WO2020063562A1 - 突变型单链人凝血因子viii融合蛋白及其制备方法与用途 - Google Patents
突变型单链人凝血因子viii融合蛋白及其制备方法与用途 Download PDFInfo
- Publication number
- WO2020063562A1 WO2020063562A1 PCT/CN2019/107432 CN2019107432W WO2020063562A1 WO 2020063562 A1 WO2020063562 A1 WO 2020063562A1 CN 2019107432 W CN2019107432 W CN 2019107432W WO 2020063562 A1 WO2020063562 A1 WO 2020063562A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fusion protein
- human
- fviii
- amino acid
- acid sequence
- Prior art date
Links
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 130
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 127
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 title claims abstract description 29
- 102000057593 human F8 Human genes 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title abstract description 11
- 108010054218 Factor VIII Proteins 0.000 claims abstract description 81
- 102000001690 Factor VIII Human genes 0.000 claims abstract description 81
- 229960000301 factor viii Drugs 0.000 claims abstract description 75
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 35
- 102000011022 Chorionic Gonadotropin Human genes 0.000 claims abstract description 16
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims abstract description 16
- 229940084986 human chorionic gonadotropin Drugs 0.000 claims abstract description 15
- 101800005309 Carboxy-terminal peptide Proteins 0.000 claims description 62
- 210000004027 cell Anatomy 0.000 claims description 53
- 230000000694 effects Effects 0.000 claims description 44
- 208000032843 Hemorrhage Diseases 0.000 claims description 32
- 208000034158 bleeding Diseases 0.000 claims description 30
- 230000000740 bleeding effect Effects 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 30
- 230000035772 mutation Effects 0.000 claims description 28
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 25
- 208000009292 Hemophilia A Diseases 0.000 claims description 23
- 238000004587 chromatography analysis Methods 0.000 claims description 20
- 102100026735 Coagulation factor VIII Human genes 0.000 claims description 18
- 201000003542 Factor VIII deficiency Diseases 0.000 claims description 18
- 230000014509 gene expression Effects 0.000 claims description 18
- 150000001413 amino acids Chemical class 0.000 claims description 16
- 238000000746 purification Methods 0.000 claims description 12
- 108020003175 receptors Proteins 0.000 claims description 11
- 102000005962 receptors Human genes 0.000 claims description 11
- 239000013598 vector Substances 0.000 claims description 10
- 108020004414 DNA Proteins 0.000 claims description 9
- 230000013595 glycosylation Effects 0.000 claims description 9
- 238000006206 glycosylation reaction Methods 0.000 claims description 9
- 238000011282 treatment Methods 0.000 claims description 9
- 238000005571 anion exchange chromatography Methods 0.000 claims description 8
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 8
- 102220315697 rs1553622313 Human genes 0.000 claims description 8
- 238000001042 affinity chromatography Methods 0.000 claims description 7
- 210000004962 mammalian cell Anatomy 0.000 claims description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 239000002808 molecular sieve Substances 0.000 claims description 6
- 230000002265 prevention Effects 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 230000002209 hydrophobic effect Effects 0.000 claims description 5
- 230000002829 reductive effect Effects 0.000 claims description 5
- 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 description 4
- 108060003951 Immunoglobulin Proteins 0.000 claims description 4
- 238000000855 fermentation Methods 0.000 claims description 4
- 230000004151 fermentation Effects 0.000 claims description 4
- 102000018358 immunoglobulin Human genes 0.000 claims description 4
- 108010088751 Albumins Proteins 0.000 claims description 3
- 102000009027 Albumins Human genes 0.000 claims description 3
- 102000004338 Transferrin Human genes 0.000 claims description 3
- 108090000901 Transferrin Proteins 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 230000007812 deficiency Effects 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 102200124454 rs80356507 Human genes 0.000 claims description 3
- 239000012581 transferrin Substances 0.000 claims description 3
- 102100029880 Glycodelin Human genes 0.000 claims description 2
- 101000585553 Homo sapiens Glycodelin Proteins 0.000 claims description 2
- 238000003306 harvesting Methods 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 102200072304 rs1057519530 Human genes 0.000 claims description 2
- 230000002269 spontaneous effect Effects 0.000 claims description 2
- 208000031169 hemorrhagic disease Diseases 0.000 claims 2
- 206010010356 Congenital anomaly Diseases 0.000 claims 1
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 claims 1
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 claims 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 claims 1
- 230000004071 biological effect Effects 0.000 abstract description 13
- 238000001727 in vivo Methods 0.000 abstract description 10
- 238000000338 in vitro Methods 0.000 abstract description 5
- 230000002035 prolonged effect Effects 0.000 abstract description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 27
- 108090000623 proteins and genes Proteins 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 20
- 238000012360 testing method Methods 0.000 description 19
- 239000003814 drug Substances 0.000 description 16
- 241000699670 Mus sp. Species 0.000 description 14
- 229940079593 drug Drugs 0.000 description 14
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 14
- 229920000053 polysorbate 80 Polymers 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 14
- 239000011780 sodium chloride Substances 0.000 description 14
- 230000006870 function Effects 0.000 description 11
- 241000700159 Rattus Species 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 8
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 8
- 239000012636 effector Substances 0.000 description 8
- 239000013604 expression vector Substances 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 230000001965 increasing effect Effects 0.000 description 8
- 108091005804 Peptidases Proteins 0.000 description 7
- 239000004365 Protease Substances 0.000 description 7
- 108090000190 Thrombin Proteins 0.000 description 7
- 230000015271 coagulation Effects 0.000 description 7
- 238000005345 coagulation Methods 0.000 description 7
- 229920000642 polymer Polymers 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 229960004072 thrombin Drugs 0.000 description 7
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 6
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 6
- 210000001367 artery Anatomy 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 239000003114 blood coagulation factor Substances 0.000 description 6
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 6
- 239000013613 expression plasmid Substances 0.000 description 6
- 125000003147 glycosyl group Chemical group 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000003981 vehicle Substances 0.000 description 6
- 108091026890 Coding region Proteins 0.000 description 5
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 5
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 229960000485 methotrexate Drugs 0.000 description 5
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 5
- 108010047303 von Willebrand Factor Proteins 0.000 description 5
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 4
- 101150074155 DHFR gene Proteins 0.000 description 4
- 239000007995 HEPES buffer Substances 0.000 description 4
- 208000031220 Hemophilia Diseases 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 102000035195 Peptidases Human genes 0.000 description 4
- 108010022394 Threonine synthase Proteins 0.000 description 4
- 229940028633 afstyla Drugs 0.000 description 4
- 210000004204 blood vessel Anatomy 0.000 description 4
- 230000024203 complement activation Effects 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 230000002950 deficient Effects 0.000 description 4
- 102000004419 dihydrofolate reductase Human genes 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 239000012149 elution buffer Substances 0.000 description 4
- 230000002439 hemostatic effect Effects 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 230000004952 protein activity Effects 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 206010002091 Anaesthesia Diseases 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000037005 anaesthesia Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 239000012501 chromatography medium Substances 0.000 description 3
- 239000003593 chromogenic compound Substances 0.000 description 3
- 229940105778 coagulation factor viii Drugs 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 239000000356 contaminant Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 210000005036 nerve Anatomy 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 230000002947 procoagulating effect Effects 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108010079356 FIIa Proteins 0.000 description 2
- 108010014173 Factor X Proteins 0.000 description 2
- 108091006020 Fc-tagged proteins Proteins 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 2
- 108010079345 Follicle Stimulating Hormone Proteins 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
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 238000012313 Kruskal-Wallis test Methods 0.000 description 2
- 102000009151 Luteinizing Hormone Human genes 0.000 description 2
- 108010073521 Luteinizing Hormone Proteins 0.000 description 2
- 230000004989 O-glycosylation Effects 0.000 description 2
- 102000011923 Thyrotropin Human genes 0.000 description 2
- 108010061174 Thyrotropin Proteins 0.000 description 2
- 230000003044 adaptive effect Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000002429 anti-coagulating effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 229940028334 follicle stimulating hormone Drugs 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000001638 lipofection Methods 0.000 description 2
- 229940040129 luteinizing hormone Drugs 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000001543 one-way ANOVA Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical compound CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 206010060964 Arterial haemorrhage Diseases 0.000 description 1
- 108010019949 BAX 855 Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- VMQMZMRVKUZKQL-UHFFFAOYSA-N Cu+ Chemical compound [Cu+] VMQMZMRVKUZKQL-UHFFFAOYSA-N 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 229920002307 Dextran Polymers 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
- 108010048049 Factor IXa Proteins 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101000966481 Mus musculus Dihydrofolate reductase Proteins 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 108010000499 Thromboplastin Proteins 0.000 description 1
- 102000002262 Thromboplastin Human genes 0.000 description 1
- 101150047749 VIII gene Proteins 0.000 description 1
- 108700029631 X-Linked Genes Proteins 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 229910001413 alkali metal ion Inorganic materials 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 210000003423 ankle Anatomy 0.000 description 1
- 210000000702 aorta abdominal Anatomy 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 229940120638 avastin Drugs 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 229960000397 bevacizumab Drugs 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940015047 chorionic gonadotropin Drugs 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 229940022353 herceptin Drugs 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 210000000629 knee joint Anatomy 0.000 description 1
- 210000002414 leg Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 229950001570 octocog alfa Drugs 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000003752 saphenous vein Anatomy 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 210000003765 sex chromosome Anatomy 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 125000005629 sialic acid group Chemical group 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000003335 steric effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229960000575 trastuzumab Drugs 0.000 description 1
- 239000001393 triammonium citrate Substances 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/36—Blood coagulation or fibrinolysis factors
- A61K38/37—Factors VIII
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
- C07K2319/91—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation
Definitions
- the present invention relates to the field of fusion proteins, and more particularly, to a fusion protein of mutant human coagulation factor VIII (FVIII), and a preparation method and application thereof, in particular to the application of treating various coagulation-related diseases.
