B-Cell Epitope Mapping of Monoclonal Anti-Factor VIII C2 Domain Inhibitors: Identification of Amino Acids That Contribute Significant Antigen-Antibody Binding Affinity.

The most troublesome clinical complication that can afflict hemophilia A patients who receive factor VIII (FVIII) infusions as replacement therapy is the development of an anti-FVIII immune response, in which antibodies bind to functionally important FVIII surfaces, thereby blocking the pro-coagulant function of this important plasma protein cofactor. These antibodies, commonly referred to as “FVIII inhibitors”, bind primarily to the FVIII A2 and C2 domains and to the C-terminal region of the C1 domain, and inhibitors mapping to other regions have also been seen. There are multiple epitopes on the FVIII C2 domain, reflecting both its immunogenicity/antigenicity and its diverse roles in mediating interactions between FVIII and other molecules. For example, the C2 domain is essential for binding of FVIII to its carrier protein von Willebrand factor (VWF). Proteolytic activation to FVIIIa causes its release from VWF and subsequent binding to negatively charged membrane surfaces, e.g. on activated platelets, whereupon a region that overlaps the VWF binding site contacts the membrane. The C2 domain also interacts with thrombin and factor Xa, which both can activate FVIII. To better understand the basis for FVIII inhibition, and to better delineate functionally important FVIII surfaces, a panel of 56 murine anti-C2 monoclonal antibodies was generated. Competition ELISAs and functional assays were used to classify the antibodies into five groups corresponding to distinct regions on the C2 surface, which comprised a larger number of distinct epitopes (