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 heavy 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. Epitope-mapping studies revealed that scFv VK34 is directed against amino acid residues Arg(484)-Ile(508), 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 V(H) 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 Asp(712)-Ala(736) in the acidic region adjacent to the A2 domain. Reactivity of VK41 with a factor VIII variant in which we replaced amino acid residues Asp(712)-Ala(736) for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr(718719723) 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. (Blood. 2000;96:540-545)
van den Brink
E N
EN
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.
Turenhout
E A
EA
Bank
C M
CM
Fijnvandraat
K
K
Peters
M
M
Voorberg
J
J
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics*; D000918:Antibody Specificity; D001435:Bacteriophages / Q000235:genetics; D001666:Binding Sites, Antibody; D018604:Epitope Mapping; D004926:Escherichia coli; D005169:Factor VIII / Q000737:chemistry / Q000276:immunology*; D006467:Hemophilia A / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D057127:Single-Chain Antibodies
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; C411901:ScFv VK34; C411900:ScFv VK41; D057127:Single-Chain Antibodies; D005169:Factor VIII