Development of a Novel Recombinant Factor VIII Product through Transgene Engineering and Lentiviral-Driven Expression

The increased cost of goods associated with low-level expression of factor VIII (fVIII) is considered a significant factor in the pricing of recombinant fVIII products. Worldwide treatment of hemophilia A is limited due to the cost of fVIII products, which easily can exceed $100,000 per year. Using state-of-the-art commercial production facilities and technology, recombinant human (h)-fVIII expresses at 100 – 1000-fold lower levels than other plasma proteins. We have developed a novel recombinant fVIII product that overcomes the primary limitation to the manufacture of currently available hemophilia A pharmaceuticals. There are two components to this product:

1. a humanized highexpression hybrid-human/porcine (HP) fVIII transgene, designated ET-801i, and

2. the LENTIMAX^™ lentiviral production system.

The ET-801i transgene contains elements of porcine fVIII (p-fVIII) sequence in the A1 and A3 domains that facilitate more efficient secretion from production cell lines and is 90% identical in amino acid sequence to h-fVIII. Additionally, lentiviral introduction of the transgene allows achievement of an optimal number of transgene copies and the lentiviral vectors typically integrate into sites of active chromatin, thus facilitating high-levels of transgene transcription. In the current study, we demonstrated that high-expression porcine sequence elements enable 20 – 100- fold superior expression over any previously described fVIII transgene variant. Using a stable baby hamster kidney cell-derived expression system, ET-801i was expressed at a level indistinguishable from p-fVIII, which both were significantly greater than h-fVIII (<0.001, Mann-Whitney U test). After scale up, ET-801i was purified using the same two-step ion exchange procedure that has been used previously to purify p-fVIII and other high-expression HP-fVIII constructs. This purification scheme does not involve an immunoaffinity step, which greatly simplifies the process and downstream quality control. The purity of ET-801i was assessed to be greater than 95% by SDS-PAGE and ET-801i displayed a specific activity of 3,400 units/nmol. Treatment of ET-801i with thrombin and endoglycosidase PNGase F resulted in a decrease in M_r for the A1 and A3-C1-C2 (light chain) domain fragments. No change in the M_r of the A2 domain was observed. These data suggest correct glycosylation of ET-801i. Using an enzyme-linked immunosorbent assay, ET-801i was demonstrated to bind von Willebrand factor indistinguishably from p-fVIII. Therefore, ET-801i should display similar pharmacokinetics to recombinant p-fVIII, which has been studied in phase 1 and 2 clinical trials. Furthermore, thrombinactivated ET-801i displays the slow decay property of activated p-fVIII (T_1/2 = 6 min), which is approximately 3-fold slower than h-fVIII. It is likely that slow decay increases the in vivo functionality of activated fVIII and may allow lower dosing in hemophilia A patients leading to further cost reduction and an improved safety profile by reducing immunogenicity. These results indicate that the combined technological advancements of high-expression fVIII elements and lentiviral-driven gene transfer and expression can be utilized to provide a potentially safer recombinant fVIII at a lower cost than current h-fVIII products and thus better support patients with hemophilia A.