Multiple VH genes are used to assemble human antibodies directed toward the A3-Cl domains of factor VIII

A well-known complication of factor VIII replacement therapy in patients wi th hemophilia A is the development of inhibitory antibodies. Several studie s have demonstrated the presence of a binding site for factor VIII inhibito rs in the A3 domain. Six different human monoclonal single-chain variable d omain antibody fragments (scFv) directed toward the A3-C1 domains of factor VIII have been isolated, using phage display technology. Sequence analysis revealed that the VH domains of 2 scFv were encoded by germline gene segme nts from the V(H)1 gene family and 4 by germline gene segments belonging to the V(H)3 gene family. Epitope mapping of the scFv was performed, using a series of hybrid factor VIII/factor V light chain fragments. This analysis revealed that 5 of 6 scFv were directed against a region encompassing amino acid sequence Q1778-D1840 in the A3 domain, a previously identified bindin g site for factor VIII inhibitors. Only 2 of 5 scFv directed against amino acid sequence Q1778-D1840 inhibited the procoagulant activity of factor VII I. Our results define the properties of human antibodies directed against r egion Q1778-D1840 in the A3 domain. Binding of one, noninhibitory scFv was independent of the region Q1778-D1840, suggesting the presence of an additi onal binding site for anti-factor VIII antibodies in the A3-C1 domains of f actor VIII. (C) 2001 by The American Society of Hematology.