Synergistic Effect in Bleed Reduction By superfva and Recombinant Human FVIIa in Vivo Suggests a Novel Bypassing Strategy for Hemophilia Patients with Inhibitors

Introduction: In Hemophilia patients with inhibitors, recombinant human (rh)FVIIa-based bypassing strategy does not always achieve hemostasis indicating a clinical need for improved treatment strategies. We engineered an activated FVa variant ( super FVa) with mutations at 3 activated protein C cleavage sites (R506, R306 and R679) and an engineered interdomain disulfide bond (H609C-E1691C) connecting the A2 and A3 domains. Super FVa resisted inactivation and showed superior hemostatic properties in FVIII-deficient plasma and in mouse models of Hemophilia A compared to wild type rhFVa. Therefore, the hemostatic effects of super FVa alone and in combination with rhFVIIa in hemophilia with inhibitors were determined in more detail. Materials and Methods: Procoagulant and clot stabilizing properties of super FVa, rhFVIIa, and combinations thereof were studied in thrombin generation (endogenous thrombin potential (ETP) and peak height) and clot lysis assays in hemophilia A and normal plasma with or without high titer inhibitors. In vivo efficacy of super FVa alone and in combination with rhFVIIa for bleed reduction in hemophilia was tested in FVIII-deficient mice using a tail clip model. Results: Extensive dose-response titrations of super FVa, rhFVIIa, and combinations thereof in hemophilic or normal plasma with inhibitors indicated synergistic responses for normalization of thrombin generation or clot stabilization. For instance, at 0.4 nM rhFVIIa (1/100 of the therapeutic plasma level), the concentration of super FVa required to normalize thrombin generation was reduced 10-fold. Vice versa, rhFVIIa tested up to 40 nM only marginally improved thrombin generation, however, rhFVIIa in the presence of a low concentration of super FVa (3 nM) normalized thrombin generation at concentrations 50-fold below the therapeutic plasma level. Saturation of the synergistic effect between super FVa and rhFVIIa was evident as thrombin generation reached a plateau at ~110% of normal plasma at higher concentrations of super FVa and rhFVIIa. Similar synergistic normalization of clot lysis time was observed when super FVa and rhFVIIa were used together at low concentration. Beneficial effects of super FVa and rhFVIIa combinations were also observed in 5 individual plasma samples of hemophilia A patients with inhibitors (41-280 BU/ml). ETP and peak height improved ~1.5-3 fold when both super FVa (3 nM) and rhFVIIa (2 nM) were present compared to the individual 10 to 20-fold higher concentrations of super FVa (30 nM) or rhFVIIa (40 nM). Also, a therapeutic dose of rhFVIIa administered to 2 hemophilia patients with inhibitors (32 & 64 BU/ml) provided unremarkable improvements of thrombin generation in plasma taken before and 10 minutes post-infusion, whereas thrombin generation was fully restored upon titration of super FVa ex-vivo. In-vivo tail bleed analysis of FVIII-deficient mice showed a dose dependent reduction of bleeding by super FVa (mean blood loss was 25.9 µL/g at 10 U/kg; 9.7 µL/g at 40 U/kg and 2.5 µL/g at 200 U/kg of super FVa compared to 26.3 µL/g for saline). At the highest dose of super FVa (200 U/kg), bleed reduction was indistinguishable from the bleed reduction by rhFVIII (200 U/kg; 2.9 µL/g). Injection of 1 or 3 mg/kg rhFVIIa, reduced mean blood loss from 25.9 µL/g (saline control) to 16.8 (p=0.1) or 7.2 µL/g (p=0.003), respectively, but rhFVIIa’s effects did not reach the blood loss reduction achieved with rhFVIII (2.9 µL/g, p=0.03). However, combination of rhFVIIa (1 mg/kg) with 10 U/kg super FVa, a concentration that did not reduce bleeding by itself, significantly reduced bleeding from 16.7 to 10.2 µL/g (p=0.05). Combination of rhFVIIa (3 mg/kg) with super FVa (40 U/kg) decreased bleeding even further (1.6 µL/g; p=0.01), similar to what was observed with rhFVIII. Conclusion: The engineered super FVa variant showed synergistic procoagulant effects with rhFVIIa in vitro in hemophilic and normal plasma with inhibitors in thrombin generation and clot lysis assays. Notably, in vivo, super FVa reduced bleeding similar to rhFVIII in FVIII-deficient mice in a dose-dependent fashion, and it was able to enhance further bleed reduction when administered in combination with rhFVIIa. These results warrant further characterization of the potential therapeutic benefits of super FVa as a novel bypassing strategy either alone or in combination with rhFVIIa-based therapy in hemophilia patients with inhibitors. Disclosures von Drygalski: University of California, San Diego: University of California, San Diego Patents & Royalties. Gale: University of California, San Diego: University of California, San Diego Patents & Royalties. Griffin: The Scripps Research Institute: The Scripps Research Institute Patents & Royalties. Mosnier: The Scripps Research Institute: The Scripps Research Institute Patents & Royalties.