title: Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries.
abstract: Following replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated anti-idiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitor-specific, high-affinity anti-idiotypes can be isolated from phage-displayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.
authors.forename: Anja
authors.initials: A
authors.lastname: Schmidt
authors.forename: Kerstin
authors.initials: K
authors.lastname: Brettschneider
authors.forename: Jörg
authors.initials: J
authors.lastname: Kahle
authors.forename: Aleksander
authors.initials: A
authors.lastname: Orlowski
authors.forename: Karin
authors.initials: K
authors.lastname: Becker-Peters
authors.forename: Diana
authors.initials: D
authors.lastname: Stichel
authors.forename: Jörg
authors.initials: J
authors.lastname: Schulze
authors.forename: Markus
authors.initials: M
authors.lastname: Braner
authors.forename: Robert
authors.initials: R
authors.lastname: Tampé
authors.forename: Dirk
authors.initials: D
authors.lastname: Schwabe
authors.affiliation: Christoph Königs, Goethe University, Department of Paediatrics, Molecular Haemostasis and Immunodeficiency, 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: D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology* / Q000627:therapeutic use*; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology / Q000627:therapeutic use; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000627:therapeutic use; D000918:Antibody Specificity; D003429:Cross Reactions; D004195:Disease Models, Animal; D018604:Epitope Mapping; D005169:Factor VIII / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology*; D006467:Hemophilia A / Q000235:genetics / Q000276:immunology* / Q000628:therapy*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007108:Immune Tolerance; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use
chemical_list: D000888:Antibodies, Anti-Idiotypic; D058846:Antibodies, Monoclonal, Murine-Derived; D057134:Antibodies, Neutralizing; D019151:Peptide Library; D057127:Single-Chain Antibodies; C078147:F8 protein, human; D005169:Factor VIII
keywords: FVIII; Haemophilia A; anti-idiotypes; inhibitors; phage display