Albumin fusion of the rVWF fragment rD'D3 reduces internalisation by clearance receptor Stabilin-2.

A recombinant VWF D'D3 albumin fusion protein (rD'D3-FP) has been developed to extend the half-life of co-administered coagulation factor VIII (FVIII) for the treatment of haemophilia A. We examined the impact of albumin fusion of rD'D3 on the Stabilin-2 (STAB2) clearance receptor pathway and rD'D3-FP recycling via engagement of the neonatal Fc receptor FcRn as two potential mechanisms for rD'D3-FP half-life extension. Binding and intracellular trafficking of fluorescently labelled rD'D3-FP was examined using human 293-F cells overexpressing STAB2 or in combination with FcRn by flow cytometry and confocal microscopy. Pharmacokinetics of rD'D3-FP in FVIII knock out (KO), FcRn KO and hFcRn Tg32 transgenic mice was also assessed. We show that the interaction of rD'D3-FP and his-tagged rD'D3 with STAB2 is optimal at neutral pH but dissociates under acidic conditions. Whilst albumin fusion appears to hinder interaction of rD'D3-FP with STAB2, STAB2-mediated internalisation and lysosomal degradation of rD'D3-FP could be observed in an in vitro system. Rescue of rD'D3-FP via diversion into Rab11+ recycling endosomes was achieved with the co-expression of STAB2 and FcRn. A significant improvement in rD'D3-FP pharmacokinetics was observed compared to his-tagged rD'D3, however, only a modest improvement in rD'D3-FP half-life extension was observed in hFcRn Tg32 mice compared to FcRn KO and wild-type mice. Whilst FcRn may play a minor role in rD'D3-FP half-life extension in mice, albumin fusion may potentially reduce other rD'D3-FP clearance mechanisms. We thus hypothesise several independent mechanisms that may contribute to rD'D3-FP half-life extension in higher order species.