and heavy chain variable region were linked to human IgG1 light chain/heavy chain constant region sequences that are previously included in the vector, respectively, to form a full length light chain encoding sequence (SEQ ID NO: 7) and a full length heavy chain encoding sequence (SEQ ID NO: 8) to generate pQD-Hyg-GSeu-cHAbl8 expression vector (Figure 1). The desirable clone was obtained after transformation, PCR based identification and sequencing analysis.
Light/heavy chain variable region sequences were synthesized with the signal peptide sequence (SEQ ID NO 13) on the 5' terminal and inserted into Nhe I + Barn HI (VH) and Hind III + Xba I
(VL) restriction site of pQD-Hyg-GSeu vector by T4-ligase (NEB, U.S.) so that the light chain variable region and heavy chain variable region were linked to human IgG1 light chain/heavy chain constant region sequences that are previously included in the vector, respectively, to form a full length light chain encoding sequence (SEQ ID NO: 7) and a full length heavy chain encoding sequence (SEQ ID NO: 8) to generate pQD-Hyg-GSeu-cHAbl8 expression vector (Figure 1). The desirable clone was obtained after transformation, PCR based identification and sequencing analysis.
[00102] Establishment of CHO Cell Line Capable of Producing Metuzumab
[00103] The pQD-Hyg-GSeu-cHAbl8 expression vector were transfected in CHO K1 and CHO K1 MAGE1.5 cells (preserved in Charles River Laboratory, 510803-MCB2) by electro-transfection using Nucleofector II (from Amaxa), respectively, to generate cell line that produce cHAbl8 and cell line that produce Metuzumab.
[00104] 3x106 CHO K1 and CHO K1 MAGE1.5 cells were respectively collected by centrifuge at 500rpm for 10min at room temperature, re-suspended in 100u1 Nucleofection !
solution V and combined with 2[tg pQD-Hyg-GSeu-cHAbl8 expression vector, 2 [tg pmaxGFP
Vector. Transfer cell/DNA suspension into Amaxa cuvette and electroporated by program U-023.
A Nucleofector Program G-023 was performed for electro-transfection.
solution V and combined with 2[tg pQD-Hyg-GSeu-cHAbl8 expression vector, 2 [tg pmaxGFP
Vector. Transfer cell/DNA suspension into Amaxa cuvette and electroporated by program U-023.
A Nucleofector Program G-023 was performed for electro-transfection.
[00105] The cells treated by electro-transfection were added into a CD OPTI-CHO medium (Invitrogen) in a 6-well and desired culturing vessel and incubator for 48 hour at 37 C, 8% CO2.
The transfected neo+ cells were selected with 500 [tg/m1 Hygromycin B
(Invitrogen), and the gene was amplified in 2 rounds of L-Methionine Sulfoximine (MSX, 25 M, Sigma) pressure selection. Cell line capable of producing approximately 163 mg/L of Metuzumab was obtained using ClonePix FL (Genentix Inc).
EXAMPLE 2: Expression of cHAbl8 and Metuzumab in batch culture
The transfected neo+ cells were selected with 500 [tg/m1 Hygromycin B
(Invitrogen), and the gene was amplified in 2 rounds of L-Methionine Sulfoximine (MSX, 25 M, Sigma) pressure selection. Cell line capable of producing approximately 163 mg/L of Metuzumab was obtained using ClonePix FL (Genentix Inc).
EXAMPLE 2: Expression of cHAbl8 and Metuzumab in batch culture
[00106] CHO cells expressing cHAbl8 (CHO K 1 -cHAb18) and CHO cells expressing Metuzumab (CHO MAGE1.5-HcHAb18) were respectively thawed and cultured in serum-free medium. Initially both cell cultures were incubated at 37 C with 5% CO2. After the cell density of the Metuzumab expressing cells reached 1 x106 cells/ml (around day 4), nutrient and glucose were fed to the culture at suitable intervals (for example 2 days interval).
After cell growth reached stationary phase (around 7 days for the two cell lines in this example), culture of the Metuzumab expressing cells was switched to incubation at 31 C until the end of the culturing (around 21 days). At day 21 of cultivation, both cell cultures were harvested.
After cell growth reached stationary phase (around 7 days for the two cell lines in this example), culture of the Metuzumab expressing cells was switched to incubation at 31 C until the end of the culturing (around 21 days). At day 21 of cultivation, both cell cultures were harvested.
[00107] The harvested cell cultures were firstly filtrated through depth filter to remove cells.
Then affinity chromatography was carried out using Mabselect Sure to capture the antibody.
Finally, hydrophobic chromatography was carried out using Phenyl Sepharose High performance to further purify the antibodies. The final purity for both cHAbl8 and Metuzumab were higher than 95%.
Then affinity chromatography was carried out using Mabselect Sure to capture the antibody.
Finally, hydrophobic chromatography was carried out using Phenyl Sepharose High performance to further purify the antibodies. The final purity for both cHAbl8 and Metuzumab were higher than 95%.
[00108] Concentrations of the antibodies were then determined by ELISA protein quantification method. Through reestablishment of the cell line (vector transfection and optimal clone selection) and adopting fed-batch and low temperature culture process, antibody concentration had been greatly improved. As shown in Figure 2, the average mab titer of cHAbl8 was 23 mg/L, the average mab titer of of Metuzumab was 308 mg/L.
EXAMPLE 3: Antigen-Binding Activity of Antibodies
EXAMPLE 3: Antigen-Binding Activity of Antibodies
[00109] Measurement of Antigen-Binding Activity by Surface plasmon resonance (SPR) I
[00110] CD147 binding of selected antibodies (original mouse antibody HAb18, chimeric antibody cHAbl 8, and glycosylation modified chimeric antibody Metuzumab) were confirmed by SPR analysis (shown in Figure 3A and Figure 3B).
[00111] The affinity (KD) for the binding of human CD147 antigen to selected antibodies was measured by Surface plasmon resonance analysis on a multi-SPR array system (ProteOn XPR36TM, Bio-Rad). Data analysis was performed using ProteOn Manager Software (Bio-Rad).
Briefly, the running buffer PBST (0.005% Tween-20 in PBS) was used continuously throughout the entire experiment at 25 C. A ProteOn GLC sensor chips (Bio-Rad) was activated for 6min using a mixture of 0.2M EDC and 5mM sulfo-NHS at a flow rate of 20 111 /min, followed by diluting 260 Ill of 10 vtg/mL each antibody in 10mM acetate buffer at pH 4.5.
The surface of the sensor chip was then deactivated with 150 jtl of 1M Ethanolamine Hydrochloride (pH 8.5). The sensor surface was regenerated through a wash (about 3 min) with HBS-EP buffer until the baseline was restored. A reference cell without antibodies was prepared by a similar procedure. 5 different concentrations of human CD147 antigen (0.375, 0.75, 1.5, 3, and 6 nM) were injected in channels 1 to 5, respectively, at flow rate of 50 1,11/min, 3min. A
reference channel was prepared in the same manner without injection of CD147 antigen. The SPR
binding responses were collected and analyzed using the ProteOn data manager program.
Briefly, the running buffer PBST (0.005% Tween-20 in PBS) was used continuously throughout the entire experiment at 25 C. A ProteOn GLC sensor chips (Bio-Rad) was activated for 6min using a mixture of 0.2M EDC and 5mM sulfo-NHS at a flow rate of 20 111 /min, followed by diluting 260 Ill of 10 vtg/mL each antibody in 10mM acetate buffer at pH 4.5.
