A Novel Single Chain Recombinant Factor VIII Fc Fusion Protein Is Active In Vitro and Fully Efficacious in An In Vivo Hema Mouse Bleeding Model

Abstract 1192 Recombinant factor VIIIFc (rFVIIIFc) is comprised of a B domain deleted (BDD) rFVIII protein genetically fused to the Fc domain of human immunoglobulin G1 (IgG1). Prior to secretion from HEK 293 cells, most of the rFVIIIFc is processed into a FVIII heavy chain (HC) and light chain (LC+Fc). In circulation, rFVIIIFc is complexed with von Willebrand factor (VWF) and released upon activation in a manner that is indistinguishable from native FVIII. Spontaneous dissociation of the HC and LC is thought to contribute to the loss of FVIII activity in plasma and during storage of FVIII drug products. Here we describe a single chain non-processed isoform of rFVIIIFc (SC rFVIIIFc), which may provide superior manufacturability and enhanced stability compared to native FVIII. SC rFVIIIFc was purified from rFVIIIFc, which contains a fraction of the non-processed isoform. Compared to rFVIIIFc, SC rFVIIIFc showed equivalent chromogenic activity but approximately 60% reduced activity by the one stage (aPTT) assay. In a thrombin generation assay (TGA), SC rFVIIIFc also showed a reduced thrombin potential, peak thrombin and slope compared to rFVIIIFc. However, full activity of SC rFVIIIFc by aPTT was observed in the absence of VWF, suggesting release from VWF may be delayed due to covalent linkage of the a3 acidic region to the HC after Arg 1680 cleavage in SC rFVIIIFc, in contrast to a3 release and dissociation from fully processed FVIII. Delayed dissociation from VWF may explain the reduced activity observed in the aPTT assay and TGA, while full activity was observed in the two-stage chromogenic assay. A reduced rate of activation in the presence of VWF was confirmed in a modified chromogenic substrate assay with limiting thrombin as FVIII activator. In vivo function of SC rFVIIIFc was assessed in the HemA mouse tail vein transection (TVT) model. SC rFVIIIFc and the rFVIIIFc demonstrated equivalent in vivo efficacy in this model, with an ED 50 of 1.17 μg/kg and 1.23 μg /kg respectively when TVT was performed at 48 hours post infusion. Comparable post TVT survival curves ( p ≥ 0.65) were observed for the SC rFVIIIFc and rFVIIIFc at each tested dose level (Figure 1), indicating that SC rFVIIIFc was equally effective as rFVIIIFc despite its lower apparent aPTT activity. The delayed in vitro activation of SC rFVIIIFc in the presence of vWF therefore appears to have no significant impact on its in vivo efficacy. Thus, SC rFVIIIFc represents a novel and efficacious isoform of rFVIIIFc with potential clinical applications. Further studies will be required to demonstrate enhanced product stability in the context of this Fc fusion protein. Disclosures: Buyue: Biogen Idec Hemophilia: Employment. Liu: Biogen Idec Hemophilia: Employment. Reidy: Biogen Idec Hemophilia: Employment. Patarroyo-White: Biogen Idec Hemophilia: Employment. Kamphaus: Biogen Idec Hemophilia: Employment. Slein: Biogen Idec Hemophilia: Employment. Pierce: Biogen Idec Hemophilia: Employment. Sommer: Biogen Idec Hemophilia: Employment. Peters: Biogen Idec Hemophilia: Employment.