Rational design of a fully active, long-acting PEGylated factor VIII for hemophilia A treatment.

A long-acting factor VIII (FVIII) as a replacement therapy for hemophilia A would significantly improve treatment options for patients with hemophilia A. To develop a FVIII with an extended circulating half-life, but without a reduction in activity, we have engineered 23 FVIII variants with introduced surface-exposed cysteines to which a polyethylene glycol (PEG) polymer was specifically conjugated. Screening of variant expression level, PEGylation yield, and functional assay identified several conjugates retaining full in vitro coagulation activity and von Willebrand factor (VWF) binding.PEGylated FVIII variants exhibited improved pharmacokinetics in hemophilic mice and rabbits. In addition, pharmacokinetic studies in VWF knockout mice indicated that larger molecular weight PEG may substitute for VWF in protecting PEGylated FVIII from clearance in vivo. In bleeding models of hemophilic mice, PEGylated FVIII not only exhibited prolonged efficacy that is consistent with the improved pharmacokinetics but also showed efficacy in stopping acute bleeds comparable with that of unmodified rFVIII. In summary site-specifically PEGylated FVIII has the potential to be a long-acting prophylactic treatment while being fully efficacious for on-demand treatment for patients with hemophilia A.