Prevalence and epitope specificity of non-neutralising antibodies in a large cohort of haemophilia A patients without inhibitors

Summary

Antibodies (inhibitors and non-neutralising antibodies [NNA]) directed against factor VIII (FVIII) remain the main iatrogenic complication in haemophilia A (HA) patients. Inhibitors reduce FVIII procoagulant properties, whereas NNA are directed against non-functional epitopes. NNA are poorly studied and their prevalence, epitope specificity and physiopathology inadequately defined. The aim of this study was first to evaluate NNA prevalence in a French retrospective multicentric series of 210 patients without inhibitors, then to determine their epitope specificity (against the heavy chain [HC] or the light chain [LC] of FVIII) and particularly to assess the prevalence of anti-B domain NNA using specifically designed x-MAP assays. NNA occurred in 18.1% of patients (38/210) and their prevalence was not influenced by the severity of the disease. Among the 38 patients with NNA, 73.7% had anti-FVIII Abs against the HC, 13.2% against the LC and 13.2% had anti-FVIII Abs against both chains. There is thus a clear immuno-dominance of the HC of FVIII in the epitope profile of NNA, whatever the severity of HA. The proportion of NNA that recognised the B domain was 18.4% (n=7/38). A multivariate analysis did not highlight differences in NNA occurrence between patients treated with recombinant FVIII or with plasma-derived FVIII (19.6% vs. 14.9%, p=0.53).

Keywords

Haemophilia A - factor VIII - x-MAP technology - non-neutralising antibodies - epitope mapping

^* AL and PL contributed equally to this study.

