title: Epitope mapping via selection of anti-FVIII antibody-specific phage-presented peptide ligands that mimic the antibody binding sites.
abstract: The most serious complication in today's treatment of congenital haemophilia A is the development of neutralising antibodies (inhibitors) against factor VIII (FVIII). Although FVIII inhibitors can be eliminated by immune tolerance induction (ITI) based on repeated administration of high doses of FVIII, 20-30% of patients fail to become tolerant. Persistence of FVIII inhibitors is associated with increased morbidity and mortality. Data from recent studies provide evidence for a potential association between ITI outcome and epitope specificity of FVIII inhibitors. Nevertheless the determination of epitopes and their clinical relevance has not yet been established. In this study a general strategy for the identification of anti-FVIII antibody epitopes in haemophilia A patient plasma was to be demonstrated. Phage-displayed peptide libraries were screened against anti-FVIII antibodies to isolate specific peptides. Peptide specificity was confirmed by FVIII-sensitive ELISA binding. Peptide residues essential for antibody binding were identified by mutational analysis and epitopes were predicted via FVIII homology search. The proposed mapping strategy was validated for the monoclonal murine antibody (mAb) 2-76. Binding studies with FVIII variants confirmed the location of the predicted epitope at the level of individual amino acids. In addition, anti-FVIII antibody-specific peptide ligands were selected for 10 haemophilia A patients with FVIII inhibitors. Detailed epitope mapping for three of them showed binding sites on the A2, A3 and C2 domains. Precise epitope mapping of anti-FVIII antibodies using antibody-specific peptide ligands can be a useful approach to identify antigenic sites on FVIII.
authors.forename: Joerg
authors.initials: J
authors.lastname: Kahle
authors.forename: Aleksander
authors.initials: A
authors.lastname: Orlowski
authors.forename: Diana
authors.initials: D
authors.lastname: Stichel
authors.forename: Karin
authors.initials: K
authors.lastname: Becker-Peters
authors.forename: Ali
authors.initials: A
authors.lastname: Kabiri
authors.forename: John F
authors.initials: JF
authors.lastname: Healey
authors.forename: Kerstin
authors.initials: K
authors.lastname: Brettschneider
authors.forename: Anja
authors.initials: A
authors.lastname: Naumann
authors.forename: Anna K
authors.initials: AK
authors.lastname: Scherger
authors.forename: Pete
authors.initials: P
authors.lastname: Lollar
authors.forename: Dirk
authors.initials: D
authors.lastname: Schwabe
authors.affiliation: Christoph Königs, MD PhD, University Hospital Frankfurt, Department of Paediatrics, Clinical and Molecular Haemostasis, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany, Tel.: +49 69 6301 83030, Fax: +49 69 6301 83991, E-mail: christoph.koenigs@kgu.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Königs
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001666:Binding Sites, Antibody; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry; D005169:Factor VIII / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000276:immunology; D057809:HEK293 Cells; D006467:Hemophilia A / Q000097:blood* / Q000188:drug therapy* / Q000276:immunology; D006801:Humans; D007107:Immune System; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013552:Swine
chemical_list: D000939:Epitopes; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D005169:Factor VIII
keywords: Haemophilia A; anti-FVIII antibodies; epitope mapping; inhibitors; phage display