- FVIII mutant human coagulation factor VIII
- Coagulation factor VIII also known as anti-hemophilia factor
- FVIII Coagulation factor VIII
- a large number of studies on the molecular genetics of FVIII have shown that the absence of FVIII in the X-linked genes of the sex chromosome can lead to hemophilia type A. According to statistics, the prevalence of hemophilia A in male population is 1/5000, accounting for more than 80% of the total number of hemophilia.
- the currently commonly used treatment for hemophilia A is replacement therapy, which supplements FVIII that is lacking in patients with hemophilia.
- the mature FVIII consists of light and heavy chains with a molecular weight of approximately 280 kDa and has a domain of A1-A2-B-A3-C1-C2. Intracellular proteolysis of Arg-1648 residues in the B domain produces a heterogeneous heavy chain (A1-A2-B) of 90-200 kDa and a light chain of 80 kDa (domain A3-C1-C2) .
- the heavy and light chains are combined into heterodimers through divalent metal ion-dependent links.
- the dimers of the heavy and light chains bind von Willebrand (vWF) with high affinity to protect them from premature degradation.
- vWF von Willebrand
- FVIII is hydrolyzed and cleaved by activated coagulation factors FX (FXa) and thrombin FII (FIIa) at the Arg372 and Arg740 positions in the heavy chain and Arg1689 position in the light chain, resulting in the release of vWF factor and the production of activated FVIII II Polymer (FVIIIa), in the presence of Ca 2+ , it forms a tight complex with the activated coagulation factors FIX (FIXa) and FX on the surface of phospholipids. FX is then activated by FIXa, and the activated FX is dissociated from the complex.
- FXa activated coagulation factors
- FIIa thrombin FII
- thrombinogen is converted to thrombin, which converts fibrinogen directly to fibrin.
- FVIII can enhance the catalytic efficiency of FIXa to activate FX by several orders of magnitude.
- FVIII Due to the complex structure and large molecular weight of FVIII protein, FVIII is very unstable and easily inactivated during the process of raw material plasma collection and recombinant purification. FVIII is relatively short-lived. Especially in aqueous solutions, factors such as storage temperature, inorganic salt ions, trace amounts of proteases, and other proteins involved in coagulation (especially vWF and albumin) will affect the stability of FVIII molecules. FVIII freeze-dried product must be safe and sterile during injection after reconstitution, and the infusion must be completed within a specified time, generally no more than 4 hours. On the other hand, violent shaking during lyophilization and reconstitution can also cause protein structure damage and inactivate FVIII. Therefore, the development of FVIII molecules with better stability can improve the flexibility of FVIII products in clinical applications, which is very important for greatly improving the safety of patients and the quality of life.
- the B domain of FVIII contains 18 N glycosylation sites, has no known function in coagulation and has no homology with other proteins, and FVIII molecules with deleted B-domain still have good procoagulant activity. Eaton et al. Disclosed that a FVIII molecule with 766 amino acids (797 to 1562) deleted from the central B domain region maintained its biological activity. In addition, the expression of FVIII in mammalian cells was higher than Full-length molecules, and exhibit faster and higher activation rates than full-length molecules (Eaton et al., Biochemistry, 1986, 25: 8343-8347).
- Afstyla is a new recombinant single-chain human FVIII product. It is currently the only single-chain coagulation factor product approved for the fusion of light and heavy chains of hemophilia A. Because of its strong affinity for vascular pseudohemophilic factor (VWF), it has higher molecular stability and longer duration of efficacy. In clinical studies, Afstyla has excellent hemostatic and bleeding prevention effects in both prophylaxis and on-demand treatment. In both regimens, the dosage of Afstyla is lower. It also has very good security. Compared with the standard care drug Octocog alfa, the protein configuration of Afstyla is more stable and the effect is relatively durable, but it still needs to be injected 2-3 times a week.
- WO2013106789 discloses a chimeric polypeptide (FVIIIFc) comprising a FVIII portion and an Fc portion.
- the terminal half-life of the chimeric FVIII polypeptide is twice as long as that of rFVIII, and the frequency of administration of preventive treatment is twice a week. To maintain the level of FVIII activity at 1-3%.
- CTP is a short peptide derived from the carboxy terminus of the ⁇ -subunit of human chorionic gonadotropin (hCG) and contains multiple O-glycosylation sites. This negatively charged, highly sialylated peptide is covalently linked to the C-terminus of other proteins, which can resist the clearance of the kidney, thereby extending the half-life of the target protein linked to it in the body.
- hCG human chorionic gonadotropin
- the present invention creatively uses a CTP polypeptide having multiple O-glycosyl sites as part of a linking peptide for linking single-chain FVIII and Fc fragments, rather than placing it at the C-terminus as a fusion ligand, because it has
- the natural glycosylation site can not only further extend the half-life of the fusion protein and improve bioavailability, but also cooperate with the conventional flexible GS flexible linker peptide to form a stable three-dimensional conformation, which promotes the independent folding of single-chain FVIII and Fc segments.
- the correct three-dimensional conformation greatly reduces the steric hindrance effect of the fusion ligand Fc on the single-chain FVIII, and maintains its high biological activity.
- the protection of the CTP glycosyl side chain can reduce the sensitivity of the linker peptide to proteases.
- the invention provides a recombinant mutant single-chain coagulation factor VIII Fc fusion protein, which has a prolonged in vivo active half-life and a biological activity similar to that of recombinant FVIII.
- the present invention provides a method for efficiently and stably expressing the fusion protein.
- the fusion protein expressed by the method has high yield, good stability during preparation and storage, and its biological activity and marketed recombinant FVIII factors. Similar advantages.
- the inventors of the present application surprisingly found that the stability of the constructed recombinant mutant single-chain FVIII fusion protein was substantially enhanced, the fusion protein could prevent protease cleavage in the cell, and the obtained fusion protein showed better after purification Stability, showing good bioavailability when applied subcutaneously.
- the present invention relates to a recombinant mutant single-chain coagulation factor VIII fusion protein.
- the fusion protein contains a single-chain human FVIII (scFVIII) having a deleted B-domain in sequence from the N-terminus to the C-terminus.
- CTP rigid unit carboxy terminal peptide rigid unit of at least one human chorionic gonadotropin beta subunit (hereinafter referred to as (CTP) n , preferably, n is 1,2, 3, 4, or 5) and an extended half-life portion (eg, an immunoglobulin Fc segment, albumin, transferrin, or PEG, preferably a human IgG Fc variant (denoted as vFc)).
- CTP rigid unit expressed as (CTP) n , preferably, n is 1,2, 3, 4, or 5
- an extended half-life portion eg, an immunoglobulin Fc segment, albumin, transferrin, or PEG, preferably a human IgG Fc variant (denoted as vFc)
- the fusion protein is represented as scFVIII-L-CTP-vFc.
- the scFVIII has amino acids 765 to 1651 deleted compared to the full-length human wild-type FVIII amino acid sequence shown in SEQ ID NO: 1; specifically, the scFVIII has the amino acid sequence described in SEQ ID NO: 2 .
- the flexible peptide linker is preferably non-immunogenic, and generates a sufficient spatial distance between scFVIII and Fc to minimize the steric effect between each other.
- a flexible peptide linker consisting of two or more amino acid residues is used and is selected from the following amino acids: Gly (G), Ser (S), Ala (A), and Thr (T).
