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

Abstract

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.

Figure 1

Schematic depiction of N8-GP before and after thrombin activation. N8-GP corresponds to FVIII (turoctocog alfa) PEGylated with a 40-kDa PEG on the O-linked glycan in the 21-aa B-domain. After cleavage with thrombin, the activated molecule has the same primary structure as native FVIIIa.

Figure 2

Reduced LRP binding of N8-GP. LRP was immobilized to a CM5 chip in a Biacore T100 instrument to 4000 to 5000 resonance units (RUs), and (A) FVIII (turoctocog alfa), (B) N8-GP, or (C) receptor-associated protein (RAP) at 12.5 nM, 3.13 nM, 0.78 nM, and 0.2 nM allowed to associate for 400 seconds and dissociate for 600 seconds using a flow of 10 µL/min.

Figure 3

Binding and uptake of N8-GP in human monocyte-derived dendritic cells. (A-B) Monocytes were differentiated into dendritic cells by 5 days of culturing with granulocyte macrophage–colony-stimulating factor and interleukin 4 before incubating with ¹²⁵I–N8-GP or ¹²⁵I-FVIII for 24 hours at 4°C. Unspecific binding determined by adding 4 µM unlabeled N8-GP or FVIII was subtracted. (C) Internalization was assessed at 37°C for up to 3 hours using concentrations at K_d (ie, 110 nM ¹²⁵I–N8-GP and 70 nM ¹²⁵I-FVIII). Data are mean and standard deviation from 4 (A) or 3 (B-C) experiments.

Figure 4

Acute effect of N8-GP in a tail bleeding model in FVIII-deficient mice. The mice received N8-GP or FVIII (Advate) intravenously at the indicated doses, and tail bleeding was measured after clipping 4 mm of the tail tip 5 minutes after dosing. Dose-response curves of total bleeding time (A) and blood loss (B) are shown for N8-GP (closed circles, black line) and FVIII (open diamonds, dotted lines). A blood loss of 1000 nmol hemoglobin approximates 0.125 mL whole blood using a hemoglobin concentration of 8 mmol/L. Control animals receiving the vehicle only are shown with open circles. Data are mean ± standard error of the mean (SEM) of n = 12 mice per group.

Figure 5

Prolonged effect of N8-GP in a tail bleeding model in FVIII-deficient mice. The mice were dosed intravenously with 200 U/kg of N8-GP (open bars) or FVIII (Advate, solid bars), and total bleeding time (A) and blood loss (B) were measured in the tail bleeding model after 5 minutes and 1, 2, or 3 days after treatment. The gray bars correspond to FVIII-deficient mice, and the hatched bars to normal mice (C57BL/6). Data are mean ± SEM of n = 6 to 12 mice per group. ***P < .001 indicates significant difference from FVIII-deficient mice. *P < .05 and **P < .01 indicate significant differences between mice treated with FVIII and N8-GP.

Figure 6

Prolonged effect of N8-GP in a FeCl3-induced injury model in FVIII-deficient mice. Occlusion time after FeCl₃-induced injury was measured 5 minutes (acute effect), 24 hours, 48 hours, 60 hours, 72 hours, and 96 hours after dosing of 280 U/kg N8-GP (open circles) or FVIII (Advate; closed circles). The dotted line indicates the observation period for the experiments; during this period, no occlusion was recorded in the vehicle-treated mice. Mean and SEM of n = 6 to 10 mice per group are shown. *P < .01; **P < .05 N8-GP compared with FVIII at individual time points.

Figure 7

Prolonged effect of N8-GP in a joint bleeding model in FVIII-deficient mice. N8-GP (A, open bars; B, open circles) or FVIII (Advate) (A, solid bars; B, solid circles) were administered intravenously to the mice at 280 U/kg, and needle-induced joint bleeding in the knee induced at the time points indicated after dosing. The gray bar (A) or gray circle (B) correspond to FVIII-deficient mice, and the hatched bar (A) and solid square (B) to normal mice (C57BL/6). The bleeding was evaluated 24 hours after injury, and VBS (A) and change in joint diameter (B) determined. A VBS of 3 indicates tense and distorted knees, whereas 0 designates a virtually nonaffected knee. Data are mean and SEM of 10 mice per group at time points 5 minutes, 24 hours, 72 hours, and 88 hours, and 15 mice per group at 36 to 60 hours after injury. *P < .05; **P < .01; and *** P < .001 indicate significant difference between treated and nontreated FVIII-deficient mice. Above the bar in (A), ***P < .001 indicates significant difference between FVIII-deficient and C57BL/6 control mice.