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A factor VIII-nanobody fusion protein forming an ultra-stable complex with VWF: effect on clearance & antibody formation

Vincent Muczynski, Caterina Casari, François Moreau, Gabriel Aymé, Charlotte Kawecki, Paulette Legendre, Valerie Proulle, Olivier D. Christophe, Cécile V. Denis and Peter J. Lenting

Key points

  • The fusion between factor VIII and anti-VWF nanobodies increases affinity for VWF 25-fold without compromising FVIII activity.

  • Stabilized VWF binding results in a 2-fold enhanced circulatory survival of FVIII and reduced anti-FVIII antibody formation.

Abstract

Von Willebrand factor (VWF) modulates factor VIII (FVIII) clearance and the anti-FVIII immune response. Despite the high affinity that defines the FVIII/VWF interaction, association/dissociation kinetics dictates 2-5% FVIII being present as free protein. To avoid free FVIII when studying the FVIII-VWF complex in vivo, we designed a FVIII-nanobody fusion protein, with the nanobody part being directed against VWF. This fusion protein, designated FVIII-KB013bv, had a 25-fold higher affinity compared to B-domainless FVIII (BDD-FVIII) for VWF. In vitro analysis revealed full cofactor activity in one-stage clotting and chromogenic assays (activity/antigen ratio 1.0±0.3 and 1.1±0.3, respectively). In vivo, FVIII-013bv displayed a two-fold increased mean residence time compared to BDD-FVIII (3.0h versus 1.6h). In a tail clip-bleeding assay performed 24h after FVIII infusion, blood loss was significantly reduced in mice receiving FVIII-KB013bv versus BDD-FVIII (15±7 microliter versus 194±146 microliter; p=0.0043). Unexpectedly, when examining anti-FVIII antibody formation in FVIII-deficient mice, the immune-response towards FVIII-KB013bv was significantly reduced compared to BDD-FVIII (1/8 versus 14/16 mice produced anti-FVIII antibodies after treatment with FVIII-KB013bv and BDD-FVIII, respectively). Our data show that a stabilized interaction between FVIII and VWF is associated with a prolonged survival of FVIII and a reduced immune response against FVIII.

  • Submitted January 24, 2018.
  • Accepted July 25, 2018.