Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain

One of the major binding sites for factor VIII inhibitors is located within the A2 domain. In this study, phage display technology was used to isolate 2 human monoclonal antibodies, termed VK34 and VK41, directed toward the h eavy chain of factor VIII. The V-H domain of a single-chain variable domain antibody fragment (scFv) VK34 is encoded by germline gene segment DP-10, E pitope-mapping studies revealed that scFv VK34 is directed against amino ac id residues Arg(489)-Ile(508) , a previously identified binding site for fa ctor VIII inhibitors in the A2 domain. ScFv VK34 inhibited factor VIII acti vity with a titer of 280 BU/mg. The V-H domain of VK41 was encoded by germl ine gene segment DP-47, A phage corresponding to VK41 competed with a monoc lonal antibody for binding to amino acid residues Asp(712)-Ala(736) in the acidic region adjacent to the A2 domain. Reactivity of VK41 with a factor V III variant in which we replaced amino acid residues Asp(712)-Ala(736) for the corresponding region of heparin cofactor II was strongly reduced. In ad dition, substitution of Tyr(718,719,723) for Phe abrogated binding of VK41 to factor VIII. ScFv VK41 did not inhibit factor VIII activity. This study not only defines the primary structure of human anti-factor VIII antibodies reactive with the A2 domain, it also describes an antibody with an epitope not previously identified in the antibody repertoire of hemophilia patient s with an inhibitor, (C) 2000 by The American Society of Hematology.