title: Two classes of germline genes both derived from the V(H)1 family direct the formation of human antibodies that recognize distinct antigenic sites in the C2 domain of factor VIII.
abstract: Most plasmas from patients with inhibitors contain antibodies that are reactive with the C2 domain of factor VIII. Previously, we have shown that the variable heavy chain (V(H)) regions of antibodies to the C2 domain are encoded by the closely related germline gene segments DP-10, DP-14, and DP-88, which all belong to the V(H)1 gene family. Here, we report on the isolation and characterization of additional anti-C2 antibodies that are derived from V(H) gene segments DP-88 and DP-5. Competition experiments using murine monoclonal antibodies CLB-CAg 117 and ESH4 demonstrated that antibodies derived from DP-5 and DP-88 bound to different sites within the C2 domain. Epitope mapping studies using a series of factor VIII/factor V hybrids revealed that residues 2223 to 2332 of factor VIII are required for binding of the DP-10-, DP-14-, and DP-88-encoded antibodies. In contrast, binding of the DP-5-encoded antibodies required residues in both the amino- and carboxy-terminus of the C2 domain. Inspection of the reactivity of the antibodies with a series of human/porcine hybrids yielded similar data. Binding of antibodies derived from germline gene segments DP-10, DP-14, and DP-88 was unaffected by replacement of residues 2181 to 2243 of human factor VIII for the porcine sequence, whereas binding of the DP-5-encoded antibodies was abrogated by this replacement. Together these data indicate that antibodies assembled from V(H) gene segments DP-5 and the closely related germline gene segments DP-10, DP-14, and DP-88 target 2 distinct antigenic sites in the C2 domain of factor VIII.
authors.affiliation: Department of Plasma Proteins, CLB, Amsterdam, the Netherlands.
authors.forename: Edward N
authors.initials: EN
authors.lastname: van den Brink
authors.forename: Wendy S
authors.initials: WS
authors.lastname: Bril
authors.forename: Ellen A M
authors.initials: EA
authors.lastname: Turenhout
authors.forename: Marleen
authors.initials: M
authors.lastname: Zuurveld
authors.forename: Niels
authors.initials: N
authors.lastname: Bovenschen
authors.forename: Marjolein
authors.initials: M
authors.lastname: Peters
authors.forename: Thynn Thynn
authors.initials: TT
authors.lastname: Yee
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Lewis
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Ortel
authors.forename: Pete
authors.initials: P
authors.lastname: Lollar
authors.forename: Dorothea
authors.initials: D
authors.lastname: Scandella
authors.forename: Jan
authors.initials: J
authors.lastname: Voorberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D019143:Evolution, Molecular; D005169:Factor VIII / Q000737:chemistry / Q000276:immunology*; D005803:Genes, Immunoglobulin / Q000276:immunology*; D006467:Hemophilia A / Q000276:immunology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007518:Isoantibodies / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D005810:Multigene Family; D017434:Protein Structure, Tertiary
chemical_list: D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007518:Isoantibodies; D005169:Factor VIII