Towards Restoring Coagulation in Hemophilia By Antibody-Mediated Inhibition of Antithrombin

Currently hemophilia A is mainly treated with factor VIII (FVIII). However, 20-25% of the hemophiliacs develop inhibiting antibodies (inhibitors) against FVIII. Hemophiliacs with inhibitors are treated with high dose FVIII or with therapies that bypass the need for FVIII such as activated FVII (rFVIIa, NovoSeven) or activated prothrombin concentrate. These have some disadvantages including rapid clearance, price and a risk of thrombo-embolism.

In hemophilia, the balance between coagulation and anticoagulation is shifted towards the latter. This balance can be restored by administration of clotting factors but also by reducing anticoagulation mechanisms. Antithrombin (AT) constitutes a main anticoagulant mechanism as it inhibits thrombin, FXa and other activated clotting factors. In silico studies as well as observations that AT deficiency reduces bleeding in hemophilic mice and humans strongly support the concept that lowering functional AT levels bypasses the need for FVIII to generate thrombin. A Phase 1 clinical trial by Alnylam Pharmaceuticals showed that lowering AT activity by RNA-interference can boost thrombin generation in hemophilia. However, RNA-interference is an experimental therapy with unknown long term effects and is not suitable for acute treatment of bleeding as it takes weeks to become effective.

We hypothesize that monoclonal antibodies (mAbs) that block AT activity can be used as FVIII bypass therapy. Such a mAb-based therapy is suitable for on demand and for prophylactic therapy, is relatively cheap and does not require administration of activated clotting factors.

We have generated a panel of mouse anti-AT mAbs exhibiting varying binding characteristics to human AT. To assess the inhibitory activity of anti-AT mAbs, FXa activity assay was performed after FXa was incubated with normal plasma supplemented with the different anti-AT mAbs. Five anti-AT mAbs were identified that inhibit AT activity at around equimolar ratio. Inhibitory anti-AT mAbs were able to dose-dependently enhance thrombin generation in normal and hemophilic plasma. Heparin caused a marked prolongation of aPTT of normal and FVIII-deficient plasma, which was almost completely normalized by addition of the inhibitory mAb anti-AT 5G1.1, but not by anti-AT 6E7.2, indicating that these mAbs exert their inhibitory activity via different mechanisms.

These data provide initial in vitro proof-of-concept that inhibitory anti-AT mAbs can be used to improve coagulation and may be used as FVIII bypass therapy.