The surface of the sensor chip was then deactivated with 150 jtl of 1M Ethanolamine Hydrochloride (pH 8.5). The sensor surface was regenerated through a wash (about 3 min) with HBS-EP buffer until the baseline was restored. A reference cell without antibodies was prepared by a similar procedure. 5 different concentrations of human CD147 antigen (0.375, 0.75, 1.5, 3, and 6 nM) were injected in channels 1 to 5, respectively, at flow rate of 50 1,11/min, 3min. A
reference channel was prepared in the same manner without injection of CD147 antigen. The SPR
binding responses were collected and analyzed using the ProteOn data manager program.
[00112] Two sets of experiments were performed, in the first set of experiments, the human CD147 binding affinity of HAbl8 and Metuzumab were compared (data shown in Figure 3A), in the second set of experiments the human CD147 binding affinity of cHAb18 and Metuzumab were compared (data shown in Figure 3B). The calculated affinities of antibodies by SPR
analysis for Metuzumab and HAb18 (I(1): 0.321 nmol/L and 0.362 nmol/L, respectively) can be considered equal. The calculated affinities of antibodies by SPR analysis for Metuzumab and cHAb 18 (KD: 0.429 nmol/L and 0.475 nmol/L, respectively) can be considered equal. In other words, HAb 18 and Metuzumab, Metuzumab and cHAb 18 all exhibited similar CD147 binding ability.
analysis for Metuzumab and HAb18 (I(1): 0.321 nmol/L and 0.362 nmol/L, respectively) can be considered equal. The calculated affinities of antibodies by SPR analysis for Metuzumab and cHAb 18 (KD: 0.429 nmol/L and 0.475 nmol/L, respectively) can be considered equal. In other words, HAb 18 and Metuzumab, Metuzumab and cHAb 18 all exhibited similar CD147 binding ability.
[00113] Measurement of Antigen-Binding Activity by flow cytometry
[00114] A549 cells (106 cells) were incubated with the Metuzumab (HcHAb18) and cHAb 18 antibody, respectively, in PBS with 0.1% BSA at 4 C for 1 h. After three washes in PBS with 0.1% BSA , cells were incubated with the FITC-conjugated Rabbit anti-Human IgG
Fe Secondary Antibody (Pierce) at 4 C in the dark for 45 min. Cells were then washed three times i and suspended in PBS for analysis using a FACS Calibur flow cytometer (BD, New Jersey, U.S.). Relative antigen expression is reported as median fluorescence intensity (MFI).
Fe Secondary Antibody (Pierce) at 4 C in the dark for 45 min. Cells were then washed three times i and suspended in PBS for analysis using a FACS Calibur flow cytometer (BD, New Jersey, U.S.). Relative antigen expression is reported as median fluorescence intensity (MFI).
[00115] As shown in Figure 4, both Metuzumab and cHAb 1 8 bound to CD147+ A549 cells, and both antibodies exhibited similar CD147-binding ability.
EXAMPLE 4: Measurement of Antigen-Binding specificity of Metuzumab
EXAMPLE 4: Measurement of Antigen-Binding specificity of Metuzumab
[00116] To test the specificity of Metuzumab (HcHAb18), tissue cross reactivity of Metuzumab was evaluated in FFPE tissue sections from a selected panel of human cancer tissue arrays (Chaoying Biotechnology Co., LTD, China.). Slides were microwaved for 10 min in 10mM sodium citrated (pH 6.0), cooled for 30 min at 25 C. After incubating for 10 min in 3%
H202 to block endogenous peroxidase, and blocking for lhr in 10% goat serum, slides were incubated overnight in a humidity chamber with biotinylated Metuzumab (30 g/ml). Binding was visualized with streptavidin-conjugated peroxidase (ZsBio, China) and DAB
detection system (ZsBio, China). Sections were analyzed using a bright-field microscope.
The staining results were evaluated by 2 experienced pathologist in a blinded manner and classified into four categories: of 0 (no visible staining), 1+ (light brown), 2+ (mid-brown), and 3+ (dark brown), respectively, with the same intensity covering more than 75% of the staining area.
H202 to block endogenous peroxidase, and blocking for lhr in 10% goat serum, slides were incubated overnight in a humidity chamber with biotinylated Metuzumab (30 g/ml). Binding was visualized with streptavidin-conjugated peroxidase (ZsBio, China) and DAB
detection system (ZsBio, China). Sections were analyzed using a bright-field microscope.
The staining results were evaluated by 2 experienced pathologist in a blinded manner and classified into four categories: of 0 (no visible staining), 1+ (light brown), 2+ (mid-brown), and 3+ (dark brown), respectively, with the same intensity covering more than 75% of the staining area.
[00117] As shown in Figure 5 and Table 1, Metuzumab specifically bound to tumor tissues, including lung cancer (60/68), ovarian cancer (9/12), stomach cancer (18/23), esophagus cancer (30/32), breast cancer (36/40), cervical cancer (17/20), liver cancer (17/20), and colon cancer (41/50). The staining pattern of the reference HAbl8 was similar to that of Metuzumab in cancer tissues, suggesting that the staining detected is specific for CD147.
Table 1. Immuno-histo-chemical Detection of CD147 by Metuzumab in Human Cancer Arrays Pathology number Staining level Positive rate Sensitivity Specificity 0 1+ 2+ 3+ response rate Lung cancer 68 8 5 23 32 88.24% 88.24%
91.67%
Normal lung 12 10 2 0 0 8.33%
Ovary cancer 12 3 2 3 4 75% 75.00%
100%
Normal ovary 12 12 0 0 0 0%
gastric cancer 23 5 1 7 10 78.26% 78.26%
80.95%
normal stomach 21 17 3 1 0 19.05%
Esophagus cancer 32 2 6 13 11 93.75% 93.75%
81.25%
Normal esophagus 16 13 3 0 0 18.75%
Mammary cancer 40 4 6 11 19 90.00% 90.00%
77.50%
Mammary tissue 40 31 5 2 2 22.50%
I
cervical cancer 20 3 2 5 10 85.00% 85.00%
100%
Normal cervix uteri 4 4 0 0 0 0%
Liver cancer 20 3 11 6 85.00% 85.00%
75.00%
Normal liver 4 3 0 1 0 25.00%
Colon cancer 50 9 8 26 7 82.00% 82.00%
84.00%
Normal colon 50 42 5 2 1 16.00%
EXAMPLE 5: Analysis of oligosaccharide and monosaccharide profiles of Antibodies
Table 1. Immuno-histo-chemical Detection of CD147 by Metuzumab in Human Cancer Arrays Pathology number Staining level Positive rate Sensitivity Specificity 0 1+ 2+ 3+ response rate Lung cancer 68 8 5 23 32 88.24% 88.24%
91.67%
Normal lung 12 10 2 0 0 8.33%
Ovary cancer 12 3 2 3 4 75% 75.00%
100%
Normal ovary 12 12 0 0 0 0%
gastric cancer 23 5 1 7 10 78.26% 78.26%
80.95%
normal stomach 21 17 3 1 0 19.05%
Esophagus cancer 32 2 6 13 11 93.75% 93.75%
81.25%
Normal esophagus 16 13 3 0 0 18.75%
Mammary cancer 40 4 6 11 19 90.00% 90.00%
77.50%
Mammary tissue 40 31 5 2 2 22.50%
I
cervical cancer 20 3 2 5 10 85.00% 85.00%
100%
Normal cervix uteri 4 4 0 0 0 0%
Liver cancer 20 3 11 6 85.00% 85.00%
75.00%
Normal liver 4 3 0 1 0 25.00%
Colon cancer 50 9 8 26 7 82.00% 82.00%
84.00%
Normal colon 50 42 5 2 1 16.00%
EXAMPLE 5: Analysis of oligosaccharide and monosaccharide profiles of Antibodies
[00118] Release of N-glycans were carried out following standard procedures (Ciucanu et al., 1984. A simple rapid method for the permethylation of carbohydrates.
