Abstract
Recombinant factor VIII Fc (rFVIIIFc) is a fusion protein consisting of a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC), we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF), with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc.
Highlights
Hemophilia A is an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII) activity [1, 2]
Chelation of the divalent cations of FVIII was used to disrupt the heavy chain (HC):light chain (LC) interactions, creating three distinct molecules that were separated through a combination of anion exchange and an affinity chromatography steps, and the divalent cations were reintroduced in specific concentrations to allow for the re-establishment of the HC:LC interactions
single chain (SC) rFVIIIFc was analyzed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1A, lane 2) and the protein was detected at approximately 220 kDa, consistent with the predicted molecular weight for SC rFVIIIFc, and only trace amounts of processed rFVIIIFc (LC-Fc and HC) were detected
Summary
Hemophilia A is an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII) activity [1, 2]. Prophylaxis is considered the standard of the care [1, 2], compliance with the regimen is hampered by the short half-life (,12 hours) of FVIII that requires dosing every other day or three times per week by intravenous injection to maintain a minimum plasma level of 1% of normal coagulation factor activity [3, 4]. RFVIIIFc consists of a single molecule of B-domain deleted (BDD) rFVIII covalently linked to the dimeric human Fc region from IgG1 with no intervening linker sequence. RFVIIIFc is expressed as two polypeptide chains, one chain consisting of the Fc domain (hinge, CH2 and CH3) of human IgG1, the other chain consisting of BDD rFVIII fused to the same Fc region. The B domain deletion is created by fusing Ser 743 (S743) to Gln 1638 (Q1638) with respect to the full length FVIII sequence resulting in a 14 amino acid sequence from the original B domain [10]
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