• References

• 1 Hoyer LW. Molecular pathology and immunology of factor VIII (hemophilia A and factor VIII inhibitors). Hum Pathol 1987; 18: 153-161.
• 2 Mann KG. Biochemistry and physiology of blood coagulation. Thromb Haemost 1999; 82: 165-174.
• 3 Lollar P, Parker CG. Subunit structure of thrombin-activated porcine factor VIII. Biochemistry 1989; 28: 666-674.
• 4 Vehar GA, Keyt B, Eaton D. et al. Structure of human factor VIII. Nature 1984; 312: 337-342.
• 5 Toole JJ, Pittman DD, Orr EC. et al. A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. Proc Natl Acad Sci USA 1986; 83: 5939-5942.
• 6 Lavigne-Lissalde G, Schved JF, Granier C. et al. Anti-factor VIII antibodies: a 2005 update. Thromb Haemost 2005; 94: 760-769.
• 7 Lacroix-Desmazes S, Moreau A. Sooryanarayana et al. Catalytic activity of antibodies against factor VIII in patients with hemophilia A. Nat Med 1999; 5: 1044-1047.
• 8 Kazatchkine MD, Sultan Y, Burton-Kee EJ. et al. Circulating immune complexes containing anti-VIII antibodies in multi-transfused patients with haemophilia A. Clin Exp Immunol 1980; 39: 315-320.
• 9 Di Giambattista M, Branckaert T, Laub R. Mapping of natural anti-factor VIII antibodies in plasma pools from healthy donors: use of rationally designed synthetic peptides. Biologicals 2001; 29: 229-232.
• 10 Kasper CK, Pool JG. Letter: Measurement of mild factor VIII inhibitors in Bethesda units. Thromb Diath Haemorrh 1975; 34: 875-876.
• 11 Verbruggen B. Diagnosis and quantification of factor VIII inhibitors. Haemophilia 2010; 16: 20-24.
• 12 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.
• 13 Hoyer LW, Hemophilia A. N Engl J Med. 1994 330. 38-47.
• 14 Dazzi F, Tison T, Vianello F. et al. High incidence of anti-FVIII antibodies against non-coagulant epitopes in haemophilia A patients: a possible role for the half-life of transfused FVIII. Br J Haematol 1996; 93: 688-693.
• 15 Ling M, Duncan EM, Rodgers SE. et al. Low detection rate of antibodies to non-functional epitopes on factor VIII in patients with hemophilia A and negative for inhibitors by Bethesda assay. J Thromb Haemost 2003; 1: 2548-2553.
• 16 Vincent AM, Lillicrap D, Boulanger A. et al. Non-neutralizing anti-FVIII antibodies: different binding specificity to different recombinant FVIII concentrates. Haemophilia 2009; 15: 374-376.
• 17 Krudysz-Amblo J, Parhami-Seren B, Butenas S. et al. Quantitation of anti-factor VIII antibodies in human plasma. Blood 2009; 113: 2587-2594.
• 18 Lavigne-Lissalde G, Lacroix-Desmazes S, Wootla B. et al. Molecular characterization of human B domain-specific anti-factor VIII monoclonal antibodies generated in transgenic mice. Thromb Haemost 2007; 98: 138-147.
• 19 Bovenschen N, Rijken DC, Havekes LM. et al. The B domain of coagulation factor VIII interacts with the asialoglycoprotein receptor. J Thromb Haemost 2005; 3: 1257-1265.
• 20 Lavigne-Lissalde G, Tarrade C, Lapalud P. et al. Simultaneous detection and epitope mapping of anti-factor VIII antibodies. Thromb Haemost 2008; 99: 1090-1096.
• 21 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.
• 22 Martin PG, Sukhu K, Chambers E. et al. Evaluation of a novel ELISA screening test for detection of factor VIII inhibitory antibodies in haemophiliacs. Clin Lab Haematol 1999; 21: 125-128.
• 23 Blanco AN, Peirano AA, Grosso SH. et al. An ELISA system to detect anti-factor VIII antibodies without interference by lupus anticoagulants. Preliminary data in hemophilia A patients. Haematologica 2000; 85: 1045-1050.
• 24 Sahud MA, Pratt KP, Zhukov O. et al. ELISA system for detection of immune responses to FVIII: a study of 246 samples and correlation with the Bethesda assay. Haemophilia 2007; 13: 317-322.
• 25 Vianello F, Radossi P, Tison T. et al. Prevalence of anti-FVIII antibodies in severe haemophilia A patients with inversion of intron 22. Br J Haematol 1997; 97: 807-809.
• 26 Gilles JG, Saint-Remy JM. Healthy subjects produce both anti-factor VIII and specific anti-idiotypic antibodies. J Clin Invest 1994; 94: 1496-1505.
• 27 Lorenzo JI, Lopez A, Altisent C. et al. Incidence of factor VIII inhibitors in severe haemophilia: the importance of patient age. Br J Haematol 2001; 113: 600-603.
• 28 van der Bom JG, Mauser-Bunschoten EP, Fischer K. et al. Age at first treatment and immune tolerance to factor VIII in severe hemophilia. Thromb Haemost 2003; 89: 475-479.
• 29 Gouw SC, van der Bom JG, van den Berg MH. Treatment-related risk factors of inhibitor development in previously untreated patients with hemophilia A: the CANAL cohort study. Blood 2007; 109: 4648-4654.
• 30 Chalmers EA, Brown SA, Keeling D. et al. Early factor VIII exposure and subsequent inhibitor development in children with severe haemophilia A. Haemophilia 2007; 13: 149-155.
• 31 Scandella D, Mondorf W, Klinge J. The natural history of the immune response to exogenous factor VIII in severe haemophilia A. Haemophilia 1998; 4: 546-551.
• 32 Scandella D, Mattingly M, Prescott R. A recombinant factor VIII A2 domain poly-peptide quantitatively neutralizes human inhibitor antibodies that bind to A2. Blood 1993; 82: 1767-1775.
• 33 Scandella D, Gilbert GE, Shima M. et al. Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site. Blood 1995; 86: 1811-1819.
• 34 Shima M, Scandella D, Yoshioka A. et al. A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine. Thromb Haemost 1993; 69: 240-246.
• 35 Batlle J, Gomez E, Rendal E. et al. Antibodies to factor VIII in plasma of patients with hemophilia A and normal subjects. Ann Hematol 1996; 72: 321-326.
• 36 Hay CR, Ludlam CA, Colvin BT. et al. Factor VIII inhibitors in mild and moderate-severity haemophilia A. UK Haemophilia Centre Directors Organisation. Thromb Haemost 1998; 79: 762-766.
• 37 Fay PJ, Haidaris PJ, Smudzin TM. Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit. J Biol Chem 1991; 266: 8957-8962.
• 38 Pipe SW. Functional roles of the factor VIII B domain. Haemophilia 2009; 15: 1187-1196.
• 39 Schwaab R, Brackmann HH, Meyer C. et al. Haemophilia A: mutation type determines risk of inhibitor formation. Thromb Haemost 1995; 74: 1402-1406.
• 40 Astermark J, Voorberg J, Lenk H. et al. Impact of inhibitor epitope profile on the neutralizing effect against plasma-derived and recombinant factor VIII concentrates in vitro. Haemophilia 2003; 9: 567-572.
• 41 Franchini M, Lippi G. Von Willebrand factor-containing factor VIII concentrates and inhibitors in haemophilia A. A critical literature review. Thromb Haemost 2010; 104: 931-940.