BACKGROUND:The X-linked bleeding disorder hemophilia B, caused by mutation(s) in the coagulation factor (F)IX 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 (i.v.) injection remains. OBJECTIVES:The study aimed to characterize rIX-FP variants with anticipated enhanced specific activity, which would leverage rIX-FP's superior pharmacokinetic profile with beneficial characteristics for subcutaneous (s.c.) administration. METHODS:Two rIX-FP variants, R338L ("Padua variant") and R338L/E410K, were characterized in vitro. Pharmacokinetic profiles of FIX antigen and activity levels were evaluated in FIX-deficient mice after i.v. and s.c. administration of these variants (dosing based on antigen levels). The efficacy of the most promising variant was tested after i.v. and s.c. administration (dosing based on activity) in a tail clip bleeding model. A marketed wild-type (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 with rIX(WT)-FP, while FXIa-mediated activation was the fastest for rIX(WT)-FP and rIX(R338L)-FP. Compared with rIX(WT)-FP and rIX(R338L/E410K)-FP, rIX(R338L)-FP exhibited higher FIX activity exposure after i.v. and s.c. administration and demonstrated comparable efficacy with rIX(WT)-FP in reducing bleeding time and blood loss in FIX-deficient mice requiring ∼4 times lower protein amount. CONCLUSION:rIX(R338L)-FP was shown to be a promising candidate for s.c. administration, exhibiting increased specific activity combined with higher activity-based exposure and indicating efficacy at a lower protein dose.

Introduction: Hemophilia B (HB) is a X-linked bleeding disorder characterized by the deficiency in coagulation factor IX (FIX). Replacement treatment with recombinant human FIX (rFIX) is a safe approach to prevent bleeding in HB patients. There is an emerging concept that FIX resides extravascularly, by potentially binding to collagen IV in the subendothelial basement membrane, where it contributes to hemostasis. Our work evaluates hemostatic efficacy of extravascular FIX using animal models of tail clip bleeding and ferric chloride (FeCl3)-induced thrombus formation. Methods: The pharmacokinetic (PK) profile of wildtype rFIX (rFIXWT) and rFIX mutants which are stated to have an enhanced (rFIXK5R) or reduced (rFIXK5A) binding to components of the extravascular space (EVS) was evaluated in C57BL/6J HB mice (B6.129P2-F9). Blood and liver samples were collected at 0.08, 24, and 72 hours after tail vein administration of rFIX proteins (n=3-5 per timepoint). Plasma activity levels of rFIX were determined by a one stage clotting assay (OSCA). The content of FIX in the liver sections was analyzed using immunofluorescence staining. The direct binding of rFIX to collagen IV was evaluated for the first-time using surface plasma resonance (SPR). The in vivo efficacy of these proteins in HB mice was compared in tail clip bleeding and FeCl3-induced thrombus formation models. Results: PK studies in HB mice showed that the total exposure was higher for the rFIXK5A mutant which exhibited a dose normalized (dn) AUC0-last of 0.442±0.025 IU/mL*h/IU, followed by lower dnAUC0-last of rFIXWT (0.069±0.023 IU/mL*h/IU) and rFIXK5R (0.056±0.031 IU/mL*h/IU). The predicted dose normalized maximum concentrations (dnCmax) at 5 minutes post administration were comparable between rFIXWT (0.013±0.003 IU/mL/IU), rFIXK5R (0.013±0.001 IU/mL/IU) and slightly higher for rFIXK5A (0.018±0.001 IU/mL/IU). At 5 minutes following intravenous injection, all three rFIX proteins were detected in liver sections with similar mean fluorescence intensities (MFI). However, at 24 hours following intravenous injection, rFIXK5R had higher MFI compared to rFIXK5A and rFIXWT groups (rFIXK5R vs. rFIXWT p<0.001). rFIXK5R was detectable in the liver tissue up to 72 hours even in the absence of circulating FIX antigen levels (Figure 1). Though rFIXK5A was detected in the plasma until 336 hours, the presence in liver tissue was observed only at 5 minutes. Human FIX and murine collagen IV co-staining in liver sections of rFIX treated HB mice showed an overlap of FIX staining mostly with collagen IV-stained regions. However, there were certain liver vascular beds stained specifically for collagen IV with no detectable FIX in that region. To further investigate this, direct binding of FIX to collagen IV was evaluated in SPR and no binding was observed between collagen IV and rFIX wildtype or rFIX mutant proteins. In the tail clip bleeding model, rFIXK5R displayed significant hemostatic protection against bleeding incidence for up to 72 hours post intravenous administration, whereas for rFIXK5A this was only observed at the 15 minutes time point. In the mesenteric artery thrombus model, the time to occlusion of the artery was comparable in both rFIXK5R and rFIXWT treated groups. In contrast, rFIXK5A lacked the ability to form occlusive clots and was comparable to vehicle group. In line with this observation, rFIXWT and rFIXK5R treated HB mice developed significantly larger clots. In contrast, rFIXK5A mutant with decreased binding to components of the EVS exhibited smaller clots compared to rFIXK5R and rFIXWT. Conclusion: The results of the PK study confirmed a longer exposure of rFIXK5A in plasma compared to rFIXK5R, as expected from decreased binding affinity of rFIXK5A to components of the EVS. The histological evaluations of the harvested liver tissues confirmed a longer accumulation of rFIXK5R, which is expected to have increased binding affinity to components of the EVS. The efficacy studies revealed that the ability of FIX to bind to components of the EVS has an impact on the hemostatic response in HB mouse models. Using two different in vivo models of hemostasis, we demonstrate that mutated rFIX protein with enhanced binding to components of the EVS (rFIXK5R) confers prolonged hemostatic efficacy while the duration of efficacy of rFIXK5A was relevantly shorter after intravenous administration of the recombinant proteins. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

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.

Introduction: The recessive X-linked bleeding disorder Hemophilia B is caused by a mutation in the coagulation factor (F) IX gene leading to partial or total loss of its function. Preventive treatment with replacement long-acting FIX is an attractive option for patients to reduce administration frequency and prevent bleeding. New recombinant FIX therapeutics like the albumin-fused FIX (rFIX-FP) or the Fc-fused FIX (rFIX-Fc) enable longer half-life in circulation and thus less frequent administration, as compared to non-fused FIX (rFIX). Studies in FIX knockout (KO) mice were conducted to characterize the effect of the modifications on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the different recombinant FIX products.

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.

Introduction: Current state of the art treatment of severe Hemophilia A is prophylactic or on-demand replacement of the deficient factor VIII (FVIII) using plasma derived or recombinant factors [Keeling 2008; Srivastava 2012; Wong 2011]. The compliance to existing therapies is hampered by the short half-life of available FVIII products necessitating multiple intravenous injections per week for adequate prophylaxis [Bjorkman 2009; Sheridan 2011]. Thus, FVIII products with improved pharmacokinetic profiles are currently under development or have recently been made available [Oldenburg 2014]. FVIII's pharmacokinetic (PK) properties are believed to be strongly dictated by its binding to von Willebrand Factor (VWF) [Lenting 2007; Dasgupta 2007; Saenko 2006]. The aim of this study was to compare the binding affinity of different recombinant FVIII (rFVIII) products to plasma derived VWF (pd VWF), and characterize their PK properties in different animal species.

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.