Human Factor VIII Variants with Diminished PACE-Furin Cleavage Exhibit Increased Biological Activity and Therapeutic Efficacy

During intracellular processing, factor VIII (FVIII) undergoes proteolysis at multiple sites with the most predominant cleavage at a Paired basic Amino acid Cleaving Enzyme (PACE)-furin cleavage site at the carboxy-terminus of the B-domain at residue 1648 to give rise to two polypeptide chains, the heavy chain and the light chain. Through a metal ion dependent association, these chains form a heterodimer that is the secreted form of the protein. Previously, we observed that canine B-domain deleted FVIII (cFVIII-BDD) is secreted primarily as a single chain (SC) molecule (170kD) while human FVIII-BDD (hFVIII-BDD) is secreted predominantly as a heterodimer. In addition, cFVIII-BDD is more stable and has higher biological activity. Amino acid sequence analysis revealed a single amino acid difference between cFVIII (1645-HHQR-1648) and hFVIII (1645-RHQR-1648) at the PACE-furin cleavage recognition site. Characterization of hFVIII-R1645H demonstrated that this residue is responsible for the secretion of cFVIII predominantly as a single polypeptide chain and its inherent increased stability and specific activity. Furthermore, hFVIII-R1645H has enhanced hemostatic effects upon vascular injury and in the setting of AAV delivery was associated with increased circulating levels compared to wild type hFVIII-BDD. These data suggest that there is suboptimal cleavage of cFVIII and hFVIII-R1645H by PACE-furin. Here we tested whether deletion of part or all of the PACE-furin recognition sequence may further decrease the efficiency of cleavage at the site resulting in a larger portion of single SC molecules and thus may confer increased stability compared to hFVIII-R1645H. A series of hFVIII-BDD deletion variants (n=5) of the PACE-furin cleavage recognition site (1645-1648) were generated: del1645, del1645-46, del1645-47, del1645-48 and del1648. Stable BHK cell lines were established for each variant and protein was purified using ion exchange chromatography. On an SDS-PAGE gel under reducing conditions the deletion variants migrate in a similar manner, however, the proportion of the hFVIII in the single chain form compared to hFVIII-BDD (15% SC) was 3.5-fold higher for del1645-46 (57% SC), del1645-47 (53% SC) and del1645-48 (48% SC) which is similar to R1645H (52% SC). The single deletion variants on SDS-PAGE were 2-fold higher (del1645, 34% SC) or identical (del1648, 15% SC) to hFVIII-BDD. In a one-stage aPTT assay, all variants had activity similar to hFVIII-BDD. While in the two-stage aPTT assay, three variants (del1645-46, del1645-47, del1645-48) had 2-fold higher activity than hFVIII-BDD. Variants del1645 and del1648 had similar activity to hFVIII-BDD. Using an A2 domain dissociation clotting-based assay, we found that del1645-46, del1645-47, del1645-48 and del1648 were more stable following thrombin (IIa) activation compared to hFVIII-BDD suggesting that the A2 domain of these variants may dissociate more slowly than the wild type human A2 domain following IIa cleavage. These findings explain, at least in part, the superior clotting activity determined in multiple assays. The hFVIII PACE-furin variants were also introduced into an adeno-associated viral vector serotype 8 (AAV8-hAAT-hFVIII-BDD) for liver targeted delivery. Each AAV8-hAAT-hFVIII-P/F variant was delivered to hemophilia A mice (5x1011vector genomes/mouse)(n=6/group). At 4 weeks post vector administration, the hFVIII expression of del1645-47 was 3-fold higher than hFVIII-BDD followed by del1645, del1645-48, del1645-46 while del1648 was similar to hFVIII-BDD. In summary, these in vitro data demonstrate that deletion of residues 1645-46, 1645-47 or 1645-48 of the PACE-furin cleavage recognition site confers 2-3-fold higher biological activity than wild type hFVIII-BDD. In vivo these variants result in 2-3 fold higher levels of hFVIII expression after AAV delivery compared to wild type hFVIII-BDD. Together these data suggest that the hFVIII-P/F deletion variants have enhanced hemostatic function and are superior to wild type hFVIII. In the setting of gene-based therapeutics for hemophilia A, these novel variants provide a unique strategy for increasing factor VIII expression which will allow the use of a lower vector dose which is a critical improvement for translation of this approach. Disclosures Nguyen: Pfizer, Inc.: Research Funding. Camire: Pfizer: Patents & Royalties, Research Funding. Sabatino: Spark Therapeutics: Research Funding; Pfizer, Inc.: Research Funding.