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

• Published in: Blood Vol. 96; no. 2; pp. 540 - 545
• Main Authors: VAN DEN BRINK, E. N, TURENHOUT, E. A. M, BANK, C. M. C, FIJNVANDRAAT, K, PETERS, M, VOORBERG, J
• Format: Journal Article
• Language: English
• Published: Washington, DC The Americain Society of Hematology 15.07.2000
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - genetics; Antibody Specificity; Bacteriophages - genetics; Binding Sites, Antibody; Biological and medical sciences; Epitope Mapping; Escherichia coli; Factor VIII - chemistry; Factor VIII - immunology; Hematologic and hematopoietic diseases; Hemophilia A - immunology; Humans; Immunoglobulin Fragments - chemistry; Immunoglobulin Fragments - genetics; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Medical sciences; Molecular Sequence Data; Peptide Library; Platelet diseases and coagulopathies; Recombinant Proteins - chemistry; Recombinant Proteins - immunology; Single-Chain Antibodies; Human; Blood coagulation; Antigenic determinant; Molecular structure; Antibody; Factor VIII; Hemopathy; Monoclonal antibody; Hemophilia A; Genetic disease; Phage display; Inhibitor; Molecular domain; Coagulopathy
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V96.2.540

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)