Abstract 2247Hemophilia A is an X-linked recessive disorder that is caused by a deficiency or defect of factor VIII (fVIII) coagulant protein. The major complication of treatment is the development of anti-fVIII antibodies (inhibitors) in approximately 20–30% of patients with severe hemophilia A. The majority of these inhibitors are directed against the A2 or C2 domains (Prescott R et al. Blood 1997). This study examines the structural and functional diversity of the humoral immune response to the A2 domain of human fVIII.A panel of 24 murine anti-A2 monoclonal antibodies (MAbs) produced in our laboratory plus MAb413 (American Red Cross) and GMA012 (Green Mountain, Burlington, VA) were used in this study. Previous studies have shown that anti-C2 MAbs produced from murine anti-fVIII hybridomas had a similar spectrum of epitopes to those found in inhibitor patient plasmas (Meeks SL et al. Blood 2008). A competition sandwich ELISA with immobilized anti-A2 primary MAb, human fVIII, biotinylated anti-A2 secondary MAb and streptavidin–alkaline phosphatase conjugate for detection was used to determine overlapping epitopes. Each antibody was used as both a capture and detection antibody. Antibody pairs were classified as having non-overlapping or overlapping epitopes based on whether the binding of the secondary antibody was present or absent, respectively. Porcine/human hybrid fVIII proteins were employed in a direct ELISA to fine map the epitopes of the anti-A2 MAbs. The results of both the competition and human/porcine mapping ELISAs were compiled into a Venn diagram describing overlapping epitopes for all MAbs. Functional mapping of the MAbs included fVIII inhibitor titers by modified Bethesda assay, inhibition in a purified intrinsic Xase assay, and inhibition of thrombin cleavage of fVIII. Thrombin activation assays were run with varying concentrations of MAbs, and fVIII cleavage by thrombin was analyzed by SDS-PAGE.The competition ELISA results demonstrated 7 non-overlapping epitopes on the A2 domain of human fVIII (Figure 1). In addition, the human/porcine mapping ELISA revealed that the epitopes of the anti-A2 MAbs covered the majority of the A2 domain. The inhibitor titers of the anti-A2 MAbs ranged from non-inhibitory to 40,000 Bethesda units (BU)/mg IgG (Table). The inhibitory MAbs displayed both type I (greater than 95% inhibition at saturating MAb concentrations) and type II-(incomplete inhibition at saturating MAb concentrations) behavior. MAb413, a group D MAb, noncompetitively inhibits factor VIIIa cofactor activity without affecting thrombin cleavage. 2–54, a group G MAb, inhibits thrombin cleavage of both heavy and light chains. In contrast, 1D4, which overlaps groups B, E, and F, only inhibited light chain cleavage. [Display omitted] Overall these results indicate that the humoral immune response to the A2 domain of fVIII is complex in terms of both structural and functional epitopes. These anti-A2 MAbs were found to target 7 non-overlapping epitopes spanning the majority of the A2 domain. Elucidation of the structural and functional complexity of the anti-A2 repertoire will lead to a better understanding of the pathogenicity of A2 inhibitors.Table:A2 MAb CharacteristicsMAbInhibitor Titer (BU/mg)GroupStructural EpitopeB25100A444–5082G10500B468–484G323000C468–508MAb41321,000D484–5082–934E541–604B664000F604–7402–5433,000G508–541, 604–740 Disclosures:No relevant conflicts of interest to declare.