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 (^superFVa) 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. ^SuperFVa 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 ^superFVa alone and in combination with rhFVIIa in hemophilia with inhibitors were determined in more detail.

Materials and Methods: Procoagulant and clot stabilizing properties of ^superFVa, 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 ^superFVa 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 ^superFVa, 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 ^superFVa 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 ^superFVa (3 nM) normalized thrombin generation at concentrations 50-fold below the therapeutic plasma level. Saturation of the synergistic effect between ^superFVa and rhFVIIa was evident as thrombin generation reached a plateau at ~110% of normal plasma at higher concentrations of ^superFVa and rhFVIIa. Similar synergistic normalization of clot lysis time was observed when ^superFVa and rhFVIIa were used together at low concentration.

Beneficial effects of ^superFVa 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 ^superFVa (3 nM) and rhFVIIa (2 nM) were present compared to the individual 10 to 20-fold higher concentrations of ^superFVa (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 ^superFVa ex-vivo.

In-vivo tail bleed analysis of FVIII-deficient mice showed a dose dependent reduction of bleeding by ^superFVa (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 ^superFVa compared to 26.3 µL/g for saline). At the highest dose of ^superFVa (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 ^superFVa, 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 ^superFVa (40 U/kg) decreased bleeding even further (1.6 µL/g; p=0.01), similar to what was observed with rhFVIII.

Conclusion: The engineered ^superFVa 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, ^superFVa 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 ^superFVa as a novel bypassing strategy either alone or in combination with rhFVIIa-based therapy in hemophilia patients with inhibitors.