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PEDS Advance Access originally published online on April 8, 2005
Protein Engineering Design and Selection 2005 18(3):111-118; doi:10.1093/protein/gzi021
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org

Improvement of Fc–erythropoietin structure and pharmacokinetics by modification at a disulfide bond

Jeffrey C. Way1, Scott Lauder, Beatrice Brunkhorst, Su-Ming Kong, An Qi, Gordon Webster, Islay Campbell, Sue McKenzie, Yan Lan, Bo Marelli, Lieu Anh Nguyen, Steven Degon, Kin-Ming Lo and Stephen D. Gillies

EMD Lexigen Research Center Corp., 45A Middlesex Turnpike, Billerica, MA 01821, USA

1 To whom correspondence should be addressed. E-mail: jway{at}emdlexigen.com

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.

Keywords: erythropoietin/disulfide bond engineering/pharmacokinetics/protein design

Received February 10, 2004; revised February 27, 2005; accepted March 9, 2005.

Edited by Mark Zoller


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