A Factor VIII Fusion with an Inactivated FIX Generates a Biologically Active Molecule with Delayed Clearance in a FVIII/VWF Knockout Mouse Model

Background: Developing a delayed clearance factor VIII for the treatment of haemophilia A is of importance due to the cost and inconvenience associated with current FVIII products which require several intravenous injections each week to maintain therapeutic FVIII levels. While there are a number of extended half-life rFVIII products in development, they only have a ~1.8-fold extension of half-life due to the dominant influence of von Willebrand factor (VWF).

Objective: To generate a long-acting rFVIII through fusion to inactive FIX allowing intermolecular binding of FVIII and FIX as well as a dynamic equilibrium with FVIII being free to bind endogenous FIXa. We hypothesized that the fusion to inactive FIX will protect FVIII from proteolytic inactivation, create steric hindrance for binding to VWF and FVIII clearance receptors and thereby delay the in vivo clearance of FVIII.

Methods: We cloned a novel rFVIII fusion molecule linked through its C-terminus to an inactivated factor IX designated 13A1FVIII. 13A1FVIII was expressed in mammalian cells (CHO) and protein expression confirmed by western blotting. Biological activity was tested using a chromogenic assay and thrombin generation test and clearance examined in VWF/FVIII double knock out mice.

Results: 13A1FVIII protein purification was achieved by affinity based chromatography with >95% purity as judged by silver staining. Purified 13A1FVIII bioactivity was confirmed in vitro using a chromogenic FVIII assay and an activated partial thromboplastin time (aPTT) test. In a PK study using VWF/FVIII double knock out mice, 13A1FVIII exhibited a reduced plasma clearance over Advate rFVIII with terminal half-life of 32hrs vs 12hrs, respectively. The observed 2.7-fold increase in half-life was achieved independent of VWF.

Conclusions: A fusion of FVIII to inactive FIX retains biological activity in two in vitro tests of haemostasis. The fusion protein also demonstrates delayed plasma clearance and an increase in circulating half-life in a haemophiliac mouse model. The results suggest it is possible to delay FVIII clearance independent of VWF using this fusion protein approach.