Binding of therapeutic Fc-fused factor VIII to the neonatal Fc receptor at neutral pH associates with poor half-life extension.

Fusion of therapeutic proteins to the Fc fragment of human immunoglobulin (Ig) G1 promotes their neonatal Fc receptor (FcRn)-mediated recycling and subsequent extension in circulating half-life. However, different Fc-fused proteins, as well as antibodies with different variable domains but identical Fc, may differ in terms of extension in half-life. Here we compared the binding behavior to FcRn of Fc-fused FVIII, Fc-fused FIX and two human monoclonal HIV-1 broadly-neutralizing IgG1, m66.6 and VRC01 with identical Fc. While all molecules bound FcRn at acidic pH, only rFVIIIFc and m66.6 interacted with FcRn at neutral pH. In silico modeling predicted a role for charged residues in the C1 and C2 domains of FVIII, and in the variable domains of m66.6, in the neutral binding to FcRn. Accordingly, mutations of key positively charged amino-acids in the FVIII C1C2 domains decreased the binding of the protein to FcRn at pH 7.4 in vitro and increased the half-life of rFVIIIFc in von Willebrand factor- knockout mice. Our findings suggest that the removal of positively charged patches on Fc-fused proteins to ameliorate FcRn recycling without affecting therapeutic efficacy, may improve their pharmacokinetic properties.