Site-Directed Pegylation of BDD FVIII (BAY 94-9027) Does Not Alter the Ability of the Molecule to Generate Thrombin, Activate Factor X or Become Inactivated By Activated Protein C

Introduction: BAY 94-9027 is a rationally designed B Domain Deleted (BDD) FVIII (Mei , B. et al Blood 2010). A single 60 kDa PEG molecule was attached to amino acids 1804 to increase its circulating half-life, reduce the exposure to epitopes reported to cause immunogenicity in the A3 domain (Shima M. Int. J. Haematol. 2006), while preserving full biological function. The PEGylation technology allows for an extended duration of action with the goal of reducing the number of infusions needed while maintaining protection from bleeds. In a recent clinical trial the molecule has shown significant efficacy for prophylactic treatment in hemophilia A patients (NCT01775618). The objective of this study was to further characterize in vitro BAY 94-9027 using Xase kinetics, activated protein C-dependent inactivation of FVIIIa, and thrombin generation assays.

Methods and Results: Biochemical assays to determine apparent binding of the Factor IXa and Factor VIIIa complex and the Factor X activation kinetics revealed identical in vitro properties of BAY 94-9027 to the BDD FVIII comparator, Refacto AF^Ò. The molecules also performed similarly in activated protein C (aPC) FVIIIa inactivation studies.

BAY 94-9027 was further characterized by thrombin generation assay (TGA) in human hemophilic plasma. The TGAs were triggered using 1pM TF or 100pM XIa to simulate extrinsic and intrinsic activation. With XIa activation no differences were seen in peak thrombin, ETP or lag time between BDD FVIII and BAY 94-9027. The lag time, peak and ETP values also remained similar using TF activation. The XIa initiated TGA gives a more potent thrombin generation response compared to TF and is more sensitive to FVIII activity as judged by the left shift of the dose response curves. The TGAs were repeated while titrating thrombomodulin (TM) to generate aPC. Both BAY 94-9027 and BDD FVIII showed similar inactivation profile in the TM-TGA.

Conclusion: The data demonstrates that the rationally designed site specific PEGylation of FVIII in BAY 94-9027 does not alter the in vitro coagulation properties, tested by the ability of BAY 94-9027 to generate thrombin, activate FX or inactivation by aPC. BAY 94-9027 represents the next generation of treatment in hemophilia, using PEGylation technology to reduce the number of infusions while maintaining FVIII activity.