- the flexible peptide linker comprises G and S residues.
- the length of the linker peptide is very important for the activity of the fusion protein.
- the peptide linker may preferably comprise a general formula for an amino acid sequence formed by a combination of (GS) a (GGS) b (GGGS) c (GGGGS) d cycle units, where a, b, c and d are greater than Or an integer equal to 0, and a + b + c + d ⁇ 1.
- the peptide linker may preferably include the following sequence:
- the CTP rigid unit is selected from a full-length sequence or a fragment thereof composed of amino acids 113 to 145 of the carboxy terminus of the human chorionic gonadotropin beta subunit, and specifically, the CTP rigid unit comprises, for example, SEQ ID NO: The amino acid sequence shown in 3 or its truncated sequence.
- the random curl of the CTP rigid linker peptide with multiple glycosylation sites relative to the flexible linker peptide can form a stable three-dimensional conformation, which promotes the independent folding of scFVIII and Fc segments to form the correct three-dimensional conformation without affecting the respective organisms. active.
- the protection of the CTP glycosyl side chain can reduce the sensitivity of the linker peptide to proteases.
- the CTP rigid unit includes at least 2 glycosylation sites; for example, in a preferred embodiment of the present invention, the CTP rigid unit includes 2 glycosylation sites.
- the CTP The rigid unit contains 10 amino acids at the end of SEQ ID NO: 3N, namely SSSS * KAPPPS *; or the rigid unit of CTP contains 14 amino acids at the end of SEQ ID NO: 3C, namely S * RLPGPS * DTPILPQ; for another example, another embodiment
- the CTP rigid unit includes three glycosylation sites.
- the CTP rigid unit includes 16 amino acids at the SEQ ID NO: 3N terminus, that is, SSSS * KAPPPS * LPSPS * R; for another example, others
- the CTP rigid unit comprises 4 glycosylation sites.
- the CTP rigid unit comprises 28, 29, 30, 31, 32 or 33 amino acids and starts from human chorionic gonadotropin
- the beta subunit is at position 113, 114, 115, 116, 117, or 118 and ends at position 145.
- the CTP rigid unit contains 28 amino acids at SEQ ID NO: 3N terminus, that is, SSSS * KAPPPS * LPSPS * RLPGPS * DTPILPQ.
- * represents a glycosylation site.
- Each possibility represents a separate embodiment of the invention.
- the CTP rigid unit provided by the present invention is at least 70% homologous to the natural CTP amino acid sequence; in other embodiments, the CTP rigid unit provided by the present invention is at least 80% homologous to the natural CTP amino acid sequence; In other embodiments, the CTP rigid units provided by the present invention are at least 90% homologous to the natural CTP amino acid sequence; in other embodiments, the CTP rigid units provided by the present invention are at least 95% homologous to the natural CTP amino acid sequence.
- the fusion protein includes one of the aforementioned CTP rigid units. In other embodiments of the present invention, the fusion protein includes two or more of the above-mentioned CTP rigid units, and preferably includes two, three, four, or five of the above-mentioned CTP rigid units.
- the extended half-life portion is preferably an immunoglobulin IgG, IgM, IgA Fc fragment; more preferably an Fc fragment from human IgG1, IgG2, IgG3, or IgG4 and variants thereof; further, the human IgG Fc variant comprises At least one amino acid modification in human IgG type Fc, and the variant has reduced effector function (ADCC and / or CDC effect) and / or enhanced binding affinity to neonatal receptor FcRn.
- human IgG and Fc variants can be selected from the group:
- vFc ⁇ 1 human IgG1 hinge region, CH2, and CH3 regions (including the amino acid sequence shown in SEQ ID NO: 4) containing Leu234Val, Leu235Ala, and Pro331Ser mutations;
- vFc ⁇ 2-1 human IgG2 hinge region, CH2 and CH3 regions containing Pro331Ser mutation (as shown in SEQ ID NO: 5);
- vFc ⁇ 2-2 human IgG2 hinge region, CH2 and CH3 regions containing Thr250Gln and Met428Leu mutations (such as the amino acid sequence shown in SEQ ID NO: 6);
- vFc ⁇ 2-3 human IgG2 hinge region, CH2 and CH3 regions (including the amino acid sequence shown in SEQ ID NO: 7) containing Pro331Ser, Thr250Gln and Met428Leu mutations;
- (v) vFc ⁇ 4 human IgG4 hinge region, CH2 and CH3 regions containing Ser228Pro and Leu235Ala mutations (as shown in SEQ ID NO: 8).
- the IgG Fc variants provided by the present invention include, but are not limited to, the five variants described in (i) to (v), and may also be the combination or superposition of mutation sites of two types of functional variants between IgG isotypes,
- the variant as described in (iv) above is a new combination variant of IgG2Fc obtained by superimposing the mutation sites in (ii) and (iii).
- the Fc variant (vFc) in the fusion protein of the present invention contains the hinge region, CH2 and CH3 regions of human IgG such as human IgG1, IgG2 and IgG4.
- This CH2 region contains amino acid mutations at positions 228, 234, 235, and 331 (determined by the EU counting system). These amino acid mutations are believed to reduce the effector function of Fc.
- Human IgG2 does not bind Fc ⁇ R, but shows extremely weak complement activity.
- the Fc ⁇ 2 variant with the Pro331Ser mutation should have lower complement activity than the native Fc ⁇ 2 and still be an Fc ⁇ R non-binding element.
- IgG4 Fc is defective in activating the complement cascade, and its binding affinity to Fc ⁇ R is about one order of magnitude lower than IgG1.
- Fc ⁇ 4 variants with a Leu235Ala mutation should exhibit minimal effector function.
- Fc ⁇ 1 with Leu234Val, Leu235Ala, and Pro331Ser mutations also showed reduced effector functions compared to native Fc ⁇ 1.
- These Fc variants are more suitable for preparing FVIII fusion proteins than natural human IgG and Fc.
- the 250 and 428 positions contain amino acid mutations that increase the binding affinity of the Fc region to the neonatal receptor FcRn, thereby further extending the half-life (Paul R et al., J. Biol Chem, 2004, 279: 6213 -6216); the above two types of functional variants are combined or superimposed with each other to obtain a new combined variant, which reduces the effector function and prolongs its half-life.
- the Fc variants of the present invention include but are not limited to mutations at the above-mentioned several sites.
- amino acid sequence of the fusion protein is shown in SEQ ID NO: 9;
- a DNA encoding the above-mentioned fusion protein is provided.
- the DNA sequence of the fusion protein is shown in SEQ ID NO: 10.
- a vector comprising the above-mentioned DNA.
- a host cell comprising the above-mentioned vector or transfected with the above-mentioned vector.
- the host cell is a CHO-derived cell line DXB-11.
- a pharmaceutical composition includes a pharmaceutically acceptable carrier, excipient or diluent, and an effective amount of the above-mentioned fusion protein.
- a method for preparing or producing the fusion protein from a mammalian cell line comprising the following steps:
- step (d) Harvesting the fermentation broth obtained in step (c), and separating and purifying the fusion protein.
- the CHO-derived cell line in step (a) is DXB-11.
- the cell culture may be performed by a batch, perfusion or fed-batch culture method.
- step (d) the fusion protein is purified by four-step chromatography, which are affinity chromatography, hydrophobic chromatography, anion exchange chromatography, and molecular sieve chromatography, respectively.
- the present invention further gives preferred conditions in combination with Example 5.
- the activity of the fusion protein prepared by the above method is> 6000 IU / mg.
- fusion protein in the manufacture of a medicament for the prevention or treatment of a bleeding disease or event caused by FVIII deficiency or functional deficiency.
- the disease includes type A (or type A) hemophilia.
- type A or type A hemophilia.
- the fusion protein of the present invention plays a role in controlling or preventing bleeding.
- the mutant single-chain FVIII fusion protein constructed by the present invention has an Fc segment that is non-cleavable, that is, by mutating the complement and receptor binding domains of the Fc fragment, regulating the binding affinity of Fc to the corresponding receptor, reducing or eliminating ADCC and CDC effects, while only retaining the Fc segment to extend the half-life of the active protein in vivo, but does not produce cytotoxicity.
- the mutant single-chain FVIII fusion protein provided by the present invention comprises a rigid CTP polypeptide having multiple glycosyl side chains, and it can form a stable three-dimensional conformation with respect to the random coil of a flexible linker peptide such as (GGGGS) n.
- This "blocking" effect promotes the independent folding of FVIII and Fc segments to form the correct three-dimensional conformation without affecting each other's biological activity.
