Figure 1.
Non-Reducing SDS-PAGE analysis and schematic of FVIII variants.
(A) SC rFVIIIFc R1645A/R1648A, SC rFVIIIFc, fully processed rFVIIIFc and rFVIIIFc [10] were compared by non-reducing SDS-PAGE. (B) Schematic presentation of SC rFVIIIFc, fully processed rFVIIIFc, and the common thrombin-activated form.
Table 1.
Specific activity of SC rFVIIIFc compared to fully processed rFVIIIFc and rFVIIIFc by chromogenic and one-stage (aPTT) assays (n = 3).
Figure 2.
Thrombin generation profile of FVIII variants.
Activity determination was based on equal chromogenic activity. Representative curves with selected concentrations are shown in (A) 1 IU/mL (B) 0.25 IU/mL (C) 0.0625 IU/mL. Select parameters are shown in (D) as peak thrombin and (E) as ETP.
Figure 3.
Activity within FXa generation assays.
(A) Affinity of FIXa to different FVIII molecules on synthetic phospholipids surface (B) FXa generation rates for Xase Complexes formed with different FVIII molecules. A representative curve is shown. All results are listed in Table 2.
Table 2.
Comparison of FIXa binding to FVIIIa within Xase complex and FX interaction with Xase complex assembled with FVIIIa on synthetic phospholipids (n = 6, Mean ± SD) [10].
Table 3.
Affinities of SC rFVIIIFc and rFVIIIFc for VWF by SPR (n = 6, Mean ± SD) [10].
Figure 4.
Thrombin-mediated release of activated FVIII variants from VWF.
(A) Schematic of the optical biosensor method developed to evaluate the dependence of VWF release rates on thrombin concentration. A detailed description is provided in Materials and Methods. (B–D) Determination of thrombin concentrations corresponding to half-maximal release rates (EC50) for (B) rFVIIIFc, (C) SC rFVIIIFc, and (D) BDD rFVIII. For BDD rFVIII, ordinate values were normalized on a molar basis to account for the molecular weight difference between rFVIIIFc and SC rFVIIIFc (220 kDa), and BDD rFVIII (170 kDa).
Figure 5.
PK profiles of SC rFVIIIFc and rFVIIIFc in HemA mice (single dose 250 IU/kg).
Results shown are mean ± SD from 4 mice per treatment at each time point. The PK parameter estimates are summarized in Table 4.
Figure 6.
SC rFVIIIFc and the rFVIIIFc demonstrated equivalent in vivo efficacy in a HemA mouse bleeding model.
Tail-bleeding of HemA mice was induced via transection of one of the lateral veins at 48 hr post i.v. dosing of SC rFVIIIFc or rFVIIIFc (0.46, 1.38 and 4.6 µg/kg). Mice were monitored for survival and re-bleeding for 24 hr post injury. (A) Schematic experimental design of TVT model. (B) 24 hr post TVT survival curves (C) 24 hr post TVT re-bleeding curves from experimental animals. (D) The linear regression curve of the percentage of protection (survival rate) versus the log (base 10) of the dose was plotted. The ED50 was extrapolated from the curves.
Table 4.
Summary of PK parameters for SC rFVIIIFc and rFVIIIFc.