Epitope mapping via selection of anti-FVIII antibody-specific phagepresented peptide ligands that mimic the antibody binding sites

Joerg Kahle; Aleksander Orlowski; Diana Stichel; Karin Becker-Peters; Ali Kabiri; John F. Healey; Kerstin Brettschneider; Anja Naumann; Anna Katharina Scherger; Pete Lollar; Dirk Schwabe; Christoph Königs

doi:10.1160/TH14-01-0101

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2015; 113(02): 396-405
DOI: 10.1160/TH14-01-0101

New Technologies, Diagnostic Tools and Drugs

Schattauer GmbH

Epitope mapping via selection of anti-FVIII antibody-specific phagepresented peptide ligands that mimic the antibody binding sites

Joerg Kahle

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

,

Aleksander Orlowski

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

,

Diana Stichel

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

,

Karin Becker-Peters

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

,

Ali Kabiri

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

,

John F. Healey

²Aflac Cancer & Blood Disorders Center, Department of Pediatrics, Children’s Healthcare of Atlanta and Emory University, Atlanta, Georgia; USA

,

Kerstin Brettschneider

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

,

Anja Naumann

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

,

Anna Katharina Scherger

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

,

Pete Lollar

²Aflac Cancer & Blood Disorders Center, Department of Pediatrics, Children’s Healthcare of Atlanta and Emory University, Atlanta, Georgia; USA

,

Dirk Schwabe

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

,

Christoph Königs

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

› Author Affiliations

Abstract

Summary

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.

Keywords

Haemophilia A - anti-FVIII antibodies - inhibitors - epitope mapping - phage display

Full Text

References

References
1 Mannucci PM, Tuddenham EG. The hemophilias—from royal genes to gene therapy. N Engl J Med 2001; 344: 1773-1779.
2 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.
3 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.
4 Dimichele D. The North American Immune Tolerance Registry: contributions to the thirty-year experience with immune tolerance therapy. Haemophilia 2009; 15: 320-328.
5 Hay CR, DiMichele DM, International Immune Tolerance S. The principal results of the International Immune Tolerance Study: a randomized dose comparison. Blood 2012; 119: 1335-1344.
6 Greninger DA, Saint-Remy JM, Jacquemin M. et al. The use of factor VIII/von Willebrand factor concentrate for immune tolerance induction in haemophilia A patients with high-titre inhibitors: association of clinical outcome with inhibitor epitope profile. Haemophilia 2008; 14: 295-302.
7 van Helden PM, Kaijen PH, Mauser-Bunschoten EP. et al. Domain specificity of factor VIII inhibitors during immune tolerance induction in patients with haemophilia A. Haemophilia 2010; 16: 892-901.
8 Fulcher CA, de Graaf Mahoney S, Zimmerman TS. FVIII inhibitor IgG subclass and FVIII polypeptide specificity determined by immunoblotting. Blood 1987; 69: 1475-1480.
9 Prescott R, Nakai H, Saenko EL. et al. The inhibitor antibody response is more complex in hemophilia A patients than in most nonhemophiliacs with factor VIII autoantibodies. Recombinate and Kogenate Study Groups. Blood 1997; 89: 3663-3671.
10 Lollar P. Pathogenic antibodies to coagulation factors. Part one: factor VIII and factor IX. J Thromb Haemost 2004; 02: 1082-1095.
11 Villard S, Piquer D, Raut S. et al. Low molecular weight peptides restore the pro-coagulant activity of factor VIII in the presence of the potent inhibitor antibody ESH8. J Biol Chem 2002; 277: 27232-27239.
12 Villard S, Lacroix-Desmazes S, Kieber-Emmons T. et al. Peptide decoys selected by phage display block in vitro and in vivo activity of a human anti-FVIII inhibitor. Blood 2003; 102: 949-952.
13 Negi SS, Braun W. Automated detection of conformational epitopes using phage display Peptide sequences. Bioinformat Biol Insights 2009; 03: 71-81..
14 Moreau V, Granier C, Villard S. et al. Discontinuous epitope prediction based on mimotope analysis. Bioinformatics 2006; 22: 1088-1095.
15 Spiegel Jr. PC, Jacquemin M, Saint-Remy JM. et al. Structure of a factor VIII C2 domain-immunoglobulin G4kappa Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII. Blood 2001; 98: 13-19.
16 Muhle C, Schulz-Drost S, Khrenov AV. et al. Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display. Thromb Haemost 2004; 91: 619-625.
17 Kessel C, Konigs 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.
18 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.
19 Ngo JC, Huang M, Roth DA. et al. Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex. Structure 2008; 16: 597-606.
20 Jacquemin MG, Desqueper BG, Benhida A. et al. Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor. Blood 1998; 92: 496-506.
21 Gringeri A, Musso R, Mazzucconi MG. et al. Immune tolerance induction with a high purity von Willebrand factor/VIII complex concentrate in haemophilia A patients with inhibitors at high risk of a poor response. Haemophilia 2007; 13: 373-379.
22 Van Regenmortel MH. From absolute to exquisite specificity. Reflections on the fuzzy nature of species, specificity and antigenic sites. J Immunol Methods 1998; 216: 37-48.
23 Jin L, Fendly BM, Wells JA. High resolution functional analysis of antibody-antigen interactions. J Mol Biol 1992; 226: 851-865.
24 Nguyen PC, Lewis KB, Ettinger RA. et al. High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plas-mon resonance. Blood 2014; 123: 2732-2739.
25 Funk WD, MacGillivray RT, Mason AB. et al. Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein. Biochemistry 1990; 29: 1654-1660.
26 Healey JF, Parker ET, Barrow RT. et al. The humoral response to human factor VIII in hemophilia A mice. J Thromb Haemost 2007; 05: 512-519.
27 Doering C, Parker ET, Healey JF. et al. Expression and characterization of rec-ombinant murine factor VIII. Thromb Haemost 2002; 88: 450-458.

Supplementary Material

Online Supplementary Material (PDF)