- CTP contains multiple O-modified oligosaccharide groups. Negatively charged, highly sialylated CTP can resist the clearance of the kidney and further extend the half-life of the fusion protein.
- the protective effect of the CTP glycosyl side chain can reduce the connection The sensitivity of the peptide to proteases makes it difficult for the fusion protein to be degraded in the junction region.
- the fusion protein of the present invention has good in vitro stability regardless of fermentation, purification and storage.
- the activity of the mutant single-chain FVIII fusion protein was significantly higher than that between Arg1648 and Glu1649 (encoded according to the full-length sequence of human wild-type FVIII SEQ ID ID: 1) at 25 ° C for 7 days.
- Chain FVIII Fc fusion protein drug, and loss of activity is less than 20%.
- the method for preparing the fusion protein provided by the present invention has a high yield. After 14 days of cultivation in a 300 ml shake flask, the cumulative yield can reach at least 200 mg / L, which can be scaled up to achieve large-scale industrial production.
- CTP is a short peptide derived from the carboxy terminus of the ⁇ -subunit of human chorionic gonadotropin (hCG).
- FSH follicle stimulating hormone
- LH luteinizing hormone
- TSH thyroid stimulating hormone
- hCG chorionic gonadotropin
- Natural CTP contains 37 amino acid residues, has 4 O-glycosylation sites, and terminates in sialic acid residues. Negatively charged, highly sialylated CTP can resist the clearance effect of the kidney, thereby prolonging the half-life of the circulation in the body (Fares, et al., Proc. Natl, Acad, Sci USA, 1992, 89 (10): 4304-4308).
- the present invention creatively links at least one CTP polypeptide to a flexible linker peptide of appropriate length as a linker peptide for linking FVIII to an extended half-life portion (eg, an immunoglobulin Fc fragment).
- the present invention has found that by adding a CTP peptide between FVIII and an Fc variant, it is equivalent to adding a segment of rigid linker peptide.
- This aspect ensures that the N-terminally fused FVIII does not affect the binding site of the Fc variant and FcRn, thereby affecting the half-life; in addition, the Protein A binding site of Fc is important for the purification step in the preparation process, and CTP is connected to ensure N- The end-fused FVIII will not "cover" its binding site with protein A, so it is possible to choose a cheaper and more suitable filler to purify the fusion protein and reduce the purification cost.
- CTP also makes the Fc fragment of about 25kDa size not interfere with the correct folding of the N-terminally fused FVIII, resulting in a decrease or loss of its biological activity / function.
- Rigid CTP polypeptides with multiple glycosyl side chains compared to the random coils of flexible linker peptides such as (GGGGS) n, can form stable stereo conformations. This "blocking" effect promotes the independent folding of FVIII and Fc segments. Correct three-dimensional conformation without affecting each other's biological activity.
- the protection of the CTP glycosyl side chain can reduce the sensitivity of the linker peptide to the protease, making the fusion protein difficult to be degraded in the linker region.
- the Fc element is derived from the constant region Fc fragment of the immunoglobulin IgG, which plays an important role in eliminating the immune defense of the pathogen.
- Fc-mediated effector function of IgG is exerted through two mechanisms: (1) binding to cell surface Fc receptors (Fc ⁇ Rs), digestion of pathogens by phagocytosis or lysis or killer cells through antibody-dependent cytotoxicity (ADCC) pathway , Or (2) Binding to C1q of the first complement component C1, triggering the complement-dependent cytotoxicity (CDC) pathway, thereby lysing the pathogen.
- IgG1 and IgG3 can effectively bind Fc ⁇ Rs, IgG4 and Fc ⁇ Rs have low binding affinity, and IgG2 and Fc ⁇ Rs have low binding assays, so human IgG2 has almost no ADCC effect.
- human IgG1 and IgG3 can effectively bind C1q to activate the complement cascade.
- Human IgG2 binds to C1q relatively weakly, while IgG4 does not bind to C1q (Jefferis et al., Immunol Rev. 1998, 163: 59-76), so human IgG2 CDC effect is also weak.
- Fc variant with enhanced affinity for neonatal receptor (FcRn) FcRn
- the plasma half-life of IgG depends on its binding to FcRn, which generally binds at pH 6.0 and dissociates at pH 7.4 (plasma pH). Through the study of the binding sites of the two, the binding site of IgG to FcRn was modified to increase its binding capacity at pH 6.0. Mutations in some residues of the human Fcy domain that have been shown to be important for binding to FcRn can increase serum half-life. Mutations in T250, M252, S254, T256, V308, E380, M428, and N434 have been reported to increase or decrease FcRn binding affinity (Roopenian et al., Nat. Rview Immunology 7: 715-725, 2007). Korean Patent No.
- KR 10-1027427 discloses trastuzumab (Herceptin, Genentech) variants with increased FcRn binding affinity, and these variants comprise a member selected from the group consisting of 257C, 257M, 257L, 257N, 257Y, 279Q, One or more amino acid modifications of 279Y, 308F, and 308Y.
- Korean Patent Publication No. KR 2010-0099179 provides variants of bevacizumab (Avastin, Genentech) and these variants show increased in vivo by amino acid modifications contained in N434S, M252Y / M428L, M252Y / N434S, and M428L / N434S half life.
- Hinton et al. also found that T250Q and M428L 2 mutants increased binding to FcRn by 3 and 7 times, respectively. Mutation at two sites simultaneously increased binding 28-fold. In rhesus monkeys, M428L or T250QM / 428L mutants show a two-fold increase in plasma half-life (Paul R. Hinton et al., J Immunol, 2006, 176: 346-356). For more mutation sites contained in Fc variants with enhanced binding affinity to the neonatal receptor (FcRn), see Chinese invention patent CN201280066663.2.
- the fusion protein gene of the present invention is prepared by an artificial synthesis method with optimized codons.
- those skilled in the art can conveniently prepare the encoding nucleic acid of the present invention by various known methods. These methods are not limited to artificial synthesis or traditional subcloning. For specific methods, see J. Sambrook, "Molecular Cloning Experiment Guide”.
- a nucleic acid coding sequence of the present invention is constructed by a method of synthesizing a nucleotide sequence in sections and performing subcloning.
- the present invention also provides an expression vector for mammalian cells, comprising the sequence encoding the fusion protein of the present invention and an expression control sequence operatively linked thereto.
- the term "operably linked” or “operably linked” refers to a condition in which certain parts of a linear DNA sequence can regulate or control the activity of other parts of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, it is operably linked to a coding sequence.
- telomeres For mammalian cell expression vectors, commercially available vectors such as, but not limited to, pcDNA3, pIRES, pDR, pBK, pSPORT, and the like can be used for eukaryotic cell system expression. Those skilled in the art can also select a suitable expression vector according to the host cell.
- restriction map of a known empty-loaded expression vector those skilled in the art can insert the coding sequence of the fusion protein of the present invention into a suitable restriction site by restriction enzyme splicing and splicing according to conventional methods to prepare the present invention.
- Recombinant expression vector Recombinant expression vector.
- the invention also provides a host cell expressing the fusion protein of the invention, which contains the coding sequence of the fusion protein of the invention.
- the host cell is preferably a eukaryotic cell, such as, but not limited to, a CHO cell, a COS cell, a 293 cell, an RSF cell, and the like.
- the cell is a CHO cell, which can better express the fusion protein of the present invention, and a fusion protein with good activity and stability can be obtained.
- the present invention also provides a method for preparing the fusion protein of the present invention by using recombinant DNA technology, the steps include:
- the coding sequence can be introduced into the host cell using a variety of known techniques in the art, such as, but not limited to, calcium phosphate precipitation, liposome transfection, electroporation, microinjection, virus infection method, and alkali metal ion method.
- the fusion protein obtained by the above preparation can be purified to a substantially uniform nature, such as a single or specific band on SDS-PAGE electrophoresis.
- the expression supernatant is first concentrated.
- the concentrated solution can be further purified by gel chromatography, or purified by ion exchange chromatography. For example, anion exchange chromatography or cation exchange chromatography.
- the gel matrix can be agarose, dextran, polyamide and other commonly used media for protein purification.
- the Q- or SP- group is a more ideal ion-exchange group.
- hydroxyapatite adsorption chromatography, metal chelation chromatography, hydrophobic interaction chromatography and reversed-phase high-performance liquid chromatography can be used to further refine and purify the purified product. All the above purification steps can be used in different combinations to ultimately achieve a substantially uniform protein purity.
- the expressed fusion protein can also be purified using an affinity chromatography column containing a specific antibody, receptor or ligand of the fusion protein. Depending on the characteristics of the affinity column used, conventional methods, such as high-salt buffer, pH change, and other methods can be used to elute the fusion polypeptide bound to the affinity column.
