Genetic Fusion to Factor XIII B-Subunit Improves the Pharmacokinetic Properties of Factor IX

Abstract

Background: Hemophilia B is a X-linked recessive bleeding disorder characterized by coagulation factor IX (FIX) deficiency. FIX concentrates are used in the prevention and treatment of bleeding episodes in patients with hemophilia B. Prophylaxis is currently considered as the optimal care for severe hemophilia. For patients and their families one of the major problems with prophylaxis is the need for frequent venepunctures. The half-life of standard FIX concentrates is approximately 18 h, which requires 2 or 3 intravenous infusions per week to achieve bleeding prevention. Prolonging the half-life of FIX can therefore reduce the frequency of infusions. Recently, extended half-life FIX concentrates have been developed using two major strategies: i) (glyco) PEGylation(1), ii) genetic fusion to immunoglobulin Fc fragments(2), or to albumin(3). These technologies may have some potential disadvantages such as reduced specific activity, unknown long-term adverse effects of PEG exposure and immunological reactivity which are currently under investigation.The aim of the present study was to develop a new strategy to prolong half-life of recombinant human FIX molecule (rFIX) using an innovative approach of fusion.Factor XIII (FXIII) is a protransglutaminase that circulates in plasma as a heterotetramer composed of two catalytic A subunits and two B subunits (FXIIIB). FXIIIB subunits act as a carrier protein that stabilizes the structure of the A subunits and protects FXIII from proteolysis. It has been reported that a primary deficiency of FXIIIB resulted in the secondary rapid loss of A subunit from the plasma (4). Thus, FXIII-B is responsible for the long half-life of the FXIII molecule (10 to 12 days).We designed a new rFIX molecule fused to coagulation FXIII subunit B (FXIIIB) and separated by a short linker sequence containing an activated Factor X-cleavable site (rFIX-LXa-FXIIIB). Thus, we expected that the carrier protein will be removed as soon as traces of (activated factor X) FXa will be generated, leaving rFIX free to perform its enzymatic role in the tenase complex.Methods: Recombinant FIX-LXa-FXIIIB molecules were expressed in human hepatic Huh-7 cell line and purified via a two-step protocol prior to in vitro and in vivo biochemical and pharmacokinetic characterizations.Results: rFIX-LXa-FXIIIB was correctly expressed by Huh-7 cells at the expected molecular weight of 150 kDa and presented similar procoagulant activity compared to rFIX-WT (106.5 UI/dL ; p> 0.05). We showed that the LXa linker was cleavable by FXa, releasing free rFIX molecule before its activation. After cleavage, rFIX was fully activable by activated FXI. Both rFIX-WT and rFIX-LXa-FXIIIB showed similar thrombin generation capacity both in vitro and in vivo after injection in FIX-deficient mice (Area under the thrombin generation curve =300nM.min ; p>0.05).The half-life (T1/2) of rFIX-LXa-FXIIIB molecule was determined in wild-type mice and rats. In mice, as commercially available extended half-life FIX products, the improvement of the half-life was very limited and in rats, it was 2.5 fold longer compared to rFIX-WT (T1/2 10h vs 4h ; p<0.05).A potential advantage of this new molecule is its capacity to bind to fibrinogen via FXIIIB. It has been recently reported that excess plasma FXIIIB2 is not free, but rather circulates bound to fibrinogen (5). We can hypothesize that the rFIX-LXa-FXIIIB fusion protein can circulate bound to fibrinogen and might therefore enhance/accelerate the transformation of fibrinogen into fibrin. In vivo experiments using mice saphenous vein bleeding model are currently conducted in our laboratory to test this hypothesis.Conclusion: We report here a new fusion protein strategy using FXIIIB. The fusion protein has normal specific activity and normal thrombin generating capacity, associated with improved pharmacokinetics in small animal models. These encouraging results suggest that genetic fusion of rFIX to FXIIIB might be a valuable concept to improve the half-life of FIX molecules. The binding capacity of this novel fusion protein to fibrinogen might be an additional advantage that may improve haemostatic capacity of the molecule.1. Negrier C et al. Blood. 2011;118:2695-701.2. Peters RT et al. Blood. 2010;115:2057-64.3. Metzner HJ et al. Thromb Haemost. 2009;102:634-44.4. Miloszewski K et al. Br J Haematol. 1970;19:685-90.5. Byrnes JR et al. Blood. 2016 Oct 13;128:1969-1978 DisclosuresLe Quellec:LFB: Honoraria; Novo Nordisk: Honoraria; Shire: Honoraria. Negrier:Baxter: Research Funding; Alnylam: Research Funding; Bayer: Honoraria, Research Funding, Speakers Bureau; Biogen/SOBI: Honoraria, Research Funding, Speakers Bureau; CSL Behring: Honoraria, Research Funding, Speakers Bureau; Inspiration: Research Funding; Novo Nordisk: Honoraria, Research Funding, Speakers Bureau; Octapharma: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau; LFB: Honoraria, Speakers Bureau. Dargaud:BMS: Honoraria, Research Funding; Baxalta/Shire: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; LFB: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Stago: Honoraria; Octapharma: Honoraria, Research Funding; Alnylam: Research Funding.