A recombinant factor VIII A2 domain polypeptide quantitatively neutralizes human inhibitor antibodies that bind to A2

Human antibodies that inactivate coagulation factor VIII (fVIII), known as inhibitors, have been shown by immunoblotting or immunoprecipitation assays to bind predominantly to epitopes within the A2 and/or C2 domains of the fVIII protein. Because these assays simply measure antibody binding, a soluble recombinant polypeptide containing the fVIII A2 domain was used to develop a quantitative inhibitor neutralization assay for antibodies that bound only to A2 by immunoblotting assay. Antibodies from six of eight inhibitor plasmas were fully neutralized by A2 (> or = 90%), whereas two were only partially neutralized. These results established the fVIII inhibitor properties of anti-A2 antibodies. In immunoprecipitation assays, five of the eight inhibitors also had significant levels of anti-light-chain antibody. In one case, this light-chain antibody was shown to have inhibitor activity. Because it did not bind to the C2 domain, this antibody appears to define a new inhibitor epitope within the fVIII light chain. Another inhibitor, which was partially neutralized by A2, was not neutralized by the light chain, even though it contained anti-light-chain antibodies by immunoprecipitation assay. Our results show additional complexities of the immune response to fVIII.