- the invention also provides a pharmaceutical composition containing an effective amount of the fusion protein of the invention, and a pharmaceutically acceptable carrier.
- an effective amount of the fusion protein of the present invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably, the pH is about 6-8.
- the term "effective amount” or “effective dose” refers to an amount that is functional or active in humans and / or animals and acceptable to humans and / or animals.
- “Pharmaceutically acceptable” ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergies), i.e. substances with a reasonable benefit / risk ratio.
- pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent and includes a variety of adjuvants and diluents.
- Pharmaceutically acceptable carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
- the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of injections, for example, by using normal saline or an aqueous solution containing glucose and other adjuvants by conventional methods.
- the pharmaceutical composition is preferably manufactured under sterile conditions.
- the active ingredient is administered in a therapeutically effective amount.
- the pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.
- the effective amount of the fusion protein according to the present invention may vary depending on the mode of administration and the severity of the disease to be treated.
- the selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through a clinical trial).
- the factors include, but are not limited to: the pharmacokinetic parameters of the fusion protein, such as bioavailability, metabolism, half-life, etc .; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. .
- Figure 3 Effect of fusion protein SS-F8 on saphenous artery bleeding time in SD rats.
- Figure 4 Effect of fusion protein SS-F8 on plasma APTT in SD rats.
- Example 1 Construction of an expression plasmid encoding a mutant single-chain FVIII fusion protein
- the gene sequences encoding FVIII leader peptide, FVIII protein with partially deleted B-domain, flexible peptide linker, CTP rigid unit and human IgG vFc variants are all artificially optimized CHO cell preference codons and obtained by artificial synthesis.
- the 5 'and 3' ends of the full-length DNA fragment of the synthesized fusion protein each have a restriction endonuclease site, SpeI and EcoRI, respectively.
- the full-length DNA fragment is inserted between the corresponding restriction sites of the pUC57 transfer vector. DNA sequencing verified its sequence.
- the full-length gene fragment of the fusion protein obtained above was transferred from the intermediate vector to the corresponding restriction site of the self-made expression plasmid PXY1A1M to obtain a fusion protein high expression plasmid.
- the PXY1A1M plasmid contains but is not limited to the following important expression components: 1) early human cytomegalovirus promoter and enhancer required for exogenous high expression in mammalian cells; 2) dual screening markers, which have kanamycin resistance in bacteria Resistance, G418 resistance in mammalian cells; 3) mouse dihydrofolate reductase (DHFR) gene expression box, when the host cell is DHFR gene-deficient, methotrexate (MTX) can co-amplify the fusion gene And the DHFR gene (see US Patent 4,399,216).
- DHFR mouse dihydrofolate reductase
- the fusion protein expression plasmid is then transfected into a mammalian host cell line.
- the preferred host cell line is a DHFR enzyme-deficient CHO-cell (see US Patent No. 4,818,679).
- the medium was changed to a screening medium containing 0.6mg / mL G418.
- Cells were planted in a 96-well culture plate at a certain concentration (5000-10000 live cells / well) and cultured for 10-14 days until large. Discrete cell clones appeared.
- ELISA analysis was used to screen transfectants resistant to the selected drug. By limiting dilution of a 96-well culture plate, subcloning produces wells with high levels of fusion protein.
- the present invention constructs a series of mutant single-chain FVIII fusion proteins, which contain peptide linkers (Linker) of different lengths, CTP rigid units of different compositions, and IgG Fc variant (vFc) elements of several different subtypes to verify Effect of linker peptide and Fc variant on the activity of mutant single-chain FVIII fusion protein. See Table 1 for details. The amino acid sequence of each component is shown in the sequence listing.
- the expression plasmid of the above-mentioned fusion protein was transfected into a mammalian host cell line to express a mutant single-chain FVIII fusion protein.
- the preferred host cell is a DHFR-deficient CHO cell (see US Pat. No. 4,818,679).
- One preferred transfection method is electroporation, but other methods can also be used, including calcium phosphate co-sedimentation, liposome transfection, and microinjection.
- the electroporation method uses a Gene Pulser Electroporator (Bio-Rad Laboratories) set to a voltage of 300 V and a capacitance of 1050 ⁇ Fd.
- PvuI linearized expression plasmid 50 ⁇ g of PvuI linearized expression plasmid is added to 2 to 3 ⁇ 10 7 cells placed in a cuvette.
- the perforated cells were transferred to shake flasks containing 30 ml of growth medium. Two days after transfection, the medium was changed to a growth medium containing 0.6 mg / mL G418, and the cells were seeded in a 96-well culture plate at a certain concentration, and cultured for 10-12 days until large discrete cell clones appeared.
- the anti-human IgG Fc analysis method was used to screen transfectants resistant to the selected drug, and then subcloned by limiting dilution to produce wells expressing the fusion protein at high levels.
- amplification of the DHFR gene inhibited by MTX drugs is appropriate.
- the transfected fusion protein gene was co-amplified with the DHFR gene.
- Subclones that were positive for DHFR expression were diluted, and the transfectants that could grow in up to 6 ⁇ M MTX medium were screened and the secretion rate was determined.
- Cell lines with high expression of foreign proteins were selected.
- Cell lines with a secretion rate in excess of about 1 (preferably about 3) IU / 10 6 (ie millions) cells / 24 hours are subjected to adaptive suspension culture using serum-free medium, and then fused with conditioned medium protein.
- the high-yield cell strain preferably obtained in Example 2 was first subjected to serum-free acclimatization culture in a petri dish, and then transferred to a shake flask for suspension acclimatization culture. After the cells are adapted to these culture conditions, then fed-feed culture is performed in a 300ml shake flask or perfusion culture is simulated by changing the medium every day.
- the CHO-derived cell strain producing the fusion protein SS-F8 obtained from the screening in Example 2 was fed and fed in a 300 ml volume shake flask for 14 days. The cumulative yield of the expressed recombinant fusion protein reached 200 mg / L, and the viable cell density It can reach up to 15 ⁇ 10 6 cells / mL.
- 1000ml shake flask culture can also be used.
- the above-mentioned CHO-derived cell strain is changed daily in a 100 ml volume shake flask, and the cumulative daily expression of the recombinant fusion protein expressed is about 20 mg / L.
- the maximum density of live cells in the shake flask can reach 30. ⁇ 10 6 cells / mL.
- the biological activity of the recombinant fusion proteins produced by the above two methods is comparable.
- the invention mainly uses a four-step chromatography method to purify the fusion protein SS-F8.
- Affinity chromatography, anion exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography The protein purification instrument used in this example is AKTA Pure 25M from GE, USA.
- the reagents used in this example were purchased from Sinopharm Chemical Reagent Co., Ltd., purity is analytical grade).
- the first step is affinity chromatography: GE's VIII Select affinity chromatography medium is used for sample capture, concentration, and removal of some contaminants.
- the clarified fermentation broth was loaded at a linear flow rate of 50-100 cm / h, and the load was not higher than 50,000 IU / ml.
- the equilibrium buffer was used: 10 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , 0.05% Tween- 80, pH 6.8-7.2, equilibrate the chromatography column with a linear flow rate of 50-100 cm / h for 3-5 column volumes (CV), rinse unbound components; use decontamination buffer 1: 10mM HEPES, 1M NaCl, 25mM CaCl 2 , 0.05% Tween-80, pH 6.8-7.2, rinse the column with 3-5 column volumes at a linear flow rate of 50-100cm / h to remove some of the contaminants; use an equilibrium buffer: 10mM HEPES, 150mM NaCl, 25mM CaCl 2 , 0.05% Tween-80, pH 6.8-7.2, equilibrate the chromatography column with a linear flow rate of 50-100 cm / h for 3-5 column volumes (CV); then use an elution buffer: 20 mM His-HC
- anion exchange chromatography use Q-HP from Borglon or other commercially available anion exchange chromatography media (such as Q HP from GE, Toyopearl GigaCap Q-650 from TOSOH, DEAE Beads 6FF from Tianheren) , Generik MC-Q from Saifen Technology, Fractogel EMD TMAE from Merck, and Q Ceramic HyperD from Pall) were used for intermediate purification to isolate structural variants and further remove HCP, DNA and other pollutants.
