A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories.

INTRODUCTION: Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half-life. AIM: Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs). METHODS: Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full-length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N = 35) tested blinded samples using in-house assays. Data from 51 OSAs (14 activated partial thromboplastin time [aPTT] reagents) and 42 CSAs (eight kits) were analyzed. RESULTS: Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under- and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two- to three-fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations. CONCLUSION: Under- or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.

Efanesoctocog alfa elicits functional clot formation that is indistinguishable to that of recombinant factor VIII.

BACKGROUND: Factor VIII (FVIII) binding to endogenous von Willebrand factor (VWF) has constrained half-life extension of recombinant FVIII (rFVIII) products for hemophilia A. Efanesoctocog alfa (rFVIIIFc-VWF-XTEN; BIVV001) is a novel fusion protein designed to decouple FVIII from VWF in circulation and maximize half-life prolongation by XTEN® polypeptides and Fc fusion. FVIII, VWF, and platelets interact to achieve normal hemostasis. Thus, bioengineered FVIII replacement products, such as efanesoctocog alfa, require comprehensive assessment of their hemostatic potential. OBJECTIVES: We compared functional clot formation and injury-induced platelet accumulation between efanesoctocog alfa and rFVIII. PATIENTS/METHODS: The hemostatic potential of efanesoctocog alfa and rFVIII were assessed by measuring their dose-dependent effects on in vitro fibrin generation in hemophilic plasma and in vivo injury-induced platelet accumulation using intravital microscopy and repeat saphenous vein laser-induced injuries in hemophilia A mice. RESULTS: Equal concentrations of efanesoctocog alfa or rFVIII (up to 1 IU/ml) added to plasma from patients with hemophilia A elicited similar kinetics for dose-dependent fibrin polymerization between factor products. In the presence of tissue plasminogen activator (tPA), clots formed had similar stability between products. Single intravenous doses (50, 100, or 150 IU/kg) of efanesoctocog alfa or rFVIII shortly before repeat saphenous vein laser-induced injuries increased platelet accumulation over time in a dose-dependent manner in hemophilia A mice. Platelet deposition kinetics were similar between products. CONCLUSIONS: Equivalent doses of efanesoctocog alfa and rFVIII had similar efficacy in promoting fibrin clot formation and injury-induced platelet accumulation. The hemostatic potential of efanesoctocog alfa was indistinguishable from that of rFVIII.

BIVV001, a new class of factor VIII replacement for hemophilia A that is independent of von Willebrand factor in primates and mice.

Factor VIII (FVIII) replacement products enable comprehensive care in hemophilia A. Treatment goals in severe hemophilia A are expanding beyond low annualized bleed rates to include long-term outcomes associated with high sustained FVIII levels. Endogenous von Willebrand factor (VWF) stabilizes and protects FVIII from degradation and clearance, but it also subjects FVIII to a half-life ceiling of ∼15 to 19 hours. Increasing recombinant FVIII (rFVIII) half-life further is ultimately dependent upon uncoupling rFVIII from endogenous VWF. We have developed a new class of FVIII replacement, rFVIIIFc-VWF-XTEN (BIVV001), that is physically decoupled from endogenous VWF and has enhanced pharmacokinetic properties compared with all previous FVIII products. BIVV001 was bioengineered as a unique fusion protein consisting of a VWF-D'D3 domain fused to rFVIII via immunoglobulin-G1 Fc domains and 2 XTEN polypeptides (Amunix Pharmaceuticals, Inc, Mountain View, CA). Plasma FVIII half-life after BIVV001 administration in mice and monkeys was 25 to 31 hours and 33 to 34 hours, respectively, representing a three- to fourfold increase in FVIII half-life. Our results showed that multifaceted protein engineering, far beyond a few amino acid substitutions, could significantly improve rFVIII pharmacokinetic properties while maintaining hemostatic function. BIVV001 is the first rFVIII with the potential to significantly change the treatment paradigm for severe hemophilia A by providing optimal protection against all bleed types, with less frequent doses. The protein engineering methods described herein can also be applied to other complex proteins.