Carbohydr.
Res. 131, 209 217.). Analysis of protein glycosylation was performed by using HPAEC-PAD.
60x150mm CLC-ODS column (Shimpack, Japan) was used for the HPAEC-PAD, the column was equilibrate with washing buffer A (10mM sodium phosphate solution (pH3.8)) at 1.0m1/min 55 C; then 25 1 of each testing sample were added to the column; after which washing buffer B
(10mM sodium phosphate solution (pH3.8) with 0.5%n-butyl alcohol) together with washing buffer A were used for gradient wash, within 80min the ratio between washing buffer B and washing buffer A was linearly increased from 1:1 to 60:40; the wash-outs were tested with florescence detector with exciting light wavelength at 320nm and emitting light wavelength at 400nm. The oligosaccharide peaks detected were then compared with standard peaks to determine the identity of each peak.
Carbohydr.
Res. 131, 209 217.). Analysis of protein glycosylation was performed by using HPAEC-PAD.
60x150mm CLC-ODS column (Shimpack, Japan) was used for the HPAEC-PAD, the column was equilibrate with washing buffer A (10mM sodium phosphate solution (pH3.8)) at 1.0m1/min 55 C; then 25 1 of each testing sample were added to the column; after which washing buffer B
(10mM sodium phosphate solution (pH3.8) with 0.5%n-butyl alcohol) together with washing buffer A were used for gradient wash, within 80min the ratio between washing buffer B and washing buffer A was linearly increased from 1:1 to 60:40; the wash-outs were tested with florescence detector with exciting light wavelength at 320nm and emitting light wavelength at 400nm. The oligosaccharide peaks detected were then compared with standard peaks to determine the identity of each peak.
[00119] As shown in Figure 6, the three major peaks in the cHAbl8 mAb sample correspond to masses of fucosylated oligosaccharides with GOF, G1F, G1F' glycoforms. By contrast, Metuzumab mAb exhibited homogeneous glycoform comprises solely Mannose-5 N-linked oligosaccharide, with no fucose residue or xylose residue was detected. Data demonstrated accumulation of non-fucosylated in the optimized antibody from Metuzumab instead of fucosylated structures in the native chimeric cHAb18 antibody.
EXAMPLE 6: Non-Glycosylated Heavy Chain in Metuzumab
EXAMPLE 6: Non-Glycosylated Heavy Chain in Metuzumab
[00120] The Non-glycosylation heavy chain (NGHC) analysis methods were based on that described in the Beckman Coulter IgG Purity/Heterogeneity kit. Unless noted otherwise, a bare fused silica capillary 50 mm id with an effective length of 20.2 cm and total length of 30.2 cm was used for reduced analysis. Prior to sample injection and separation, the capillary was rinsed with basic wash for 3 min at 70 psi, acidic wash for 1 min at 70 psi, water wash for 1 min at 70 !
psi, and gel buffer wash for 10 min at 70 psi. Following the rinses, the capillary and electrode ends were dipped twice in separate water vials prior to sample injection at 5 kV. Another water dip was performed followed by separation at 15 kV. During separation, pressure was applied to both ends of the capillary at 20 psi. Detection was performed at 220 nm using a Pulse Distribution Analysis (PDA) detector. The percentage of NGHC in Metuzumab was measured and calculated in several independent experiments, and the percentages obtained were all below 5%.
EXAMPLE 7: Antibody-Dependent Cellular Cytotoxicity (ADCC) of Metuzumab
psi, and gel buffer wash for 10 min at 70 psi. Following the rinses, the capillary and electrode ends were dipped twice in separate water vials prior to sample injection at 5 kV. Another water dip was performed followed by separation at 15 kV. During separation, pressure was applied to both ends of the capillary at 20 psi. Detection was performed at 220 nm using a Pulse Distribution Analysis (PDA) detector. The percentage of NGHC in Metuzumab was measured and calculated in several independent experiments, and the percentages obtained were all below 5%.
EXAMPLE 7: Antibody-Dependent Cellular Cytotoxicity (ADCC) of Metuzumab
[00121] The ADCC activity was determined by measuring lactate dehydrogenase (LDH) activity in the medium using a Cytotoxicity Detection Kit (LDH; Roche) according to the manufacturer's protocols.
[00122] In vitro ADCC assays were performed with human lung cancer cell lines (NCI-H520, A549 and NCI-H446) as target cells, and human spleen cells were used as effector cells. The target cells (1 x 104 per well) were washed with PBS and pre-incubated with antibodies solution for 30 min at 37 C in serum-free RPMI-1640 supplemented with 0.1% BSA. The antibody solution (Metuzumab or the chimeric antibody cHAb 18 mAb) that has been adjusted to a concentration of 0.001, 0.01, 0.1, 1 or 10 g/ml was dispensed at 100 1.11/we11 into each well that contained the target cells, Subsequently, 100u1 of the effector cells at an effector/target ratio of 50:1 was dispensed into each well, and incubated at in a carbon dioxide incubator. The cells were incubated for an additional 24 h prior to the detection of cell death by measuring lactate dehydrogenase (LDH) activity in the medium using a Cytotoxicity Detection Kit (LDH; Roche) according to the manufacturer's protocols. The ADCC-inducing activity was calculated to the following formula: Cytotoxicity (expressed as a percentage) = (experimental cell lysis -spontaneous effector lysis - spontaneous target lysis) / (maximum target lysis - spontaneous target lysis) x 100%. All assays were performed in triplicate.
[00123] As shown in Figure 7, the ADCC-inducing activity of the Metuzumab increased by about 10-folds more than that of cHAb 18 mAb in all three cell line, EC50% of Metuzumab was 0.04 g/m1, 0.0614m1 and 0.11m/m1 in NCI-H520, A549 and NCI-H446 cells, respectively. No ADCC activity was found with the control antibody IgGI.
EXAMPLE 8: Inhibition of cancer cell invasion and metastasis by Metuzumab !
EXAMPLE 8: Inhibition of cancer cell invasion and metastasis by Metuzumab !
[00124] Wound healing and transwell invasion assays were performed. For wound healing assay, 2 x104 cells were plated in 24-well plate to form cell monolayer and allowed to reach confluence for 2 days. A wound was made in the monolayer (at time 0) by a 200 ill tip. After wash of the monolayer, Metuzumab solution was dispensed at 0.1 ig/ml, 1p.g/m1 and 10 g/m', respectively, at 200 1/well into each well. Saline was used as control. The distance between two sides of an open wound was measured under a CX71 microscope (Olympus). The distance between the two sides of the wound after 20-72 h of migration was divided by the distance at time 0 and represented on a graph. The experiments were performed in triplicates, the results were shown as mean SD.
