Antigen Binding Site Glycosylation Determines the Anticoagulant Activity of a Type II Factor VIII Antibody without Modifying Affinity or Epitope Specificity

Abstract number: P0032

Jacquemin¹ M, Lavend'homme¹ R, Van der Elst¹ L, Dewaele¹ J, Collen^1,2 D, Saint-Remy¹ JM, Dewerchin² M

^{1,2 11}Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium ¹¹Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium ²²Center for Transgene Technology and Gene Therapy, VIB, University of Leuven, Leuven, Belgium

Anti-Factor VIII (FVIII) antibodies appearing in haemophilia A patients treated with FVIII concentrates are subdivided into two categories: type I antibodies completely inhibit FVIII activity, whereas type II antibodies only partially inhibit it. Like 5% of antibodies, the type II human monoclonal antibody mAb-LE2E9 carries N-glycosylation in the variable region. Because the role of such glycosylation still is poorly understood, we investigated the effect of the carbohydrate on mAb-LE2E9 inhibitory activity in vitro and in vivo. From to 2 to 200 µg/ml, mAb-LE2E9 produced in CHO cells inhibited 75–85% FVIII activity, whereas a variant antibody, mAb-LE2E9Q, devoid of N-glycosylation site in the antigen binding site inhibited only 45% of FVIII activity. Competition experiments between LE2E9 and LE2E9Q indicated the absence of the carbohydrate did not impair LE2E9 binding to FVIII. The limited FVIII inactivation displayed by mAb-LE2E9Q raised the question whether such an antibody would exert a physiological effect in vivo. Because the native antibody is a potent antithrombotic agent, we investigated the ability of mAb-LE2E9Q to prevent thrombosis in mice carrying a type II deficiency mutation in the heparin binding site of antithrombin. Despite its limited inhibition of FVIII activity, mAb-LE2E9Q (100 µg, repeated after 3 days) efficiently prevented thrombosis in treated animals. This is the first report of an effect of carbohydrate on the activity of an antibody independent of its binding to the target antigen. Such a phenomenon may contribute to the large range in FVIII inhibition that can be observed with type II FVIII inhibitors. Unexpectedly, modification of the glycosylation of human monoclonal antibodies such as mAb-LE2E9 may provide a strategy to generate a novel type of anti-thrombotic drug with well defined anticoagulant properties.