- Q HP from GE
- Toyopearl GigaCap Q-650 from TOSOH
- DEAE Beads 6FF from Tianheren
- Generik MC-Q from Saifen Technology
- Fractogel EMD TMAE from Merck
- Q Ceramic HyperD from Pall
- equilibration buffer 20 mM His-HCl, 100 mM NaCl, 10 mM CaCl 2 , 0.02% Tween 80, pH 7.0-7.5, and rinse the chromatography column with a linear flow rate of 50-100 cm / h for 3-5 column volumes (CV);
- the target protein separated by affinity chromatography in the first step is diluted 5-10 times, and the sample is loaded after reducing the concentration of organic matter, and the load is controlled at 5000-10000IU / ml.
- Step 3 Hydrophobic Chromatography: Use Butyl HP from Borglon or other commercially available hydrophobic chromatography media (such as GE's Butyl HP, TOSOH's Toyopearl Butyl-650, Tiandiren and Butyl Beads 4FF, Saifen Technology) Generik MC30-HIC Butyl and Merck's Fractogel EMD Propyl) were used for intermediate purification to reduce the polymer content.
- hydrophobic Chromatography media such as GE's Butyl HP, TOSOH's Toyopearl Butyl-650, Tiandiren and Butyl Beads 4FF, Saifen Technology
- the second step of the anion exchange chromatography eluent still contains a certain proportion of polymers because of the various reasons for the formation of the polymers Including the polymerization of unchanged structure and the polymerization of changed structure, their biological activities differ greatly, so it will cause greater interference to the analysis of biological activity.
- the target protein is polymerized, there are differences in properties between the polymer and the monomer, including charge characteristics and hydrophobicity. We use the difference in hydrophobicity to separate the two. Since the final purification step is molecular sieve chromatography, purification is performed using Butyl HP with the goal of partially removing the polymer to less than 10%.
- Debuffer elute 3-5 column volumes (CV) at a linear flow rate not higher than 60 cm / h, collect the eluted fractions in sections, and submit them to SEC-HPLC for inspection.
- the target components with a monomer percentage greater than 90% were combined for further chromatography.
- the fourth step is molecular sieve chromatography: using GE's superdex 200 or other commercially available molecular sieve media (such as Chromdex 200 prep grade from Boglong) for separation, the goal is to reduce the polymer content to ⁇ 5%, and further reduce the key Contaminant content.
- the loading amount is not higher than 3% of the column volume, and the sample is rinsed at a linear flow rate of 20 cm / h.
- the eluted components are collected in turn, and the SEC test is combined.
- the SEC-HPLC purity results and SDS-PAGE electrophoresis results of the samples are shown in Figures 1 and 2.
- the SEC-HPLC results showed that the purity of the main peak of the purified fusion protein was more than 98%; the SDS-PAGE electrophoresis band pattern was in line with expectations.
- Example 5 In vitro determination of fusion protein in vitro activity by chromogenic substrate method
- the activity of the mutant single-chain FVIII fusion protein can be determined using a chromogenic substrate method.
- This example was determined using the Biophen FVIII: C kit (HYPHEN BioMed, Ref. 221402).
- the detection principle is as follows: When activated by thrombin, FVIII: C is combined with FIXa to form an enzyme complex in the presence of phospholipids and calcium ions. , Which in turn can activate factor X into its active form Xa.
- the factor Xa formed by activation can in turn cleave its specific chromogenic substrate (SXa-11), releasing the chromophore pNA.
- Example 6 Study on the stability of purified fusion protein
- the mutant single-chain FVIII fusion protein was stored at 25 ° C for several days, and the effect of this condition on the activity of the fusion protein was examined.
- the drug stability test box (purchased from Shanghai-Heng Scientific Instrument) was set at a test temperature of 25 ° C and a humidity of 75%.
- SS-F8 and double-stranded eight-factor control drug DS-F8 (the fusion protein retains the protease cleavage site between human wild-type FVIII Arg1648 and Glu1649, and its amino acid sequence is as shown in SEQ ID NO: 11 (Indicated)
- Each is packed in 8 pieces, each 200 ⁇ l, and stored in a drug stability test box. Take one SS-F8 and DS-F8 aliquots each, and measure the activity of the fusion protein according to the method described in Example 5 above, and record it as the activity value on the first day (d1).
- SS-F8 only decreased protein activity by about 10% after 5 days at 25 ° C, and the protein activity of DS-F8 decreased by more than 25%.
- the rate of DS-F8 activity decreased after 7 days at room temperature. It is more significant than SS-F8, and SS-F8 still retains more than 80% of protein activity after 7 days of standing, which shows that the stability of SS-F8 fusion protein is significantly better than DS-F8.
- Blood coagulation is actually an enzymatic reaction of a series of coagulation factors.
- the whole coagulation process is divided into three stages: the first stage is blood thrombin formation; the second stage is thrombin formation; the third stage is fibrin formation, of which FVIII, FIX It is an endogenous coagulation factor, and FVII is an exogenous coagulation factor.
- Bleeding time is the time required to bleed naturally to stop bleeding after the skin capillary is punctured.
- the procoagulant effect of the fusion protein is detected by observing the effect of the mutant single-chain FVIII fusion protein on the bleeding time of the saphenous artery in SD rats.
- mice Seven-week-old SD rats (purchased from Shanghai Slark Experimental Animal Co., Ltd.) were randomly divided into two groups.
- SS-F8 was administered intravenously in a single dose of 200 IU / head
- the control group was administered the same volume of physiological saline.
- the rats were induced with anesthesia. After sterilizing the surgical site, cut the skin from the knee to the arteries at the base of the leg about 1 cm from the inner ankle, cut off the subcutaneous tissue and the protective film on the blood vessel, and sequentially expose the venous blood vessels, arterial blood vessels and nerves.
- Example 8 Direct determination of biological activity of fusion protein by coagulation method
- Seven-week-old SD rats (purchased from Shanghai Slark Experimental Animal Co., Ltd.) were randomly divided into 12 groups. After inducing anesthesia in rats, under continuous anesthesia, cut with a scalpel along the midline of the abdomen, and take 10-12 ml of blood from the abdominal aorta. Take the above blood sample, centrifuge at 1500 rpm for 30 min at 20 ° C, separate the supernatant plasma, and add each into labeled 1.5 ml centrifuge tubes.
- the whole plasma and the test drugs SS-F8 and DS-F8 were respectively prepared into test samples with a concentration of 25-1000 IU / ml at a volume ratio of 6: 1, and the control group was added with a dilution solution at a volume ratio of 6: 1.
- the experimental data is expressed as mean ⁇ standard deviation (Means ⁇ SD).
- SPSS is used 18.0 software, one-way analysis of variance or student's-test; if it is not normally distributed, use the nonparametric test Kruskal-wallis test or Mann-whitney test. P ⁇ 0.05 has a significant difference, and P ⁇ 0.01 has a very significant difference.
- test drugs SS-F8 and DS-F8 have anticoagulant effects on the plasma of normal rats.
- the APTT of the test drugs DS-F8 and SS-F8 Compared with the solvent control group, the time decreased by 33.65% and 31.86% respectively; the EC 50 values of DS-F8 and SS-F8 were 139.4IU / ml and 115.6IU / ml, respectively, and the EC 50 values of the test drug SS-F8 were low.
- DS-F8 lower doses can be foreseen.
- the time of APTT is significantly shortened, and there is a certain dose-effect relationship.
- mice The acute hemostatic effect of SS-F8 in HA mice was evaluated using the Tail Clipping Bleeding Model of the VIII gene knockout homozygous hemophilia A (HA) mice.
- HA mice 6-7 week old male HA mice (from Shanghai N forcing Model Biotechnology Co., Ltd.) were selected and randomly divided into 2 groups after one week of adaptive breeding, which were the HA mouse blank vehicle control group and SS-F8 G.
- Male C57BL / 6 mice purchased from Shanghai Slark Experimental Animal Co., Ltd.
- Anesthetized mice were intraperitoneally injected with 1% sodium pentobarbital (Merck) at a dose of 7.5 ml / kg, and administered by tail vein injection.
- 1% sodium pentobarbital Merck
- mice normal control group and HA mice blank vehicle control group were intravenously administered 10 ml / kg of vehicle, and the administration group was given 112 IU / kg of SS-F8 intravenously.
- the tail was cut off at a distance of 15 mm from the end of the tail with a surgical blade, and the wound was quickly immersed in 13 mL of physiological saline preheated at 37 ° C. The timing and blood collection were started, and the total bleeding time and bleeding within 30 min after the tail was recorded. the amount.
- Blood loss (ml) (weight of centrifuge tube after blood collection (g)-weight of centrifuge tube before blood collection (g)) / 1.0.
- the comparison between the experimental groups was analyzed by T-test test, and the analysis software was Graphpad Prism 8.0, p ⁇ 0.05 was considered statistically significant. See Table 2 for detailed results.
