The human monoclonal antibody Mab-LE2E9 has been derived from a patient with mild haemophilia A (patient LE) who carries the mutation Arg2150->His and developed a high titre inhibitor following SB203580 FVIII administration, while maintaining unaltered FVIII levels [9]. Patient LE B cells were immortalized with the Epstein-Barr virus. One cell line producing an antibody to FVIII, Mab-LE2E9, was selected and cloned. Mab-LE2E9 inhibited FVIII with high specific activity (10.000 BU mg−1) [13]. By contrast, Mab-LE2E9 did

not reduce the FVIII activity present in the plasma of patients with mutated Arg2150His. Mab-LE2E9 behaves as a type II inhibitor, characterized by incomplete FVIII inactivation, even in large excess of antibody [13]. Thus far, partial inactivation of FVIII by type II inhibitor antibodies had been attributed to the interaction of FVIII with VWF. Gawryl and Hoyer demonstrated that some type II inhibitors compete with VWF for binding to FVIII [2]. Conversely, VWF is required for certain type II inhibitor antibodies to exert their activity, by

binding exclusively to FVIII complexed with VWF [4] or by reducing the rate of dissociation of activated FVIII from VWF [3]. By contrast, Mab-LE2E9 inhibited FVIII effectively in the absence of VWF. Thus, although Mab-LE2E9 competes with VWF for FVIII binding, VWF does not protect FVIII from inactivation. Mab-LE2E9 represents Epacadostat mouse therefore a novel form of type II inhibitor, the action mechanism of which is still being investigated [14]. The absence of recognition of Arg2150His FVIII suggested that the epitope recognized by Mab-LE2E9 was located on the FVIII light chain. Immunoprecipitation experiments indicated that Mab-LE2E9 binds to the C1 domain but not its mutated counterpart. Those observations identified the FVIII C1 domain as a novel target for FVIII inhibitors and suggested that alteration of B cell epitope(s)

may contribute to the higher incidence of inhibitors 上海皓元 found in mild/moderate haemophilia A patients with mutations in the carboxy-terminal end of the FVIII C1 domain [10]. In contrast to the partial neutralization of FVIII activity, the inhibition of FVIII binding to VWF is complete at concentrations of Mab-LE2E9 in slight excess to those of FVIII. When those experiments were performed, the binding of FVIII to VWF was attributed to two FVIII regions: the carboxy-terminal part of the C2 domain and the acidic part of the A3 domain [15–18]. It was therefore unexpected that Mab-LE2E9, which recognizes an epitope in the C1 domain, could interfere with FVIII binding to VWF. Those observations raised the question of whether residue Arg2150 in the C1 domain contributes to FVIII binding to VWF and prompted the study of the effect of mutations located in C1 and responsible of mild/moderate haemophilia A on FVIII binding to VWF.