[00125] For invasion assay, NCI-H520 or A549 cells were serum-starved for 12 hours in DMEM containing 0.1% FBS. Serum-starved cells were trypsinized and re-suspended in DMEM
containing 0.1% FBS and Metuzumab solution at 0.111g/ml, 1[tg/m1 and 10 1.1g/ml, respectively.
5x104 cells were added to the upper chamber of each well (6.5 mm in diameter, 8-1.tm pore size;
Corning, NY) coated with 30 mg/cm2 Matrigel (BD Bioscience). Medium containing 10% FBS
was placed in the lower compartment of the chamber. After 24 hours at 37 C, cells on the upper membrane surface were removed by careful wiping with a cotton swab, and the filters were fixed by treatment with 95% ethanol for 30 minutes and stained with 0.2% crystal violet solution for 30 minutes. Invasive cells adhering to the undersurface of the filter were then counted (five high-power fields/chamber) using a CX71 microscope (Olympus).
containing 0.1% FBS and Metuzumab solution at 0.111g/ml, 1[tg/m1 and 10 1.1g/ml, respectively.
5x104 cells were added to the upper chamber of each well (6.5 mm in diameter, 8-1.tm pore size;
Corning, NY) coated with 30 mg/cm2 Matrigel (BD Bioscience). Medium containing 10% FBS
was placed in the lower compartment of the chamber. After 24 hours at 37 C, cells on the upper membrane surface were removed by careful wiping with a cotton swab, and the filters were fixed by treatment with 95% ethanol for 30 minutes and stained with 0.2% crystal violet solution for 30 minutes. Invasive cells adhering to the undersurface of the filter were then counted (five high-power fields/chamber) using a CX71 microscope (Olympus).
[00126] As shown in Figure 8A, in the experiment with NCI-H520 cells, the migration rate of saline group was 18.8pm/hr, however, the rate was 15.0 m/hr, 12.4[1m/hr and 10.5 ,m/hr in 0.1 g/ml, 1p.g/m1 and 10 jig/m1 Metuzumab group, respectively. The migration activity decreased by 20.2% (P<0.01), 34.0% (P<0.001) and 44.1% (P<0.001) compared to the control group, respectively. All these data indicated that Metuzumab inhibits cell migration of NCI-H520 cells. In the experiments with A549 cells, the migration rate of control group was 20.11,tm/hr, however, the rate was 15.2 pm/hr, 12.4 gm/hr and 10.1 lim/hr in 0.11.tg/ml, lidg/m1 and 10 jig/ml Metuzumab group, respectively. The migration activity repressed by 24.4%
(P<0.05), 38.3% (P<0.001) and 50% (P<0.001) compared to the control group, respectively. The data in Figure 8A indicated that Metuzumab inhibits cell migration of A549 cells.
(P<0.05), 38.3% (P<0.001) and 50% (P<0.001) compared to the control group, respectively. The data in Figure 8A indicated that Metuzumab inhibits cell migration of A549 cells.
[00127] As shown in Figure 8B, compared to control groups, the invasiveness of cells repressed by 30.0% (P<0.01), 48.0% (P<0.001) and 66.2% (P<0.001) after being treated by !
11.tg/m1 and 10 g/m1 Metuzumab, respectively. The invasiveness of A549 repressed by 53.5% (P<0.01), 61.5% (P<0.001) and 74.6% (P<0.001) after being treated by 0.11.tg/ml, 1 g/m1 and 10 pg/m1 Metuzumab compared to control groups, respectively. The data in Figure 8B indicated that Metuzumab inhibits cell invasion of NCI-H520 and A549 cells.
EXAMPLE 9: Antitumor Effects of Metuzumab in Xenograft Models
11.tg/m1 and 10 g/m1 Metuzumab, respectively. The invasiveness of A549 repressed by 53.5% (P<0.01), 61.5% (P<0.001) and 74.6% (P<0.001) after being treated by 0.11.tg/ml, 1 g/m1 and 10 pg/m1 Metuzumab compared to control groups, respectively. The data in Figure 8B indicated that Metuzumab inhibits cell invasion of NCI-H520 and A549 cells.
EXAMPLE 9: Antitumor Effects of Metuzumab in Xenograft Models
[00128] The antitumor effects of Metuzumab were confirmed using a human lung cancer-derived cell line A549 xenograft models.
[00129] Animals were housed in specific pathogen-free (SPF) condition. For the localized subcutaneous xenograft mouse model, 1 x 106 NCI-H520 or A549 cells, suspended in Hank's balanced salt solution were implanted in the right dorsal flank of male SCID
mice (6-8 week old). When tumors reached a mean volume of 100 mm3, mice were randomized into treatment cohorts (n = 10 mice per group). Mice were grouped for different treatments, including Metuzumab treatment groups treated with 2 mg/kg, 10 mg/kg and 30 mg/kg Metuzumab;
chemistry treatment group treated with 2mg/kg Cisplatin and 100mg/kg Gemcitabine;
combination treatment group treated with 10 mg/kg Metuzumab with 2mg/kg Cisplatin and 100mg/kg Gemcitabine, or saline (control group) via tail vein, respectively.
Antibody injections were administered twice weekly, for 3 weeks consecutively; chemotherapeutic agents were administered once a week, for 3 weeks consecutively. Tumor size was measured twice each week for the duration of the study using calipers. Tumor volumes were determined using the following formula: (length x width2)/2. Antitumor activity was assessed by calculating inhibition ratio of tumor volumes (IRTV) based on medians by using following formula: [1-average (Ttreatment (day x)-Ttreatment (day 0))/average (Tcontrol (day x)-Tcontrol (day 0))] x 100%.
mice (6-8 week old). When tumors reached a mean volume of 100 mm3, mice were randomized into treatment cohorts (n = 10 mice per group). Mice were grouped for different treatments, including Metuzumab treatment groups treated with 2 mg/kg, 10 mg/kg and 30 mg/kg Metuzumab;
chemistry treatment group treated with 2mg/kg Cisplatin and 100mg/kg Gemcitabine;
combination treatment group treated with 10 mg/kg Metuzumab with 2mg/kg Cisplatin and 100mg/kg Gemcitabine, or saline (control group) via tail vein, respectively.
Antibody injections were administered twice weekly, for 3 weeks consecutively; chemotherapeutic agents were administered once a week, for 3 weeks consecutively. Tumor size was measured twice each week for the duration of the study using calipers. Tumor volumes were determined using the following formula: (length x width2)/2. Antitumor activity was assessed by calculating inhibition ratio of tumor volumes (IRTV) based on medians by using following formula: [1-average (Ttreatment (day x)-Ttreatment (day 0))/average (Tcontrol (day x)-Tcontrol (day 0))] x 100%.