- the median bleeding volume was 438.9 ⁇ l and 790.00 ⁇ l (p ⁇ 0.0001), and the median bleeding time was The numbers are 739s and 1800s (p ⁇ 0.0001), and the procoagulant effect is obvious, indicating that SS-F8 can be used as an effective coagulant for hemorrhage caused by hemophilia A factor VIII deficiency.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Diabetes (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Reproductive Health (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims (17)
- 一种人凝血因子VIII的融合蛋白,所述融合蛋白从N端至C端依次包含B-结构域部分缺失的突变型单链人凝血因子VIII、柔性肽接头、至少1个人绒毛膜***β亚基羧基末端肽刚性单元和延长半衰期部分;其中,所述单链人凝血因子VIII氨基酸序列如SEQ ID NO:2所示;其中,延长半衰期部分选自免疫球蛋白Fc段、白蛋白、转铁蛋白或PEG,优选人IgG Fc变体)。
- 如权利要求1所述的融合蛋白,其特征在于,所述融合蛋白是糖基化的,优选是通过在哺乳动物细胞(如,中国仓鼠卵巢细胞)中表达而糖基化的。
- 如权利要求1所述的融合蛋白,其特征在于,所述B-结构域部分缺失的突变型单链人凝血因子VIII包含如SEQ ID NO:2所示的氨基酸序列,或者所述单链人凝血因子VIII的氨基酸序列与如SEQ ID NO:2所示的氨基酸序列有至少90%的同一性。
- 如权利要求1所述的融合蛋白,其特征在于,所述柔性肽接头含有2个或多个选自G、S、A和T残基的氨基酸,优选地,所述柔性肽接头具有以(GS)a(GGS)b(GGGS)c(GGGGS)d循环单元组合形成的氨基酸序列通式,其中a,b,c和d是大于或等于0的整数,且a+b+c+d≥1,更优选地,所述柔性肽接头优选自下组:(i)GSGGGSGGGGSGGGGS;(ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS;(iii)GGGGSGGGGSGGGGSGGGGS;(iv)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS;(v)GGGSGGGSGGGSGGGSGGGS。
- 如权利要求1所述的融合蛋白,其特征在于,所述人绒毛膜***β亚基的羧基末端肽刚性单元包含如SEQ ID NO:3所示氨基酸序列或其截短的序列,其中,所述截短的序列包含至少2个糖基化位点,优选地,所述人绒毛膜***β亚基的羧基末端肽刚性单元包含以下氨基酸序列:(i)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ;(ii)SSSSKAPPPSLPSPSRLPGPSDTPILPQ;(iii)SSSSKAPPPS;(iv)SRLPGPSDTPILPQ。
- 如权利要求1所述的融合蛋白,其特征在于,所述人绒毛膜***β亚基的羧基末端 肽刚性单元与权利要求5所述融合蛋白中的羧基末端肽刚性单元氨基酸序列至少具有70%,80%,90%或95%的同一性。
- 如权利要求1所述的融合蛋白,其特征在于,所述融合蛋白包含1、2、3、4或5个人绒毛膜***β亚基的羧基末端肽刚性单元。
- 如权利要求1所述的融合蛋白,其特征在于,所述人IgG Fc变体具有降低的ADCC效应和/或CDC效应和/或与FcRn受体的结合亲和力增强,优选地,所述Fc变体选自:(i)含有Leu234Val、Leu235Ala和Pro331Ser突变的人IgG1绞链区、CH2和CH3区域;(ii)含有Pro331Ser突变的人IgG2绞链区、CH2和CH3区域;(iii)含有Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域;(iv)含有Pro331Ser、Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域;(v)含有Ser228Pro和Leu235Ala突变的人IgG4绞链区、CH2和CH3区域。
- 如权利要求1所述的融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如SEQ ID NO:9所示。
- 如权利要求1-9中任一项所述的融合蛋白,其特征在于,所述融合蛋白的活性>6000IU/mg。
- 编码如权利要求1-10中任一项所述的融合蛋白的DNA,优选所述DNA的序列如SEQ ID NO:10所示。
- 一种载体,其特征在于,包含如权利要求11所述的DNA。
- 一种宿主细胞,其特征在于,包含如权利要求12所述的载体,或者转染了权利要求12所述的载体。
- 一种药物组合物,其特征在于,包含药学上可接受的载体、赋形剂或稀释剂,以及有效剂量的如权利要求1-10中任一项所述的融合蛋白。
- 一种如权利要求1-10中任一项所述的融合蛋白的制备方法,所述方法包括:(a)将权利要求11所述编码融合蛋白的DNA序列引入CHO细胞,生成CHO衍生的细胞系;(b)筛选步骤(a)中在其生长培养基中每24小时期间内,表达超过1IU/10 6(百万)个细胞的高产细胞株;(c)培养步骤(b)筛选到的细胞株,表达融合蛋白;(d)收获步骤(c)得到的发酵液,并分离纯化融合蛋白。
- 如权利要求15所述的方法,其特征在于,所述步骤(d)中融合蛋白纯化过程包含亲和层析、疏水层析、阴离子交换层析和分子筛层析。
- 一种如权利要求1-10中任一项所述的融合蛋白在制备用于预防或治疗出血性疾病(例如,用于FVIII先天性或获得性缺乏症患者的出血性疾病的预防或治疗、血友病A患者的自发或手术性出血的预防或治疗)的药物中应用。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/280,343 US20220033476A1 (en) | 2018-09-26 | 2019-09-24 | Fusion protein of mutated single-chain human coagulation factor viii, preparation method therefor, and use thereof |
BR112021005831-1A BR112021005831A2 (pt) | 2018-09-26 | 2019-09-24 | proteína de fusão de fator viii de coagulação humana de cadeia simples mutado, método de preparação para isso e uso do mesmo |
CN201980059489.0A CN112673026A (zh) | 2018-09-26 | 2019-09-24 | 突变型单链人凝血因子viii融合蛋白及其制备方法与用途 |
EP19865552.4A EP3858865A4 (en) | 2018-09-26 | 2019-09-24 | MUTANT SINGLE CHAIN HUMAN COLOTTING FACTOR VIII FUSION PROTEIN, METHOD OF PRODUCTION THEREOF AND USE THEREOF |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811123918.X | 2018-09-26 | ||
CN201811123918.XA CN110950964B (zh) | 2018-09-26 | 2018-09-26 | 突变型单链人凝血因子viii融合蛋白及其制备方法与用途 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020063562A1 true WO2020063562A1 (zh) | 2020-04-02 |
Family
ID=69949730
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2019/107432 WO2020063562A1 (zh) | 2018-09-26 | 2019-09-24 | 突变型单链人凝血因子viii融合蛋白及其制备方法与用途 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20220033476A1 (zh) |
EP (1) | EP3858865A4 (zh) |
CN (3) | CN110950964B (zh) |
BR (1) | BR112021005831A2 (zh) |
WO (1) | WO2020063562A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4130048A4 (en) * | 2020-03-31 | 2024-04-24 | Ampsource Biopharma Shanghai Inc. | METHOD FOR EFFICIENT SEPARATION AND PURIFICATION OF RECOMBINANT HUMAN COAGULATE FACTOR VIII-FC FUSION PROTEIN |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111808170A (zh) * | 2020-06-29 | 2020-10-23 | 江苏为真生物医药技术股份有限公司 | 多肽、hla-dr蛋白及其制备方法和应用 |
WO2023227015A1 (zh) * | 2022-05-25 | 2023-11-30 | 江苏晟斯生物制药有限公司 | 具有延长的半衰期的fviii融合蛋白缀合物及其应用 |
CN116036244B (zh) * | 2023-02-24 | 2023-09-19 | 北京基科晟斯医药科技有限公司 | 培重组人凝血因子VIII-Fc融合蛋白用于治疗含抑制物的血友病A的用途 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4399216A (en) | 1980-02-25 | 1983-08-16 | The Trustees Of Columbia University | Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials |
US4818679A (en) | 1985-02-19 | 1989-04-04 | The Trustees Of Columbia University In The City Of New York | Method for recovering mutant cells |
KR20100099179A (ko) | 2007-12-26 | 2010-09-10 | 젠코어 인코포레이티드 | FcRn에 대한 변경된 결합성을 갖는 Fc 변이체 |
KR101027427B1 (ko) | 2004-11-12 | 2011-04-11 | 젠코어 인코포레이티드 | FcRn에 대하여 증가된 결합력을 갖는 Fc 변이체 |
WO2013106789A1 (en) | 2012-01-12 | 2013-07-18 | Biogen Idec Ma Inc. | Methods of reducing immunogenicity against factor viii in individuals undergoing factor viii therapy |
CN106279436A (zh) * | 2016-08-19 | 2017-01-04 | 安源医药科技(上海)有限公司 | 活化的人凝血因子vii融合蛋白及其制备方法与用途 |
CN107787328A (zh) * | 2015-05-22 | 2018-03-09 | 瑞士杰特贝林生物制品重组设备股份公司 | 用于治疗血友病的截短的血管性血友病因子多肽 |
Family Cites Families (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6103501A (en) * | 1997-11-17 | 2000-08-15 | Washington University | Single chain glycoprotein hormones comprising two β and one α subunits and recombinant production thereof |
