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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 540-545
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
Edward N. van den Brink,
Ellen A. M. Turenhout,
Christine M. C. Bank,
Karin Fijnvandraat,
Marjolein Peters, and
Jan Voorberg
From the Department of Plasma Proteins, CLB, the Laboratory for
Experimental and Clinical Immunology, Academic Medical Centre,
University of Amsterdam, and the Emma Children's Hospital AMC,
Amsterdam, The Netherlands.
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 heavy chain of factor VIII. The VH
domain of a single-chain variable domain antibody fragment (scFv) VK34
is encoded by germline gene segment DP-10. Epitope-mapping studies
revealed that scFv VK34 is directed against amino acid residues
Arg484-Ile508 , a previously identified
binding site for factor VIII inhibitors in the A2 domain. ScFv VK34
inhibited factor VIII activity with a titer of 280 BU/mg. The
VH domain of VK41 was encoded by germline gene segment
DP-47. A phage corresponding to VK41 competed with a monoclonal
antibody for binding to amino acid residues
Asp712-Ala736 in the acidic region adjacent to
the A2 domain. Reactivity of VK41 with a factor VIII variant in which
we replaced amino acid residues Asp712-Ala736
for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr718719723 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 patients with an inhibitor.
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