Enhancing the Acute Hemostatic Efficacy in Cynomolgus Monkeys By Targeting Activated Coagulation Factor VII to Platelets

Abstract

Recombinant activated factor VII (rFVIIa) is used for controlling bleeds in hemophilia A and B patients with inhibitory antibodies against factor VIII or factor IX. The hemostatic effect of rFVIIa at pharmacological doses is believed to be mediated by activating the tenase complex on platelets directly at the site of injury to promote thrombin generation. To increase the hemostatic activity of rFVIIa, we sought to target rFVIIa to platelets by fusing a single chain variable region of a monoclonal antibody against human platelet receptor αIIbβ3. The resulting fusion protein, FVII-189, displays higher affinity to human platelets and about 50-fold higher hemostatic activity than that of rFVIIa in whole blood from hemophilia A patients by rotational thromboelastometry (ROTEM). Here we investigated the safety, pharmacokinetics, and pharmacodynamics of FVII-189 in cynomolgus monkeys.First, we confirmed that FVII-189 cross-reacted to platelets from cynomolgus monkeys. When spiked in the monkey whole blood, FVII-189 could bind platelets in a dose-dependent manner, as measured by flow cytometry using fluorescently labeled antibodies against FVII, whereas platelet-binding by rFVIIa was not detectable in the same assay. More importantly, increased hemostatic activity of FVII-189 was observed by ROTEM in acquired hemophilic monkey blood where factor VIII activity was neutralized by the inhibitory antibodies.We then evaluated FVII-189 in cynomolgus monkeys following a bolus intravenous administration (n=3) at an equal molar dose to the pharmacological dose of rFVIIa (~2 nmol/kg). rFVIIa was included as a comparator. Both FVII-189 and rFVIIa appeared to be well tolerated with no abnormal clinical observations that could be attributed to the dosing. No apparent effects on platelet count in whole blood, as well as other hematology and coagulation parameters except for a temporary decrease in prothrombin time over a few hours, which was an expected pharmacological effect of rFVIIa and FVII-189. Compared to rFVIIa, FVII-189 cleared more rapidly in plasma, and distributed more to the platelets. The half-life of FVII-189 antigen on platelets was found to be several fold longer than that of rFVIIa antigen in plasma, as measured by ELISA of the platelet-rich plasma, or by flow cytometry of platelet-associated FVII-189 antigen. Despite the lower initial recovery and faster clearance of FVII-189 in plasma, directing FVII-189 to platelets resulted in much higher hemostatic activity of the whole blood 5 minute and 1 hour after dosing, as measured by ex vivo ROTEM assay.In conclusion, these results indicate that in cynomolgus monkeys, 1) FVII-189 is safe and does not affect the platelet clearance, 2) FVII-189 protein is targeted to platelets with fast clearance in plasma and prolonged half-life on platelets and 3) FVII-189 could be more efficacious in controlling acute bleeds than rFVIIa. DisclosuresTan:Biogen Idec: Employment, Equity Ownership. Salas:Biogen Idec: Employment, Equity Ownership. Chen:Biogen Idec: Employment, Equity Ownership. Ashworth:Biogen Idec: Employment, Equity Ownership. Smith:Biogen Idec: Employment, Equity Ownership. Kistanova:Biogen Idec: Employment, Equity Ownership. Moore:Biogen Idec: Employment, Equity Ownership. Acosta:Biogen Idec: Employment, Equity Ownership. Kole:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership. Peters:Biogen Idec: Employment. Jiang:Biogen Idec: Employment, Equity Ownership.