AU2004255553B2 (en) * | 2003-06-19 | 2009-08-20 | Genentech, Inc. | Compositions and methods for treating coagulation related disorders |
CN105153313A (zh) * | 2010-02-16 | 2015-12-16 | 诺沃—诺迪斯克有限公司 | 因子viii融合蛋白 |
CN102311495A (zh) * | 2010-06-30 | 2012-01-11 | 上海同科生物科技有限公司 | 新型重组人凝血因子ⅷ及其生产方法 |
US20130017997A1 (en) * | 2010-08-19 | 2013-01-17 | Amunix Operating Inc. | Factor VIII Compositions and Methods of Making and Using Same |
CN104114183A (zh) * | 2011-12-20 | 2014-10-22 | 印第安纳大学研究及科技有限公司 | 用于治疗糖尿病的基于ctp的胰岛素类似物 |
JP2016510319A (ja) * | 2012-12-28 | 2016-04-07 | アッヴィ・インコーポレイテッド | 多価結合タンパク質組成物 |
CN104693270B (zh) * | 2013-12-10 | 2018-10-16 | 清华大学 | 一种用于融合蛋白的连接肽 |
CN117106095A (zh) * | 2014-01-10 | 2023-11-24 | 比奥贝拉蒂治疗公司 | 因子viii嵌合蛋白及其用途 |
DK3177317T3 (en) * | 2014-08-04 | 2020-06-15 | Csl Ltd | Factor viii formulation |
WO2017050820A1 (en) * | 2015-09-22 | 2017-03-30 | Novo Nordisk A/S | Fviii fusion proteins |
CN107759696A (zh) * | 2016-08-19 | 2018-03-06 | 安源医药科技(上海)有限公司 | 人白介素7融合蛋白及其制备方法 |
CN106279437B (zh) * | 2016-08-19 | 2017-10-31 | 安源医药科技(上海)有限公司 | 高糖基化人凝血因子viii融合蛋白及其制备方法与用途 |
CN106256835A (zh) * | 2016-08-19 | 2016-12-28 | 安源医药科技(上海)有限公司 | 高糖基化人生长激素融合蛋白及其制备方法与用途 |
GB201614462D0 (en) * | 2016-08-24 | 2016-10-05 | Univ Sheffield | Clotting factor |
AU2017358861B2 (en) * | 2016-11-11 | 2022-02-17 | CSL Behring Lengnau AG | Truncated von Willebrand Factor polypeptides for treating hemophilia |
-
2018
- 2018-09-26 CN CN201811123918.XA patent/CN110950964B/zh active Active
- 2018-09-26 CN CN202110532012.9A patent/CN113105562B/zh active Active
-
2019
- 2019-09-24 BR BR112021005831-1A patent/BR112021005831A2/pt unknown
- 2019-09-24 WO PCT/CN2019/107432 patent/WO2020063562A1/zh unknown
- 2019-09-24 CN CN201980059489.0A patent/CN112673026A/zh active Pending
- 2019-09-24 US US17/280,343 patent/US20220033476A1/en active Pending
- 2019-09-24 EP EP19865552.4A patent/EP3858865A4/en active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4399216A (en) | 1980-02-25 | 1983-08-16 | The Trustees Of Columbia University | Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials |
US4818679A (en) | 1985-02-19 | 1989-04-04 | The Trustees Of Columbia University In The City Of New York | Method for recovering mutant cells |
KR101027427B1 (ko) | 2004-11-12 | 2011-04-11 | 젠코어 인코포레이티드 | FcRn에 대하여 증가된 결합력을 갖는 Fc 변이체 |
KR20100099179A (ko) | 2007-12-26 | 2010-09-10 | 젠코어 인코포레이티드 | FcRn에 대한 변경된 결합성을 갖는 Fc 변이체 |
WO2013106789A1 (en) | 2012-01-12 | 2013-07-18 | Biogen Idec Ma Inc. | Methods of reducing immunogenicity against factor viii in individuals undergoing factor viii therapy |
CN107787328A (zh) * | 2015-05-22 | 2018-03-09 | 瑞士杰特贝林生物制品重组设备股份公司 | 用于治疗血友病的截短的血管性血友病因子多肽 |
CN106279436A (zh) * | 2016-08-19 | 2017-01-04 | 安源医药科技(上海)有限公司 | 活化的人凝血因子vii融合蛋白及其制备方法与用途 |
Non-Patent Citations (13)
Title |
---|
DATTA-MANNAN A ET AL., MABS. TAYLOR&FRANCIS, vol. 4, 2012, pages 267 - 273 |
EATON ET AL., BIOCHEMISTRY, vol. 25, 1986, pages 8343 - 8347 |
EFFERIS R ET AL., IMMUNOL REV, vol. 163, 1998, pages 59 - 76 |
FARES F A ET AL., PROC NATL ACAD SCI USA, vol. 89, no. 10, 1992, pages 4304 - 4308 |
OLANDER RM ET AL., DEV BIOL STAND, vol. 86, 1996, pages 338 |
PAUL R ET AL., J BIOL CHEM, vol. 279, 2004, pages 6213 - 6216 |
PAUL RHINTON ET AL., J IMMUNOL, vol. 176, 2006, pages 346 - 356 |
PETERS R T ET AL., J THROMB HAEMOST, vol. 11, no. 1, 2013, pages 670 - 678 |
ROOPENIAN ET AL., NAT. RVIEW IMMUNOLOGY, vol. 7, 2007, pages 715 - 725 |
SANDBERG H ET AL., SEMIN HEMATOL, vol. 38, 2001, pages 4 - 12 |
See also references of EP3858865A4 |
SHIELDS RL ET AL., J BIOL CHEM, vol. 276, no. 9, 2001, pages 6591 - 604 |
TURECEK PL ET AL., HAMOSTASEOLOGIE, vol. 1, 2012, pages 29 - 38 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4130048A4 (en) * | 2020-03-31 | 2024-04-24 | Ampsource Biopharma Shanghai Inc. | METHOD FOR EFFICIENT SEPARATION AND PURIFICATION OF RECOMBINANT HUMAN COAGULATE FACTOR VIII-FC FUSION PROTEIN |
Also Published As
Publication number | Publication date |
---|---|
EP3858865A9 (en) | 2024-02-14 |
EP3858865A1 (en) | 2021-08-04 |
CN113105562B (zh) | 2023-12-01 |
CN112673026A (zh) | 2021-04-16 |
CN110950964B (zh) | 2021-06-18 |
CN110950964A (zh) | 2020-04-03 |
CN113105562A (zh) | 2021-07-13 |
EP3858865A4 (en) | 2023-04-19 |
US20220033476A1 (en) | 2022-02-03 |
BR112021005831A2 (pt) | 2021-07-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11472863B2 (en) | Human coagulation factor IX (FIX) fusion protein, preparation method therefor, and use thereof | |
US11471513B2 (en) | Highly glycosylated human blood-clotting factor VIII fusion protein, and manufacturing method and application of same | |
WO2020063562A1 (zh) | 突变型单链人凝血因子viii融合蛋白及其制备方法与用途 | |
US9493543B2 (en) | Factor VIII fusion protein | |
US9458223B2 (en) | Von willebrand factor variants having improved factor VIII binding affinity | |
US20130183280A1 (en) | Stabilized factor viii variants | |
CN103739712A (zh) | 具有延长的体内半衰期的因子viii,冯·维勒布兰德因子或它们的复合物 | |
WO2018032639A1 (zh) | 活化的人凝血因子vii融合蛋白及其制备方法与用途 | |
AU2012228990A1 (en) | Potent and selective inhibitors of Nav1.3 and Nav1.7 | |
US11560436B2 (en) | Anti-VWF D'D3 single-domain antibodies fuse to clotting factors |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 19865552 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112021005831 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 2019865552 Country of ref document: EP Effective date: 20210426 |
|
ENP | Entry into the national phase |
Ref document number: 112021005831 Country of ref document: BR Kind code of ref document: A2 Effective date: 20210325 |