Multiple VH genes are used to assemble human antibodies directed toward the A3-C1 domains of factor VIII

Blood. 2001 Feb 15;97(4):966-72. doi: 10.1182/blood.v97.4.966.

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.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Antibody Specificity
  • Epitopes / chemistry
  • Epitopes / immunology*
  • Factor VIII / chemistry
  • Factor VIII / immunology*
  • Gene Rearrangement, B-Lymphocyte, Heavy Chain*
  • Genes, Immunoglobulin*
  • Humans
  • Immunoglobulin Heavy Chains / genetics*
  • Immunoglobulin Light Chains / genetics
  • Immunoglobulin Variable Region / genetics*
  • Immunoglobulin Variable Region / immunology
  • Isoantibodies / chemistry
  • Isoantibodies / immunology*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Library
  • Protein Structure, Tertiary
  • Recombinant Proteins / immunology
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Surface Plasmon Resonance

Substances

  • Epitopes
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Light Chains
  • Immunoglobulin Variable Region
  • Isoantibodies
  • Peptide Library
  • Recombinant Proteins
  • Factor VIII