title: Improvement of Fc-erythropoietin structure and pharmacokinetics by modification at a disulfide bond.
abstract: Erythropoietin (Epo) is a cytokine that controls the production of red blood cells (RBCs). Epo acts continuously on RBC precursors to prevent apoptosis, so it is important to maintain high levels of Epo activity when treating anemic patients. We describe here modified human Epo [Epo(NDS)] with mutations His32Gly, Cys33Pro, Trp88Cys and Pro90Ala that result in the rearrangement of the disulfide bonding pattern from Cys29-Cys33 to Cys29-Cys88 and that, in the context of an Fc-Epo(NDS) fusion protein, lead to significantly improved properties. Fc-Epo was secreted from NS/0 myeloma cells as about 35% high molecular weight aggregates, was unstable upon removal of N-linked oligosaccharides and showed poor pharmacokinetics and little efficacy in mice. In contrast, a corresponding Fc-Epo(NDS) was secreted almost exclusively as a unit dimer, was relatively stable to removal of N-linked oligosaccharides, had much improved pharmacokinetic properties and had a significantly improved effect on RBC production. These results indicate that rearrangement of the disulfide bonding pattern in a therapeutic protein can have a significant effect on pharmacokinetics and, potentially, the dosing schedule of a protein drug.
authors.affiliation: EMD Lexigen Research Center Corp., 45A Middlesex Turnpike, Billerica, MA 01821, USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Way
authors.forename: Scott
authors.initials: S
authors.lastname: Lauder
authors.forename: Beatrice
authors.initials: B
authors.lastname: Brunkhorst
authors.forename: Su-Ming
authors.initials: SM
authors.lastname: Kong
authors.forename: An
authors.initials: A
authors.lastname: Qi
authors.forename: Gordon
authors.initials: G
authors.lastname: Webster
authors.forename: Islay
authors.initials: I
authors.lastname: Campbell
authors.forename: Sue
authors.initials: S
authors.lastname: McKenzie
authors.forename: Yan
authors.initials: Y
authors.lastname: Lan
authors.forename: Bo
authors.initials: B
authors.lastname: Marelli
authors.forename: Lieu Anh
authors.initials: LA
authors.lastname: Nguyen
authors.forename: Steven
authors.initials: S
authors.lastname: Degon
authors.forename: Kin-Ming
authors.initials: KM
authors.lastname: Lo
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Gillies
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D002417:Cattle; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D011961:Receptors, Fc / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000737:chemistry
chemical_list: D003062:Codon; D004220:Disulfides; D009844:Oligosaccharides; D010455:Peptides; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D014357:Trypsin; D003545:Cysteine