[00130] The results are shown in Figure 9A and 9B. All of treatment groups displayed significantly inhibition of tumor growth compared with the control treatment group (P <0.01). In the NCI-H520 models, the injection of 2-30 mg/kg Metuzumab resulted in a significant inhibition in the IRry (48.85%, 49.53%, 53.34%, 57.75% for chemotherapeutic agents group, the 2 mg/kg, 10 mg/kg, and 30 mg/kg Metuzumab groups, respectively; P <0.001). In A549 models, the tumor volume inhibition rates were 14.78%, 26.52%, and 50.64% in 2, 10, and 30 mg/kg Metuzumab, respectively, (P <0.01). As a result, the Metuzumab was found to exhibit antitumor effects in all of the administration groups. As shown in the result, the combination treatment group shows better tumor growth inhibition effect than any other groups. The results indicated I
that the combination of Metuzumab with chemical drugs enhanced the antitumor efficacy when compared with the mAb treatment groups or chemotherapeutic agents treatment group in preclinical models of human NSCLC. Therefore, in the combination therapy the dose of the chemotherapeutics could be decreased while decreasing the side effects of chemotherapeutics and increasing the therapeutic effect.
that the combination of Metuzumab with chemical drugs enhanced the antitumor efficacy when compared with the mAb treatment groups or chemotherapeutic agents treatment group in preclinical models of human NSCLC. Therefore, in the combination therapy the dose of the chemotherapeutics could be decreased while decreasing the side effects of chemotherapeutics and increasing the therapeutic effect.
[00131] While the invention has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.
REFERENCE
Baba, M., Inoue, M., Itoh, K., and Nishizawa, Y. (2008). Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism. Biochemical and biophysical research communications 374, 111-116.
Bian, H., Zheng, J.S., Nan, G., Li, R., Chen, C., Hu, C.X., Zhang, Y., Sun, B., Wang, X.L., Cui, S.C., et al. (2014).
Randomized trial of [131I]metuximab in treatment of hepatocellular carcinoma after percutaneous radiofrequency ablation. Journal of the National Cancer Institute 106.
Chen, H., Wang, L., Beretov, J., Hao, J., Xiao, W., and Li, Y. (2010). Co-expression of CD147/EMMPRIN with monocarboxylate transporters and multiple drug resistance proteins is associated with epithelial ovarian cancer progression. Clinical & experimental metastasis 27, 557-569.
Chen, Z.N., Mi, L., Xu, J., Song, F., Zhang, Q., Zhang, Z., Xing, J.L., Bian, H.J., Jiang, J.L., Wang, X.H., et al.
(2006). Targeting radio immunotherapy of hepatocellular carcinoma with iodine (131I) metuximab injection: clinical phase I/II trials. International journal of radiation oncology, biology, physics 65, 435-444.
Jefferis, R. (2009). Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action.
Trends in pharmacological sciences 30, 356-362.
Kataoka, H., DeCastro, R., Zucker, S., and Biswas, C. (1993). Tumor cell-derived collagenase-stimulatory factor increases expression of interstitial collagenase, stromelysin, and 72-kDa gelatinase. Cancer research 53, 3154-3158.
Kirk, P., Wilson, M.C., Heddle, C., Brown, M.H., Barclay, A.N., and Halestrap, A.P. (2000). CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression. The EMBO
journal 19, 3896-3904.
Li, Y., Xu, J., Chen, L., Zhong, W.D., Zhang, Z., Mi, L., Zhang, Y., Liao, C.G., Bian, H.J., Jiang, J.L., et al. (2009).
HAb18G (CD147), a cancer-associated biomarker and its role in cancer detection. Histopathology 54, 677-687.
Liang, Q., Han, Q., Huang, W., Nan, G., Xu, B.Q., Jiang, J.L., and Chen, Z.N.
(2014). 1-IAbl8G/CD147 regulates vinculin-mediated focal adhesion and cytoskeleton organization in cultured human hepatocellular carcinoma cells.
PloS one 9, e102496.
Orazizadeh, M., and Salter, D.M. (2008). CD147 (extracellular matrix metalloproteinase inducer-emmprin) expression by human articular chondrocytes. Iranian biomedical journal 12, 153-158.
Philp, N.J., Ochrietor, J.D., Rudoy, C., Muramatsu, T., and Linser, P.J.
(2003). Loss of MCT1, MCT3, and MCT4 expression in the retinal pigment epithelium and neural retina of the 5A11/basigin-null mouse. Investigative ophthalmology & visual science 44, 1305-1311.
Schneiderhan, W., Scheler, M., Holzmann, K.H., Marx, M., Gschwend, J.E., Bucholz, M., Gress, T.M., Seufferlein, T., Adler, G., and Oswald, F. (2009). CD147 silencing inhibits lactate transport and reduces malignant potential of pancreatic cancer cells in in vivo and in vitro models. Gut 58, 1391-1398.
Xu, J., Shen, Z.Y., Chen, X.G., Zhang, Q., Bian, H.J., Zhu, P., Xu, H.Y., Song, F., Yang, X.M., Mi, L., et al.
(2007a). A randomized controlled trial of Licartin for preventing hepatoma recurrence after liver transplantation.
Hepatology 45, 269-276.
Xu, J., Xu, H.Y., Zhang, Q., Song, F., Jiang, J.L., Yang, X.M., Mi, L., Wen, N., Tian, R., Wang, L., et al. (2007b).
HAb18G/CD147 functions in invasion and metastasis of hepatocellular carcinoma.
Molecular cancer research :
MCR 5, 605-614.
i Zeng, H.Z., Qu, Y.Q., Liang, A.B., Deng, A.M., Zhang, W.J., Xiu, B., Wang, H., and Wang, H. (2011). Expression of CD147 in advanced non-small cell lung cancer correlated with cisplatin-based chemotherapy resistance.
Neoplasma 58, 449-454.
Zhao, S., Chen, C., Liu, S., Zeng, W., Su, J., Wu, L., Luo, Z., Zhou, S., Li, Q., Zhang, J., et al. (2013). CD147 promotes MIX resistance by immune cells through up-regulating ABCG2 expression and function. Journal of dermatological science 70, 182-189.
Zou, W., Yang, H., Hou, X., Zhang, W., Chen, B., and Xin, X. (2007).
Inhibition of CD147 gene expression via RNA interference reduces tumor cell invasion, tumorigenicity and increases chemosensitivity to paclitaxel in HO-8910pm cells. Cancer letters 248, 211-218.
I
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with section 111(1) of the Patent Rules, this description contains a sequence listing in electronic form in ASCII text format (file: 95652-8seq2015-10-07v1.txt).
A copy of the sequence listing in electronic form is available from the Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are reproduced in the following table.
SEQUENCE TABLE
<110> Fourth Military Medical University <120> HUMAN-MOUSE CHIMERIC ANTI-CD147 ANTIBODY WITH NON-FUCOSYLATED
GLYCOSYLATION
<130> 95652-8 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 108 <212> PRT
<213> Mouse <400> 1 Ser Ile Val Met Thr Gin Thr Pro Thr Phe Leu Val Val Ser Ala Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gin Ser Val Ile Asn Asp Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Phe Tyr Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly 50 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gin Ala i Glu Asp Leu Ala Val Tyr Phe Cys Gin Gin Asp Tyr Ser Pro Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg <210> 2 <211> 111 <212> PRT
<213> Mouse <400> 2 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Ala Trp Met Asp Trp Val Arg Gin Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Pro Tyr Tyr Thr Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile Ile Tyr Leu Gin Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Arg Asp Ser Thr Ala Thr His Trp Gly Gin Gly Thr <210> 3 <211> 230 <212> PRT
<213> Chimeric <400> 3 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly I
Ser Ile Val Met Thr Gin Thr Pro Thr Phe Leu Val Val Ser Ala Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gin Ser Val Ile Asn Asp Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Phe Tyr Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gin Ala Glu Asp Leu Ala Val Tyr Phe Cys Gin Gin Asp Tyr Ser Pro Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys <210> 4 <211> 463 <212> PRT
<213> Chimeric <400> 4 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Ala Trp Met Asp Trp Val Arg Gin Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Pro Tyr Tyr Thr Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile Ile Tyr Leu Gin Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Arg Asp Ser Thr Ala Thr His Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser !
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 5 <211> 324 <212> DNA
<213> Mouse <400> 5 agcattgtga tgacccagac tcccacattc ctggttgtat cagcaggaga cagggttacc 60 ataacctgca aggccagtca gagtgtgatt aatgatgtag cttggtacca acagaagcca gggcagtctc ctaaactgct gatattctat gcatccaatc gcaacactgg agttcctgat cgcttcactg gcagtggata tgggacggat ttcactttca ccatcagcac tgtgcaggct gaagacctgg cagtttattt ctgtcagcag gattatagtc ctccattcac gttcggctcg gggacaaagt tggaaatcaa gcgg <210> 6 <211> 351 <212> DNA
<213> Mouse <400> 6 gaagtgaagc tggaggagtc tggaggaggc ttggtgcaac ctggaggatc catgaaactg tcttgtgttg cctctggatt cacttttagt gacgcctgga tggactgggt ccgccagtct ccagagaagg gacttgagtg ggttgctgaa attagaagca aagctaataa tcatgcacca tactatactg agtctgtgaa agggaggttc accatctcac gagatgattc caagagtatt atctacctgc aaatgaacaa cttaagagct gaagacactg gcatttatta ctgtaccagg gatagcacgg ctacccactg gggccaaggg actctggtca ctgtctctgc a i <210> 7 <211> 693 <212> DNA
<213> Chimeric <400> 7 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcag cattgtgatg acccagactc ccacattcct ggttgtatca gcaggagaca gggttaccat aacctgcaag gccagtcaga gtgtgattaa tgatgtagct tggtaccaac agaagccagg gcagtctcct aaactgctga tattctatgc atccaatcgc aacactggag ttcctgatcg cttcactggc agtggatatg ggacggattt cactttcacc atcagcactg tgcaggctga agacctggca gtttatttct gtcagcagga ttatagtcct ccattcacgt tcggctcggg gacaaagttg gaaatcaagc ggaccgtggc cgccccctcc gtgttcatct tccccccctc cgacgagcag ctgaagtccg gcaccgcctc cgtggtgtgc ctgctgaaca acttctaccc ccgggaggcc aaggtgcagt ggaaggtgga caacgccctg cagtccggca actcccagga gtccgtgacc gagcaggact ccaaggactc cacctactcc ctgtcctcca ccctgaccct gtccaaggcc gactacgaga agcacaaggt gtacgcctgc gaggtgaccc accagggcct gtcctccccc gtgaccaagt ccttcaaccg gggcgagtgc tag <210> 8 <211> 1392 <212> DNA
<213> Chimeric <400> 8 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcga agtgaagctg gaggagtctg gaggaggctt ggtgcaacct ggaggatcca tgaaactgtc ttgtgttgcc tctggattca cttttagtga cgcctggatg gactgggtcc gccagtctcc agagaaggga cttgagtggg ttgctgaaat tagaagcaaa gctaataatc atgcaccata ctatactgag tctgtgaaag ggaggttcac catctcacga gatgattcca agagtattat ctacctgcaa atgaacaact taagagctga agacactggc atttattact gtaccaggga tagcacggct acccactggg gccaagggac tctggtcact gtctctgcag cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc I
ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggccgtccta cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct ccgggtaaat ga <210> 9 <211> 29 <212> DNA
<213> SYTHESIS
<400> 9 agcattgtga tgacccagac tcccacatt <210> 10 <211> 30 <212> DNA
<213> SYTHESIS
<400> 10 ccgcttgatt tccaactttg tccccgagcc <210> 11 <211> 31 I
<212> DNA
<213> SYTHESIS
<400> 11 gaagtgaagc tggaggagtc tggaggaggc t <210> 12 <211> 31 <212> DNA
<213> SYTHESIS
<400> 12 tgcagagaca gtgaccagag tcccttggcc c <210> 13 <211> 48 <212> DNA
<213> SYTHESIS
<400> 13 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggc I
REFERENCE
Baba, M., Inoue, M., Itoh, K., and Nishizawa, Y. (2008). Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism. Biochemical and biophysical research communications 374, 111-116.
Bian, H., Zheng, J.S., Nan, G., Li, R., Chen, C., Hu, C.X., Zhang, Y., Sun, B., Wang, X.L., Cui, S.C., et al. (2014).
Randomized trial of [131I]metuximab in treatment of hepatocellular carcinoma after percutaneous radiofrequency ablation. Journal of the National Cancer Institute 106.
Chen, H., Wang, L., Beretov, J., Hao, J., Xiao, W., and Li, Y. (2010). Co-expression of CD147/EMMPRIN with monocarboxylate transporters and multiple drug resistance proteins is associated with epithelial ovarian cancer progression. Clinical & experimental metastasis 27, 557-569.
Chen, Z.N., Mi, L., Xu, J., Song, F., Zhang, Q., Zhang, Z., Xing, J.L., Bian, H.J., Jiang, J.L., Wang, X.H., et al.
(2006). Targeting radio immunotherapy of hepatocellular carcinoma with iodine (131I) metuximab injection: clinical phase I/II trials. International journal of radiation oncology, biology, physics 65, 435-444.
Jefferis, R. (2009). Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action.
Trends in pharmacological sciences 30, 356-362.
Kataoka, H., DeCastro, R., Zucker, S., and Biswas, C. (1993). Tumor cell-derived collagenase-stimulatory factor increases expression of interstitial collagenase, stromelysin, and 72-kDa gelatinase. Cancer research 53, 3154-3158.
Kirk, P., Wilson, M.C., Heddle, C., Brown, M.H., Barclay, A.N., and Halestrap, A.P. (2000). CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression. The EMBO
journal 19, 3896-3904.
Li, Y., Xu, J., Chen, L., Zhong, W.D., Zhang, Z., Mi, L., Zhang, Y., Liao, C.G., Bian, H.J., Jiang, J.L., et al. (2009).
HAb18G (CD147), a cancer-associated biomarker and its role in cancer detection. Histopathology 54, 677-687.
Liang, Q., Han, Q., Huang, W., Nan, G., Xu, B.Q., Jiang, J.L., and Chen, Z.N.
(2014). 1-IAbl8G/CD147 regulates vinculin-mediated focal adhesion and cytoskeleton organization in cultured human hepatocellular carcinoma cells.
PloS one 9, e102496.
Orazizadeh, M., and Salter, D.M. (2008). CD147 (extracellular matrix metalloproteinase inducer-emmprin) expression by human articular chondrocytes. Iranian biomedical journal 12, 153-158.
Philp, N.J., Ochrietor, J.D., Rudoy, C., Muramatsu, T., and Linser, P.J.
(2003). Loss of MCT1, MCT3, and MCT4 expression in the retinal pigment epithelium and neural retina of the 5A11/basigin-null mouse. Investigative ophthalmology & visual science 44, 1305-1311.
