The substitutions Ile2098Ser, Ser2119Tyr,

Asn2129Ser, Arg2150His and Pro2153Gln in the C1 domain significantly impaired FVIII binding to VWF [4,13]. Analysis of the binding of selected FVIII variants indicated that the affinities of the mutants were 3- to 80-fold lower than that of normal FVIII [13]. Shortly later, another group also identified a series of mutations in the FVIII C1 domain resulting in reduced FVIII binding to VWF and mild/moderate cancer metabolism inhibitor haemophilia A. Thus, mutations located in the FVIII light chain and impairing FVIII binding to VWF now appear to be a common cause of mild/moderate haemophilia A. Examination of the amino acid sequence of Mab-LE2E9 revealed a consensus N-glycosylation site AsnPheThr at residues 47–49 in the complementarity determining region (CDR) 1 of the variable region of the heavy chain (VH) [19]. To determine whether VH glycosylation played a role in the inhibitory activity of Mab-LE2E9, we produced a recombinant antibody, Mab-LE2E9Q, in which the glycosylation site was deleted by mutating Asn47 to Gln. Both native and mutated

antibodies were produced in Chinese Hamster Ovary cells. The recombinant mutated antibody bound to FVIII with an affinity identical to that of the native antibody. Similarly, the glycosylation did not change the see more stoichiometry of the reaction. However, despite their identical affinities and specificities, Mab-LE2E9 and Mab-LE2E9Q displayed strikingly different FVIII inhibitory activities. Indeed, Mab-LE2E9Q inhibited ∼40% of FVIII activity whereas Mab-LE2E9 reached a maximum inhibitory activity of ∼80–95% [19]. Glycan analysis of Mab-LE2E9 confirmed that the antibody is glycosylated. Molecular modelling of the V regions of the Fab of Mab-LE2E9 indicates that the glycosylation site at Asn47 is on an exposed loop of CDR1 away from the antigen binding site. The outer arms of the

glycan, but not the core residues, could make contact with the antigen. This provides a rationale for the higher level of inhibition of FVIII by the glycosylated antibody and for the unchanged affinity [19]. By contrast with the native antibody, Mab-LE2E9Q does not inhibit FVIII binding to VWF [19]. It is therefore MCE公司 likely that the N-glycosylation of the VH contributes by steric hindrance to inhibition of FVIII binding to VWF. Such a role of the glycan is compatible with the location of the oligosaccharides in the 3D-model of Mab-LE2E9. The observation that Mab-LE2E9 VHN-glycosylation determines the maximal inhibitory activity of the antibody offered a unique opportunity to develop an optimized anticoagulant agent targeting FVIII. Such a drug would be very helpful if it allowed avoiding or minimizing well-know risks associated with antithrombotic therapy. Thus, anti-vitamin K drugs exert their activity not only on procoagulant enzymes but also on inhibitor of the coagulation cascade such as Protein C and require monitoring.