title: Selection and characterisation of FVIII-specific single chain variable fragments.
abstract: The development of inhibitory anti-FVIII antibodies is currently the most severe complication in the treatment of haemophilia A patients. Inhibitor eradication can be achieved by immune tolerance induction (ITI). Recent findings suggest a correlation between the FVIII-specific IgG subclass distribution and the duration or outcome of ITI. To quantify FVIII-specific IgG subclasses in patients' plasma FVIII-specific IgG standards are required. Here, the isolation of FVIII-specific single chain variable fragments (scFvs) from synthetic phage display libraries and the characterisation of their FVIII domain specificity are described. The isolated scFv 1G10, which binds to the FVIII A2 domain, was cloned into the context of the four human IgG (hIgG) subclasses and expressed in mammalian cells. Purified 1G10-hIgG1, -hIgG2, -hIgG3 and -hIgG4 are used as standards to determine the absolute amounts and relative contribution of the different FVIII-specific IgG subclasses in future studies. The results from these studies will eventually add to understanding the role of the FVIII-specific IgG subclass distribution as prognostic factor for the outcome of ITI.
authors.affiliation: Christoph Königs, University Hospital Frankfurt, Goethe University, Department of Paediatrics, Molecular Haemostasis and Immunodeficiency, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany, Tel. +49/(0)69/630 18 30 30, Fax +49/(0)69/630 18 39 91, E-mail: christoph.koenigs@kgu.de.
authors.forename: A
authors.initials: A
authors.lastname: Naumann
authors.forename: A K
authors.initials: AK
authors.lastname: Scherger
authors.forename: J
authors.initials: J
authors.lastname: Neuwirth
authors.forename: A
authors.initials: A
authors.lastname: Orlowski
authors.forename: J
authors.initials: J
authors.lastname: Kahle
authors.forename: D
authors.initials: D
authors.lastname: Schwabe
authors.forename: C
authors.initials: C
authors.lastname: Königs
mesh_terms: D001777:Blood Coagulation / Q000187:drug effects*; D015195:Drug Design; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D005169:Factor VIII
keywords: FVIII inhibitors; Haemophilia A; IgG subclasses; phage display