Selection and characterisation of FVIII-specific single chain variable fragments

A. Naumann; A. K. Scherger; J. Neuwirth; A. Orlowski; J. Kahle; D. Schwabe; C. Königs

doi:10.1055/s-0037-1619801

Hämostaseologie, Inhaltsverzeichnis

Hamostaseologie 2013; 33(S 01): S39-S45
DOI: 10.1055/s-0037-1619801

Original article

Schattauer GmbH

Selection and characterisation of FVIII-specific single chain variable fragments

Selektion und Charakterisierung FVIII-spezifischer einzelkettiger Antikörperfragmente

Autoren

A. Naumann

¹Molecular Haemostasis and Immunodeficiency, Department of Paediatrics, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany
A. K. Scherger

¹Molecular Haemostasis and Immunodeficiency, Department of Paediatrics, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany
J. Neuwirth

¹Molecular Haemostasis and Immunodeficiency, Department of Paediatrics, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany
A. Orlowski

¹Molecular Haemostasis and Immunodeficiency, Department of Paediatrics, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany
J. Kahle

¹Molecular Haemostasis and Immunodeficiency, Department of Paediatrics, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany
D. Schwabe

¹Molecular Haemostasis and Immunodeficiency, Department of Paediatrics, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany
C. Königs

¹Molecular Haemostasis and Immunodeficiency, Department of Paediatrics, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany

Abstract

Summary

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.

Zusammenfassung

In der modernen Therapie von Hämophilie A-Patienten ist die Entwicklung inhibitorischer FVIII-spezifischer Antikörper die schwerwiegendste Komplikation. Toleranz für FVIII kann durch eine Immuntoleranztherapie (ITI) erreicht werden. Aktuelle Untersuchungen deuten auf einen Zusammenhang zwischen der FVIII-spezifischen IgG-Subklassenverteilung und der Dauer sowie dem Erfolg der ITI hin. Um die FVIII-spezifischen IgGSubklassen im Patientenplasma zu quantifizieren, werden FVIII-spezifische IgG-Standards benötigt.

Dafür wurden FVIII-spezifische Antikörperfragmente aus synthetischen Phagen-Bibliotheken isoliert und ihre FVIII Domänen-Spezifität charakterisiert. Der FVIII-spezifische scFv 1G10, der an die FVIII A2-Domäne bindet, wurde in den Kontext der vier humanen IgG (hIgG)-Subklassen kloniert und in Säugerzellen exprimiert. Aufgereinigte 1G10-hIgG1, -hIgG2, -hIgG3 und -hIgG4 werden als Standard verwendet, um in Studien die absoluten und relative Anteile der vier FVIII-spezifischen IgG-Subklassen zu bestimmen. Die Ergebnisse dieser Studien tragen zum weiteren Verständnis über den Zusammenhang von IgG-Subklassenverteilung und ITI-Erfolg bei.

Keywords

Haemophilia A - phage display - FVIII inhibitors - IgG subclasses

Schlüsselwörter

Hämophilie A - Phagen-Display - FVIII-Hemmkörper - IgG-Subklassen

Volltext

Referenzen

References
1 Biggs R, Austen DE, Denson KW. et al. The mode of action of antibodies which destroy factor VIII. II. Antibodies which give complex concentration graphs. Br J Haematol 1972; 23: 137-155.
2 Brackmann HH, Oldenburg J, Schwaab R. Immune tolerance for the treatment of factor VIII inhibitors--twenty years’ ‘bonn protocol’. Vox Sang 1996; 70 (Suppl. 01) 30-35.
3 Ehrenforth S, Kreuz W, Scharrer I. et al. Incidence of development of factor VIII and factor IX inhibitors in haemophiliacs. Lancet 1992; 339: 594-598.
4 Fulcher CA, de Graaf SMahoney, Roberts JR. et al. Localization of human factor FVIII inhibitor epitopes to two polypeptide fragments. Proc Natl Acad Sci USA 1985; 82: 7728-7732.
5 Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988; 53: 505-518.
6 Gitschier J, Wood WI, Goralka TM. et al. Characterization of the human factor VIII gene. Nature 1984; 312: 326-330.
7 Goletz S, Christensen P, Kristensen P. et al. Selection of large diversities of antiidiotypic antibody fragments by phage display. J Mol Biol 2002; 315: 1087-1097.
8 Healey JF, Lubin IM, Nakai H. et al. Residues 484–508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII. J Biol Chem 1995; 270: 14505-14509.
9 Hoots WK. Pathogenesis of hemophilic arthropathy. Semin Hematol 2006; 43: S18-S22.
10 Hoyer LW. Hemophilia A. N Engl J Med 1994; 330: 38-47.
11 Kehoe JW, Kay BK. Filamentous phage display in the new millennium. Chem Rev 2005; 105: 4056-4072.
12 Kessel C, Königs C, Linde R. et al. Humoral immune responsiveness to a defined epitope on factor VIII before and after B cell ablation with rituximab. Mol Immunol 2008; 46: 8-15.
13 Lenting PJ, van Mourik JA, Mertens K. The life cycle of coagulation factor VIII in view of its structure and function. Blood 1998; 92: 3983-3996.
14 Mannucci PM, Tuddenham EG. The hemophilias--from royal genes to gene therapy. N Engl J Med 2001; 344: 1773-1779.
15 Mariani G, Siragusa S, Kroner BL. Immune tolerance induction in hemophilia A: a review. Semin Thromb Hemost 2003; 29: 69-76.
16 Markovitz RC, Healey JF, Parker ET. et al. The diversity of the immune response to the A2 domain of human factor VIII. Blood 2013; 121: 2785-2795.
17 Schirrmann T, Büssow K. Transient Production of scFv-Fc Fusion Proteins in Mammalian Cells. Antibody Engineering 2010; 387-400.
18 Van Helden PM, van den Berg HM, Gouw S. et al. IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A. Br J Haematol 2008; 142: 644-652.
19 Whelan SF, Hofbauer CJ, Horling FM. et al. Distinct characteristics of antibody responses against factor VIII in healthy individuals and in different cohorts of hemophilia A patients. Blood 2013; 121: 1039-1048.