title: Multiple VH genes are used to assemble human antibodies directed toward the A3-C1 domains of factor VIII.
abstract: A well-known complication of factor VIII replacement therapy in patients with hemophilia A is the development of inhibitory antibodies. Several studies have demonstrated the presence of a binding site for factor VIII inhibitors in the A3 domain. Six different human monoclonal single-chain variable domain antibody fragments (scFv) directed toward the A3-C1 domains of factor VIII have been isolated, using phage display technology. Sequence analysis revealed that the V(H) domains of 2 scFv were encoded by germline gene segments 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 binding site for factor VIII inhibitors. Only 2 of 5 scFv directed against amino acid sequence Q1778-D1840 inhibited the procoagulant activity of factor VIII. Our results define the properties of human antibodies directed against region Q1778-D1840 in the A3 domain. Binding of one, noninhibitory scFv was independent of the region Q1778-D1840, suggesting the presence of an additional binding site for anti-factor VIII antibodies in the A3-C1 domains of factor VIII.
authors.affiliation: Department of Plasma Proteins, CLB, Amsterdam, The Netherlands.
authors.forename: E N
authors.initials: EN
authors.lastname: van den Brink
authors.forename: E A
authors.initials: EA
authors.lastname: Turenhout
authors.forename: N
authors.initials: N
authors.lastname: Bovenschen
authors.forename: B G
authors.initials: BG
authors.lastname: Heijnen
authors.forename: K
authors.initials: K
authors.lastname: Mertens
authors.forename: M
authors.initials: M
authors.lastname: Peters
authors.forename: J
authors.initials: J
authors.lastname: Voorberg
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005169:Factor VIII / Q000737:chemistry / Q000276:immunology*; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D007518:Isoantibodies / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000276:immunology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
chemical_list: D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007518:Isoantibodies; D019151:Peptide Library; D011994:Recombinant Proteins; D005169:Factor VIII