Characterization of recombinant Factor IX fusion proteins enabling subcutaneous administration.

BACKGROUND: The X-linked bleeding disorder Hemophilia B, caused by mutation(s) in the coagulation factor IX (FIX) gene, leads to partial or total loss of its function requiring lifelong FIX replacement therapy. Although new recombinant FIX (rIX) therapeutics like albumin-fusion proteins (rIX-FP) enable longer plasma half-life and thus less frequent administration, the complexity of intravenous (IV) injection remains. OBJECTIVES: The study aims to characterize rIX-FP variants with anticipated enhanced specific activity, which would leverage rIX-FP's superior pharmacokinetic (PK) profile with beneficial characteristics for subcutaneous (SC) administration. METHODS: Two rIX-FP variants, R338L ("Padua-variant") and R338L/E410K, were characterized in vitro. PK profiles of FIX antigen and activity levels were evaluated in FIX-deficient mice after IV and SC administration of these variants (dosing based on antigen levels). The efficacy of the most promising variant was tested after IV and SC administration (dosing based on activity) in a tail-clip bleeding model. A marketed wildtype (WT) rIX-FP product served as the comparator. RESULTS: Both rIX-FP variants showed a 4- to 5-fold increase in specific activity in vitro compared to rIX(WT)-FP, whilst FXIa-mediated activation was the fastest for rIX(WT)-FP and rIX(R338L)-FP. Compared to rIX(WT)-FP and rIX(R338L/E410K)-FP, rIX(R338L)-FP exhibited higher FIX activity exposure after IV and SC administration, and demonstrated comparable efficacy towards rIX(WT)-FP in reducing bleeding time and blood loss in FIX-deficient mice requiring ∼4 times lower protein amount. CONCLUSIONS: rIX(R338L)-FP was shown to be a promising candidate for SC administration, exhibiting increased specific activity combined with higher activity-based exposure, and indicating efficacy at lower protein dose.

Modulation of Extravascular Binding of Recombinant Factor IX Impacts the Duration of Efficacy in Mouse Models.

BACKGROUND: There is an emerging concept that in addition to circulating coagulation factor IX (FIX), extravascular FIX contributes to hemostasis. OBJECTIVE: Our objective was to evaluate the efficacy of extravascular FIX using animal models of tail clip bleeding and ferric chloride-induced thrombosis. METHODS: Mutant rFIX proteins with described enhanced (rFIXK5R) or reduced (rFIXK5A) binding to extracellular matrix were generated and characterized using in vitro aPTT, one-stage clotting, and modified FX assays. Using hemophilia B mice, pharmacokinetic (PK) parameters and in vivo efficacy of these proteins were compared against rFIX wild-type protein (rFIXWT) in a tail clip bleeding and FeCl3-induced thrombosis model. Respective tissue disposition of FIX was evaluated using immunofluorescence. RESULTS: In vitro characterization demonstrated comparable clotting activity of rFIX proteins. The PK profile showed that rFIXK5A displayed the highest plasma exposure compared to rFIXWT and rFIXK5R. Immunofluorescence evaluation of liver tissue showed that rFIXK5R was detectable up to 24 hours, whereas rFIXWT and rFIXK5A were detectable only up to 15 minutes. In the tail clip bleeding model, rFIXK5R displayed significant hemostatic protection against bleeding incidence for up to 72 hours postintravenous administration of 50 IU/kg, whereas the efficacy of rFIXK5A was already reduced at 24 hours. Similarly, in the mesenteric artery thrombus model, rFIXK5R and rFIXWT demonstrated prolonged efficacy compared to rFIXK5A. CONCLUSION: Using two different in vivo models of hemostasis and thrombosis, we demonstrate that mutated rFIX protein with enhanced binding (rFIXK5R) to extravascular space confers prolonged hemostatic efficacy in vivo despite lower plasma exposure, whereas rFIXK5A rapidly lost its efficacy despite higher plasma exposure.

Increased potency of recombinant VWF D'D3 albumin fusion proteins engineered for enhanced affinity for coagulation factor VIII.

