OC 21.2 - Design of a Superior Factor VIII Mimetic with Membrane Binding Properties and Built-in Reversibility

Background: Emicizumab is a bispecific antibody that augments factor X (FX) activation by factor IXa (FIXa) in the absence of factor VIIIa (FVIIIa) by binding and approximating the substrate FX and protease FIXa. Whereas the intrinsic tenase complex (FVIIIa-FIXa-FX) absolutely requires a membrane surface for assembly, emicizumab lacks any membrane binding ability itself and exhibits significant FXa generation in the absence of membranes. This major distinction likely allows FVIIIa to function at several 100-fold lower concentrations than emicizumab to greatly accelerate the activation of FX.

Aims: We sought to test whether the addition of a membrane binding feature in the monospecific FIXa binding fragment of emicizumab could alone approximate the cofactor activity of the bispecific antibody.

Methods: A single chain antibody fragment of the FIXa binding arm of emicizumab (scFv, VH9VL) was fused with Factor V C2-domain to generate VH9VLC2.

Results: In vitro kinetic analyses with purified proteins showed approximately 22-fold faster activation of FX by VH9VLC2 as compared to VH9VL. Remarkably, despite its inability to bind FX, cofactor mimetic activity of VH9VLC2 was about three-fold higher than that of emicizumab. Potent enhancement of FX activation by VH9VLC2 was also reflected in thrombin generation assays (TGA) in congenital FVIII-deficient plasma. Additionally, the cofactor mimetic activity of VH9VLC2 remained unaffected in FVIII-inhibitor plasma. These results were also replicated in two additional engineered molecules: (1) fusion of the lactadherin C2-domain instead of FV C2-domain to VH9VL, and (2) FIXa specific scFv derived from another bispecific antibody, Mim 8 instead of emicizumab derived scFv. Lastly, the C2 domain-mediated increase in cofactor activity of VH9VLC2 was effectively reversed by an antibody fragment (E9scFv) that impairs the binding of factor V to membranes.

Conclusion(s): Our results provide revealing insights into how a single chain, monospecific, membrane-anchored FVIII mimetic enhances the catalytic activity of FIXa.