Analysis of factor VIII inhibitors in a haemophilia A patient with an Arg⁵⁹³→Cys mutation using phage display

Abstract

Summary. We characterized anti-factor VIII antibodies in a mild haemophilia A patient with an Arg 593 fiCys mutation in the A2 domain, using V gene phage-display technology. All isolated single-chain variable-domain antibody fragments were directed against residues Arg 484 -Ile 508 , a binding site for factor VIII inhibitors in the A2 domain. After a further period of replacement therapy, a transient rise in inhibitor titre was observed. These antibodies were directed against the A2 domain. Activation of a pre-existing pool of B cells, which express antibodies against residues Arg 484 -Ile 508 , could explain the rapid anamnestic response.Keywords: factor VIII inhibitors, haemophilia A, phage display, B lymphocyte, immunoglobulin repertoire.A serious complication in haemophilia A treatment is the development of antibodies that neutralize factor VIII activity (inhibitors). The prevalence of factor VIII inhibitors in patients with severe haemophilia A is approximately 25% (Mannucci & Tuddenham, 2001). Factor VIII inhibitors are infrequently observed in patients with mild or moderate haemophilia A (Sultan, 1992). The lower risk of inhibitor formation in this group of patients can be explained by the presence of tolerizing amounts of circulating endogenous factor VIII. Inhibitor development in these patients commonly arises after intensive factor VIII replacement therapy and is usually a transient event (Hay et al, 1998). Interestingly, some genetic defects are frequently observed in patients with mild haemophilia A who developed inhibitors. Amino acid substitutions located at the junction between the C1 and C2 domain or located in the A2 domain of factor VIII may predispose towards the development of factor VIII inhibitors (Hay et al, 1998). We have previously reported on the presence of human alloantibodies in a patient with an Arg 593 fiCys mutation (Fijnvandraat et al, 1997). In this patient, the anti-factor VIII antibodies were directed to wild-type A2 domain and not to the A2 domain containing the Arg 593 fiCys mutation when analysed at a late stage of inhibitor formation. These findings indicate that the missense mutation Arg 593 fiCys was related to antibody development.In the present study, phage display technology was used for the characterization of anti-factor VIII antibodies in the patient. Our findings showed that peripheral IgG + B cells can express human antibodies reactive with residues Arg 484 -Ile 508 . These antibodies are distinct from the alloantibodies found in plasma. Following a period of intensive treatment with FVIII concentrate after a surgical intervention, the patient developed a transient low-titre inhibitor that cross-reacted with endogenous factor VIII. We propose that activation of pre-existing factor VIII-specific B cells underlies the rapid appearance of cross-reactive antibodies. MATERIALS AND METHODSExpression, metabolic labelling and immunoprecipitation of A2 domain variants. The wild-type factor VIII A2 domain, the A2 domain containing the Arg 593 fiCys mutation (A2-R593C) and...

Multiple B‐cell clones producing antibodies directed to the spacer and disintegrin/thrombospondin type‐1 repeat 1 (TSP1) of ADAMTS13 in a patient with acquired thrombotic thrombocytopenic purpura

“…Subsequently, FRETS-VWF73 substrate was added and the fluorescence was determined every minute for 1 h. The mean slopes of the curves from three experiments were used to calculate residual activity (and standard deviation) and were plotted against the concentration of scFv. ScFv 92-102 (C) directed against FVIII [33] was used as a control in these experiments. (B) Competition for binding of scFv I-9, I-16, I-26, and I-41, to the disintegrin/ thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/ spacer domains of ADAMTS13 in the presence of increasing amounts of full-length IgG I-9.…”

“…Thorough dialysis of scFv and IgG was necessary to remove trace amounts of buffer and salts that might disturb ADAMTS13 activity measurements. Various concentrations of scFv or IgG were preincubated with 5 μ L plasma in a total volume of 50 μ L, 5 mmol L −1 Tris pH 8.0 at 37 °C for 1 h. ScFv 92–102 directed to factor VIII was included as negative control [33]. FRETS‐VWF73 was diluted in reaction buffer according to the instructions of the manufacturer (Peptides International, Louisville, KY, USA) and 50 μ L substrate and 100 μ L reaction buffer were added to the plasma‐antibody mixtures.…”

“…Further epitope-mapping of scFv I-16 was performed by immunoprecipitation as described previously [26]. ScFv I-9 and scFv 92-102 [33] were included as controls. Five micrograms of purified scFv was coupled to Co-loaded chelating sepharose [34].…”

In non‐severe hemophilia A the risk of inhibitor after intensive factor treatment is greater in older patients: a case–control study

“…There have been previous reports of the R593C mutation and its association with inhibitor formation in patients with mild hemophilia A [14]. Most recently, in a cohort of subjects from the Netherlands, 8 out of 10 subjects with an inhibitor had the R593C mutation [3].…”

Comparative Measurement of Anti-Factor VIII Antibody by Bethesda Assay and ELISA Reveals Restricted Isotype Profile and Epitope Specificity

“…Inhibitor antibodies may also arise spontaneously either in nonhemophilic normal individuals or following immunological disorders, malignancies, postpartum, in the elderly and following some medications with an incidence of 0.2–1 per 1 million persons per year [7, 8]. A2, C2 and to a lesser extent A3 domains have been identified as the most immunogenic regions of FVIII against which inhibitor antibodies may arise [9,10,11,12]. …”