Schneiderhan, W., Scheler, M., Holzmann, K.H., Marx, M., Gschwend, J.E., Bucholz, M., Gress, T.M., Seufferlein, T., Adler, G., and Oswald, F. (2009). CD147 silencing inhibits lactate transport and reduces malignant potential of pancreatic cancer cells in in vivo and in vitro models. Gut 58, 1391-1398.
Xu, J., Shen, Z.Y., Chen, X.G., Zhang, Q., Bian, H.J., Zhu, P., Xu, H.Y., Song, F., Yang, X.M., Mi, L., et al.
(2007a). A randomized controlled trial of Licartin for preventing hepatoma recurrence after liver transplantation.
Hepatology 45, 269-276.
Xu, J., Xu, H.Y., Zhang, Q., Song, F., Jiang, J.L., Yang, X.M., Mi, L., Wen, N., Tian, R., Wang, L., et al. (2007b).
HAb18G/CD147 functions in invasion and metastasis of hepatocellular carcinoma.
Molecular cancer research :
MCR 5, 605-614.
i Zeng, H.Z., Qu, Y.Q., Liang, A.B., Deng, A.M., Zhang, W.J., Xiu, B., Wang, H., and Wang, H. (2011). Expression of CD147 in advanced non-small cell lung cancer correlated with cisplatin-based chemotherapy resistance.
Neoplasma 58, 449-454.
Zhao, S., Chen, C., Liu, S., Zeng, W., Su, J., Wu, L., Luo, Z., Zhou, S., Li, Q., Zhang, J., et al. (2013). CD147 promotes MIX resistance by immune cells through up-regulating ABCG2 expression and function. Journal of dermatological science 70, 182-189.
Zou, W., Yang, H., Hou, X., Zhang, W., Chen, B., and Xin, X. (2007).
Inhibition of CD147 gene expression via RNA interference reduces tumor cell invasion, tumorigenicity and increases chemosensitivity to paclitaxel in HO-8910pm cells. Cancer letters 248, 211-218.
I
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with section 111(1) of the Patent Rules, this description contains a sequence listing in electronic form in ASCII text format (file: 95652-8seq2015-10-07v1.txt).
A copy of the sequence listing in electronic form is available from the Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are reproduced in the following table.
SEQUENCE TABLE
<110> Fourth Military Medical University <120> HUMAN-MOUSE CHIMERIC ANTI-CD147 ANTIBODY WITH NON-FUCOSYLATED
GLYCOSYLATION
<130> 95652-8 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 108 <212> PRT
<213> Mouse <400> 1 Ser Ile Val Met Thr Gin Thr Pro Thr Phe Leu Val Val Ser Ala Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gin Ser Val Ile Asn Asp Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Phe Tyr Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly 50 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gin Ala i Glu Asp Leu Ala Val Tyr Phe Cys Gin Gin Asp Tyr Ser Pro Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg <210> 2 <211> 111 <212> PRT
<213> Mouse <400> 2 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Ala Trp Met Asp Trp Val Arg Gin Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Pro Tyr Tyr Thr Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile Ile Tyr Leu Gin Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Arg Asp Ser Thr Ala Thr His Trp Gly Gin Gly Thr <210> 3 <211> 230 <212> PRT
<213> Chimeric <400> 3 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly I
Ser Ile Val Met Thr Gin Thr Pro Thr Phe Leu Val Val Ser Ala Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gin Ser Val Ile Asn Asp Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Phe Tyr Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gin Ala Glu Asp Leu Ala Val Tyr Phe Cys Gin Gin Asp Tyr Ser Pro Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys <210> 4 <211> 463 <212> PRT
<213> Chimeric <400> 4 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Ala Trp Met Asp Trp Val Arg Gin Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Pro Tyr Tyr Thr Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile Ile Tyr Leu Gin Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Arg Asp Ser Thr Ala Thr His Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser !
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 5 <211> 324 <212> DNA
<213> Mouse <400> 5 agcattgtga tgacccagac tcccacattc ctggttgtat cagcaggaga cagggttacc 60 ataacctgca aggccagtca gagtgtgatt aatgatgtag cttggtacca acagaagcca gggcagtctc ctaaactgct gatattctat gcatccaatc gcaacactgg agttcctgat cgcttcactg gcagtggata tgggacggat ttcactttca ccatcagcac tgtgcaggct gaagacctgg cagtttattt ctgtcagcag gattatagtc ctccattcac gttcggctcg gggacaaagt tggaaatcaa gcgg <210> 6 <211> 351 <212> DNA
<213> Mouse <400> 6 gaagtgaagc tggaggagtc tggaggaggc ttggtgcaac ctggaggatc catgaaactg tcttgtgttg cctctggatt cacttttagt gacgcctgga tggactgggt ccgccagtct ccagagaagg gacttgagtg ggttgctgaa attagaagca aagctaataa tcatgcacca tactatactg agtctgtgaa agggaggttc accatctcac gagatgattc caagagtatt atctacctgc aaatgaacaa cttaagagct gaagacactg gcatttatta ctgtaccagg gatagcacgg ctacccactg gggccaaggg actctggtca ctgtctctgc a i <210> 7 <211> 693 <212> DNA
<213> Chimeric <400> 7 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcag cattgtgatg acccagactc ccacattcct ggttgtatca gcaggagaca gggttaccat aacctgcaag gccagtcaga gtgtgattaa tgatgtagct tggtaccaac agaagccagg gcagtctcct aaactgctga tattctatgc atccaatcgc aacactggag ttcctgatcg cttcactggc agtggatatg ggacggattt cactttcacc atcagcactg tgcaggctga agacctggca gtttatttct gtcagcagga ttatagtcct ccattcacgt tcggctcggg gacaaagttg gaaatcaagc ggaccgtggc cgccccctcc gtgttcatct tccccccctc cgacgagcag ctgaagtccg gcaccgcctc cgtggtgtgc ctgctgaaca acttctaccc ccgggaggcc aaggtgcagt ggaaggtgga caacgccctg cagtccggca actcccagga gtccgtgacc gagcaggact ccaaggactc cacctactcc ctgtcctcca ccctgaccct gtccaaggcc gactacgaga agcacaaggt gtacgcctgc gaggtgaccc accagggcct gtcctccccc gtgaccaagt ccttcaaccg gggcgagtgc tag <210> 8 <211> 1392 <212> DNA
<213> Chimeric <400> 8 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcga agtgaagctg gaggagtctg gaggaggctt ggtgcaacct ggaggatcca tgaaactgtc ttgtgttgcc tctggattca cttttagtga cgcctggatg gactgggtcc gccagtctcc agagaaggga cttgagtggg ttgctgaaat tagaagcaaa gctaataatc atgcaccata ctatactgag tctgtgaaag ggaggttcac catctcacga gatgattcca agagtattat ctacctgcaa atgaacaact taagagctga agacactggc atttattact gtaccaggga tagcacggct acccactggg gccaagggac tctggtcact gtctctgcag cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc I
ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggccgtccta cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct ccgggtaaat ga <210> 9 <211> 29 <212> DNA
<213> SYTHESIS
<400> 9 agcattgtga tgacccagac tcccacatt <210> 10 <211> 30 <212> DNA
<213> SYTHESIS
<400> 10 ccgcttgatt tccaactttg tccccgagcc <210> 11 <211> 31 I
<212> DNA
<213> SYTHESIS
<400> 11 gaagtgaagc tggaggagtc tggaggaggc t <210> 12 <211> 31 <212> DNA
<213> SYTHESIS
<400> 12 tgcagagaca gtgaccagag tcccttggcc c <210> 13 <211> 48 <212> DNA
<213> SYTHESIS
<400> 13 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggc I
Claims (24)
1. A nucleotide sequence comprising the sequence of SEQ ID NO: 5, and/or sequence of SEQ
ID NO: 6.
