Regular Article THROMBOSIS AND HEMOSTASIS High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance

• Amino acid residues comprising B-cell epitopes recognized by neutralizing antifactor VIII antibodies (inhibitors) have been identified. • Amino acids contributing significant antigen–antibody binding avidity are candidates for mutagenesis in the design of less antigenic proteins. Neutralizing anti-factor VIII (FVIII) antibodies that develop in patients with hemophilia A and inmurinehemophiliaAmodels, clinically termed“inhibitors,” bind to several distinct surfaces on the FVIII-C2 domain. Tomap these epitopes at high resolution, 60 recombinant FVIII-C2 proteinswere generated, each having a single surface-exposed residuemutated to alanine or a conservative substitution. The binding kinetics of these muteins to 11 monoclonal, inhibitory anti-FVIII-C2 antibodies were evaluated by surface plasmon resonance and the results compared with those obtained for wild-type FVIII-C2. Clusters of residues with significantly altered binding kinetics identified “functional” B-cell epitopes, defined as those residues contributing appreciable antigen–antibody avidity. These antibodies were previously shown to neutralize FVIII activity by interfering with proteolytic activationof FVIII by thrombin or factor Xa, or with its binding to phospholipid surfaces, von Willebrand factor, or other components of the intrinsic tenase complex. Finemappingof epitopesby surfaceplasmon resonance also indicated surfaces through which FVIII interacts with proteins and phospholipids as it participates in coagulation. Mutations that significantly altered the dissociation times/half-lives identified functionally important interactions within antigen– antibody interfaces and suggested specific sequence modifications to generate novel, less antigenic FVIII proteins with possible therapeutic potential for treatment of inhibitor patients. (Blood. 2014;123(17):2732-2739)