A bispecific nanobody for the treatment of von Willebrand disease type 1

ABSTRACT: von Willebrand disease (VWD) type 1 is a bleeding disorder characterized by a quantitative deficiency of functional von Willebrand factor (VWF). We designed a novel bispecific nanobody, named KB-V13A12, that aims to increase endogenous VWF levels by bridging it to albumin. KB-V13A12 comprises 2 single-domain antibodies, 1 targeting VWF and 1 targeting albumin. VWF bound efficiently to the albumin/KB-V13A12 complex (2.0 ± 0.4 nM) in immunosorbent assays, and binding was stable at pH 5.6 and 7.4. VWF ristocetin activity and factor VIII binding remained unaffected in the presence of a 100- to 200-fold molar excess of KB-V13A12/albumin. Humanized VWD type 1 mice were used for in vivo analysis. A single subcutaneous dose of KB-V13A12 (5 mg/kg) was associated with a nanobody half-life of 3.0 ± 0.7 days, and dose-dependently increased VWF in VWD type 1 mice 1.4- to 2.1-fold for up to 14 days. Factor VIII activity was also increased during this period. The VWF propeptide/VWF antigen ratio (a marker for VWF clearance) was significantly reduced in the presence of KB-V13A12, suggesting that delayed clearance contributes to increased VWF levels. Clearance experiments in wild-type mice using recombinant VWF preincubated with KB-V13A12 indeed confirmed a prolonged survival, while this prolongation was absent in FcRn-deficient mice. Finally, treatment with KB-V13A12 resulted in a significantly improved bleeding tendency in VWD type 1 mice when using the saphenous vein puncture model. In conclusion, KB-V13A12 is a bispecific nanobody that efficiently increases functional levels of endogenous VWF, and could be a therapeutic option to treat VWD type 1.