Development of rVIIIFc-VWF-XTEN (BIVV001)

rVIIIFc-VWF-XTEN uses several modifications of the human factor VIII molecule to extend its half-life.
(A) Stages in the synthesis of the new molecule. Construct 1 covalently attaches a D1D2DʹD3 (C1099A/C1142A) domain of VWF through immunoglobulin-G1 Fc molecules, which prevents interaction of rFVIIIFc with endogenous full-length VWF. A R1648A mutation in FVIII prevents FVIII processing into heavy and light chains. Construct 2 inserts an XTEN polypeptide into the B-domain region of factor VIII and a shorter XTEN polypeptide between DʹD3 and Fc. Construct 3 replaces the LVPR thrombin cleavage site with an FVIII acidic region 2 (a2) thrombin cleavage site. Construct 4 removes 3 amino acids from the factor VIII-XTEN junction and 9 amino acids from the B-domain linker to avoid potential MCH-Class II binding sites. 
(B) Domain structure of the new molecule, which is generated in HEK293 cells. The two polypeptide chains are introduced separately and linked by disulfide bonds in the Fc region. During intracellular processing, several modifications occur including removal of the D1D2 propeptide.