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.

Targeted erythropoietin selectively stimulates red blood cell expansion in vivo

“…Binding of 10F7-EPO R150A to huGYPA reduces its maximal plasma concentration (C max ) and increases its terminal plasma half-life. EPO pharmacokinetics can be influenced by receptor binding, glycosylation, and molecular weight, which affect clearance through receptor-mediated endocytosis by EPO-Rs, liver asialoglycoprotein receptors, and kidney filtration, respectively (13,29,34). Moreover, binding to huGYPA on mature RBCs is expected to create a sink effect (35), through which most of 10F7-EPO R150A should equilibrate with the free plasma state.…”

“…Comparisons between treatments were done using Student's t test. *P < 0.1; ***P < 0.005. and binding to asialoglycoprotein receptors should remove only a subpopulation of drug molecules (29). Finally, clearance of RBCbound drug via splenic apoptosis should be slow (36).…”

Novel Erythropoiesis-Stimulating Agents

“…This EPO-GM-CSF complex proved to be able to stimulate erythropoiesis in cynomolgus monkeys (28) but was later found to induce anti-EPO antibodies, causing severe anemia (29). Yet another approach is the genetic fusion of EPO with the Fc region of human IgG (Fc-EPO) (30). This molecular modification promotes recycling out of the cell upon endocytosis via the Fc recycling receptor (31,32), again providing an alternative mechanism for enhancing circulating half-life.…”

Development of Recombinant Erythropoietin and Erythropoietin Analogs

“…This EPO-GM-CSF complex proved to be able to stimulate erythropoiesis in cynomolgus monkeys (Coscarella et al, 1998a), but was later found to induce anti-erythropoietin antibodies, causing severe anemia (Coscarella et al, 1998b). Yet another approach is the genetic fusion of EPO with the Fc region of human immunoglobulin G (Fc-EPO) (Way et al, 2005). This molecular modification promotes recycling out of the cell upon endocytosis via the Fc recycling receptor (Capon et al, 1989;Yeh et al, 1992), again providing an alternative mechanism for enhancing circulating half-life.…”