Evidence For Flexible Tethering Of Fc To FVIII In Recombinant FVIII-Fc Fusion Protein rFVIIIFc
Authors: Leksa Nina , Liu Lucy , Goodman Allison , Chiu Po-lin , Walz Thomas , Peters Robert , Kulman John 
Published: October 2019

Abstract

Introduction
Hemophilia A is characterized by a deficiency in factor VIII (FVIII) activity, resulting in failure of blood to clot properly, leading to traumatic and spontaneous bleeding, particularly into joints and soft tissues. Current treatment strategies rely on intravenous administration of FVIII, but require frequent dosing owing to the relatively short half-life of FVIII. We previously reported the development of a recombinant FVIIIFc fusion protein (rFVIIIFc), that shows an approximate 1.5-fold increase in half-life relative to otherwise unmodified FVIII, potentially enabling less frequent dosing compared to rFVIII. Both FVIII and rFVIIIFc exhibit comparable specific activities and similar affinities for von Willebrand factor (VWF). Furthermore, hydrogen-deuterium exchange (H/Dx) analysis established structural comparability of rFVIII and the FVIII component of rFVIIIFc, while small angle x-ray scattering (SAXS) results suggested that the Fc is flexibly tethered to the C-terminus of FVIII, leaving the regions of FVIII necessary for interaction with phospholipid, VWF, FIXa, and FX binding sterically accessible. Consistent with these findings, the crystal structure of rFVIIIFc shows that the FVIII structure is unaffected by the appended Fc domain, while the Fc domain itself is not visible, suggesting conformational freedom.

Aims
The goal of this study is to further compare the structure of rFVIII with that of the FVIII component of rFVIIIFc by evaluating the affinities of a panel of anti-FVIII antibodies having structurally distinct epitopes. Additionally, we assess the conformational flexibility of the Fc component of rFVIIIFc using negative stain electron microscopy.

Methods
Thirty-nine anti-FVIII mouse monoclonal antibodies, 14 of which are being reported here for the first time, were evaluated by surface plasmon resonance (SPR) to determine their affinities for both rFVIII and rFVIIIFc. Affinities were then compared to assess whether the respective epitopes, which collectively span the 5 structural domains of FVIII, are conformationally conserved between FVIII and rFVIIIFc. Subsequently, Fab fragments were generated for two of these antibodies, GMA-8015 and ESH8, and the structures of the complexes formed between these Fabs and FVIII were visualized by negative stain EM. The structure of rFVIIIFc was determined by negative stain EM to determine the positional variability of the Fc element with respect to the FVIII component of rFVIIIFc.

Results and Conclusion
The affinities of all 39 antibodies for rFVIII and rFVIIIFc, expressed in terms of KD, spanned the picomolar to nanomolar range and each showed comparable affinities for rFVIII and rFVIIIFc. Among these are 13 antibodies which are known to compete with VWF for FVIII binding, providing further evidence that the structural determinants of VWF binding are the same in both molecules. As expected, the GMA-8015 and ESH8 Fabs bound to the A2 and C2 domains of FVIII, respectively, and were clearly visible in EM images. For rFVIIIFc, particles were grouped into 100 classes, and the resulting class averages revealed a high degree of conformational freedom between FVIII and the appended Fc domain, while the gross morphology of the FVIII component remained unchanged. Notably, rFVIII and rFVIIIFc exhibited similar affinities for ESH8, despite the close proximity of the ESH8 epitope and the Fc fusion site in the C2 domain at the C-terminus of FVIII, indicating flexible tethering between FVIII and the Fc domain. These findings provide further mechanistic insight into the observed functional similarity of FVIII and rFVIIIFc with respect to cofactor activity and VWF binding.

Disclosures:
Leksa: Biogen Idec: Employment, Equity Ownership. Liu:Biogen Idec: Employment, Equity Ownership. Goodman:Biogen Idec: Employment, Equity Ownership. Chiu:Biogen Idec: Research Funding. Walz:Biogen Idec: Honoraria, Research Funding. Peters:Biogen Idec: Employment, Equity Ownership. Kulman:Biogen Idec: Employment, Equity Ownership.