Abstract
Abstract 2247
Hemophilia 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.
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.
Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.