BACKGROUND: We have recently reported on a recombinant von Willebrand factor (VWF) D'D3 albumin fusion protein (rD'D3-FP) developed to extend the half-life of coagulation factor VIII (FVIII) for the treatment of hemophilia A. Based on predictive modelling presented in this study, we hypothesized that modifying rD'D3-FP to improve FVIII interaction would reduce exchange with endogenous VWF and provide additional FVIII half-life benefit. OBJECTIVES: The aim of this study was to identify novel rD'D3-FP variants with enhanced therapeutic efficacy in extending FVIII half-life. METHODS: Through both directed mutagenesis and random mutagenesis using a novel mammalian display platform, we identified novel rD'D3-FP variants with increased affinity for FVIII (rVIII-SingleChain) under both neutral and acidic conditions and assessed their ability to extend FVIII half-life in vitro and in vivo. RESULTS: In rat preclinical studies, rD'D3-FP variants with increased affinity for FVIII displayed enhanced potency, with reduced dose levels required to achieve equivalent rVIII-SingleChain half-life extension. In cell-based imaging studies in vitro, we also demonstrated reduced dissociation of rVIII-SingleChain from the rD'D3-FP variants within acidic endosomes and more efficient co-recycling of the rD'D3-FP/rVIII-SingleChain complex via the FcRn recycling system. CONCLUSIONS: In summary, at potential clinical doses, the rD'D3-FP variants provide marked benefits with respect to dose levels and half-life extension of co-administered FVIII, supporting their development for use in the treatment of hemophilia A.

FVIII half-life extension by coadministration of a D'D3 albumin fusion protein in mice, rabbits, rats, and monkeys.

A novel mechanism for extending the circulatory half-life of coagulation factor VIII (FVIII) has been established and evaluated preclinically. The FVIII binding domain of von Willebrand factor (D'D3) fused to human albumin (rD'D3-FP) dose dependently improved pharmacokinetics parameters of coadministered FVIII in all animal species tested, from mouse to cynomolgus monkey, after IV injection. At higher doses, the half-life of recombinant FVIII (rVIII-SingleChain) was calculated to be increased 2.6-fold to fivefold compared with rVIII-SingleChain administered alone in rats, rabbits, and cynomolgus monkeys, and it was increased 3.1-fold to 9.1-fold in mice. Sustained pharmacodynamics effects were observed (ie, activated partial thromboplastin time and thrombin generation measured ex vivo). No increased risk of thrombosis was observed with coadministration of rVIII-SingleChain and rD'D3-FP compared with rVIII-SingleChain alone. At concentrations beyond the anticipated therapeutic range, rD'D3-FP reduced the hemostatic efficacy of coadministered rVIII-SingleChain. This finding might be due to scavenging of activated FVIII by the excessive amount of rD'D3-FP which, in turn, might result in a reduced probability of the formation of the tenase complex. This observation underlines the importance of a fine-tuned balance between FVIII and its binding partner, von Willebrand factor, for hemostasis in general.

Non-clinical pharmacokinetics and pharmacodynamics of rVIII-SingleChain, a novel recombinant single-chain factor VIII.

INTRODUCTION: rVIII-SingleChain (CSL627), a novel recombinant coagulation factor VIII (FVIII), is under investigation in a phase I/III clinical programme (AFFINITY) for the treatment of haemophilia A. Non-clinical studies were conducted to investigate the pharmacokinetic/pharmacodynamic profile of rVIII-SingleChain in comparison with full-length recombinant FVIII. MATERIALS AND METHODS: Binding affinity of rVIII-SingleChain for von Willebrand factor was investigated by surface plasmon resonance analysis. The pharmacokinetic profile of rVIII-SingleChain was compared with a marketed full-length recombinant FVIII concentrate (Advate(®)) in haemophilia A mice, von Willebrand factor knock-out mice, Crl:CD (SD) rats, rabbits and cynomolgus monkeys. Systemic FVIII activity or antigen levels were recorded. Procoagulant activity was measured in an FeCl3-induced arterial occlusion model and by recording thrombin generation activity (ex vivo) after administration of 200-250 IU/kg rVIII-SingleChain or full-length FVIII to haemophilia A mice. RESULTS: rVIII-SingleChain displayed a high affinity for von Willebrand factor (KD=44 pM vs. 139 pM for full-length recombinant FVIII). In all animal species tested, rVIII-SingleChain had more favourable pharmacokinetic properties than full-length recombinant FVIII: clearance was decreased and area under the curve and terminal half-life were enhanced vs. full-length recombinant FVIII, while in vivo recovery and volume of distribution were equivalent. rVIII-SingleChain showed a prolonged thrombin generation potential and prolonged procoagulant activity vs. full-length recombinant FVIII in an FeCl3-induced arterial occlusion model. CONCLUSIONS: rVIII-SingleChain had a higher affinity for von Willebrand factor than full-length recombinant FVIII and displayed favourable pharmacokinetic/pharmacodynamic properties in non-clinical models.