A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models.

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model.

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide.

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

Administration of recombinant FVIIa (rFVIIa) to concizumab-dosed monkeys is safe, and concizumab does not affect the potency of rFVIIa in hemophilic rabbits.

Essentials Hemophilia patients on concizumab prophylaxis may need rFVIIa to treat breakthrough bleeds. Effect and safety of concizumab + rFVIIa were tested in vitro and in vivo. Concizumab + rFVIIa had no additive effects on bleeding in hemophilic rabbits. High steady-state levels of concizumab did not affect the safety of rFVIIa in cynomolgus monkeys. SUMMARY: Background Concizumab is a monoclonal antibody (mAb) against tissue factor pathway inhibitor (TFPI), currently in clinical development as a subcutaneous prophylactic therapy for hemophilia A/B with and without inhibitors. In patients with inhibitors, the treatment choice for breakthrough bleeding will comprise bypassing agents, e.g. activated recombinant FVIIa (rFVIIa) or activated prothrombin complex concentrates. Objectives To explore the effect and safety of concizumab and rFVIIa when they are simultaneously present. Methods Human blood made hemophilic with a FVIII antibody was spiked with increasing concentrations of concizumab, rFVIIa, or concizumab and rFVIIa in combination, and this was followed by thrombin generation test or thromboelastography. Blood loss in hemophilic rabbits was measured when concizumab, rFVIIa or concizumab + rFVIIa was administered either before or during cuticle bleeding. In a safety study, cynomolgus monkeys were exposed to high steady-state concizumab concentrations and given three doses of rFVIIa, and then subjected to full necropsy and histopathological examination. Results In human blood, concizumab + rFVIIa had more pronounced procoagulant effects under hemophilic conditions than the sum of individual responses. In contrast, concizumab + rFVIIa had no additional effects on blood loss in hemophilic rabbits as compared with rFVIIa or concizumab alone. In cynomolgus monkeys, the macroscopic and microscopic pathological examinations revealed no thrombi or other signs of excessive coagulation activation. Both rFVIIa and concizumab caused increases in thrombin-antithrombin and D-dimer concentrations; this effect tended to be additive with concomitant administration. Conclusions Concizumab did not affect the potency or safety of rFVIIa in vivo. These results support a clinical evaluation of rFVIIa at standard dose (90 µg kg-1 ) to treat breakthrough bleeds in concizumab clinical trials.