ID NO: 6.
2. A vector comprising the nucleotide sequence of claim 1.
3. A host cell comprising the vector of claim 2.
4. An antibody that binds to extracellular region of human CD147, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ
ID NO: 2, and the antibody contains a glycoform lacking both fucose residues and xylose residues.
ID NO: 2, and the antibody contains a glycoform lacking both fucose residues and xylose residues.
5. The antibody of claim 4, wherein the antibody further comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 1.
6. The antibody of claim 4, wherein the antibodies or fragments thereof comprise a predominant portion of glycoforms comprising N-linked oligosaccharides of Mannose-5.
7. The antibody of claim 4, wherein the antibodies or fragments thereof comprises solely a glycoform comprising N-linked Marmose-5.
8. The antibody of claim 5, wherein the antibody-has a glycosylation profile as analyzed by High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) that is substantially equivalent to that of Metuzumab as shown in Figure 6.
9. The antibody of claim 4, wherein the antibody is obtained from acetyl-glucosamine transferase deficient cell line.
10. The antibody of claim 9, wherein the cell line is CHO cell line.
11. The antibody of claim 4, wherein the ADCC activity of the antibody is at least 2 times, 5 times, 10 times or 20 times higher than the ADCC activity of an antibody that contains a glycoform with fucose residues, xylose residues, or both.
12. A pharmaceutical composition comprising the antibody of claim 5, or fragment thereof, and a pharmaceutically acceptable vehicle or excipient.
13. The pharmaceutical composition of claim 12, further comprising chemotherapeutic agent.
14. The pharmaceutical composition of claim 13, wherein the chemotherapeutic agent is selected from gemcitabine, cisplatin, paclitaxel, navelbine.
15. A method for producing the antibody of claim 5, or fragment thereof, comprising:
obtaining nucleotide sequence encoding for a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and/or nucleotide sequence encoding for a light chain variable region having the amino acid sequence of SEQ ID NO: 1;
constructing an vector comprising the nucleotide sequence encoding for the antibody;
transfecting the vector into an acetyl-glucosamine transferase deficient cell line;
culturing the transfected cell line in media;
obtaining the antibody from the culture.
obtaining nucleotide sequence encoding for a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and/or nucleotide sequence encoding for a light chain variable region having the amino acid sequence of SEQ ID NO: 1;
constructing an vector comprising the nucleotide sequence encoding for the antibody;
transfecting the vector into an acetyl-glucosamine transferase deficient cell line;
culturing the transfected cell line in media;
obtaining the antibody from the culture.
16. The method of claim 15, wherein culturing the transfected cell line in media comprises culturing the transfected cell line at 36-38°C for a first period and then at 30-32°C for a second period.
17. The method of claim 16, wherein the first period is 4-10 days .
18. The method of claim 16, wherein the second period is 11-21 days.
19. The method of claim 15, wherein the expression level of the antibody by the cell line is at least 100 mg/L, at least 200 mg/L, or at least 300 mg/L.
20. The method of claim 15, wherein the percentage of non-glycosylated heavy chain calculated based on the total amount of the antibody obtained is less than 10%, 7%, 5% or 3%.
21. The method of claim 15, wherein the cell line is acetyl-glucosamine -transferase-deficient CHO cell line.
22. A method for treating human CD147 expression-related disease in a subject in need thereof, comprising administering of an effective amount of the antibody of claim 5 to the subject and thereby treating the disease.
23. The method of claim 22, further comprising administering a chemotherapeutic agent to the subject.
24. The method of claim 22, wherein the disease is lung cancer.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2908331A CA2908331A1 (en) | 2015-10-07 | 2015-10-07 | Human-mouse chimeric anti-cd147 antibody with non-fucosylated glycosylation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2908331A CA2908331A1 (en) | 2015-10-07 | 2015-10-07 | Human-mouse chimeric anti-cd147 antibody with non-fucosylated glycosylation |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2908331A1 true CA2908331A1 (en) | 2017-04-07 |
Family
ID=58468368
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2908331A Pending CA2908331A1 (en) | 2015-10-07 | 2015-10-07 | Human-mouse chimeric anti-cd147 antibody with non-fucosylated glycosylation |
Country Status (1)
Country | Link |
---|---|
CA (1) | CA2908331A1 (en) |
-
2015
- 2015-10-07 CA CA2908331A patent/CA2908331A1/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11723971B2 (en) | Antibodies to TIGIT | |
KR102658660B1 (en) | BCMA targeting antibodies and uses thereof | |
CN111434692B (en) | anti-CLD18A2 nano antibody and application thereof | |
TWI679021B (en) | Afucosylated anti-fgfr2iiib antibodies | |
EP2760891B1 (en) | Hybrid constant regions | |
ES2923397T3 (en) | Cancer Treatment Using a Humanized Anti-CD19 Chimeric Antigen Receptor | |
EP3431102A1 (en) | Cell injury inducing therapeutic drug for use in cancer therapy | |
DK2199390T3 (en) | ANTI-EphA2 ANTIBODY | |
KR20180030899A (en) | PD-L1 (&quot; Programmed Killing-Ligand 1 &quot;) antibody | |
TW202003562A (en) | Anti-claudin 18.2 (CLDN18.2) antibodies | |
JP2022541349A (en) | Antigen-binding protein that specifically binds to MAGE-A | |
TW201446794A (en) | Effective targeting of primary human leukemia using anti-CD123 chimeric antigen receptor engineered T cells | |
CN110035773A (en) | Novel anti-CTLA 4 antibody | |
CA3176246A1 (en) | Antibodies to tigit | |
JP2022543387A (en) | Modified bispecific anti-CD3 antibodies | |
US20240059774A1 (en) | Anti-ctla-4 antibody and use thereof | |
CN112823166A (en) | Conditionally active chimeric antigen receptors for modified T cells | |
US9963506B2 (en) | Human-mouse chimeric anti-CD147 antibody with non-fucosylated glycosylation | |
JP7477127B2 (en) | Uses of Anti-CTLA-4 Antibodies | |
JP7472405B2 (en) | Anti-CTLA-4 antibody | |
CA2908331A1 (en) | Human-mouse chimeric anti-cd147 antibody with non-fucosylated glycosylation | |
EP4082570A1 (en) | Anti-ctla-4 antibody and use thereof | |
JP2022529502A (en) | Anti-CD38 antibody and formulation | |
RU2815823C2 (en) | Isolated bispecific antibody that specifically binds to cd47 and pd-l1 | |
TW202216764A (en) | Anti-CTLA-4 antibody and use thereof having CTLA-4 binding activity depending on the concentration of an adenosine-containing compound |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20200928 |
|
EEER | Examination request |
Effective date: 20200928 |
|
EEER | Examination request |
Effective date: 20200928 |
|
EEER | Examination request |
Effective date: 20200928 |
|
EEER | Examination request |
Effective date: 20200928 |
|
EEER | Examination request |
Effective date: 20200928 |
|
EEER | Examination request |
Effective date: 20200928 |