PB0225 - An engineered factor X variant as a novel by-passing agent for hemophilia

Background: In hemophilia with inhibitors, by-passing agents are aimed at increasing thrombin generation efficiency by restoring or boosting the activation of factor X (FX). However, although activated FX (FXa) should be the ideal therapeutic molecule, it is rapidly inhibited and displays very short half-life in vivo. In this context, the availability of a by-passing FX zymogen molecule would be of great relevance, with potentially low immunogenicity due to the presence of circulating FX in patients.

Aims: To develop a novel hyper-activatable engineered FX variant (FXHA-BP, patent application in progress) able to by-pass the defective intrinsic pathway in hemophilia plasma.

Methods: The expression plasmid for the FXHA-BP variant was produced by site-directed mutagenesis of wild-type FX (FXwt) cDNA and transiently transfected into HEK293 cells. Secreted FX protein levels were evaluated by ELISA. Activity was assessed by PT-based (FX-deficient plasma) and aPTT-based (hemophilia A plasma) assays.

Results: The FXHA-BP variant was secreted at appreciable levels from transiently transfected HEK293 cells. As compared to FXwt, the specific activity of FXHA-BP was slightly increased in FX-deficient plasma (1.5±0.4 fold; p=0.0414) in PT-based assays. Importantly, upon intrinsic activation in aPTT-based assays, addition of 500 ng/ml FXHA-BP to hemophilia A plasma significantly shortened coagulation times (105±2 sec) over the baseline (118±4 sec; p< 0.0001), whereas addition of an equivalent amount of FXwt, used as control, resulted in negligible effects (117±4 sec; p< 0.0001). These data demonstrated the by-passing activity of the FXHA-BP variant even at the relatively low concentrations obtained after transient expression.

Conclusion(s): Through rational engineering we created a novel improved FX variant acting as a by-passing agent in hemophilia plasma. The FXHA-BP molecule will be further engineered to prolong its half-life, with the aim of developing an extended half-life by-passing variant that might represent a novel therapeutic tool for hemophilia with inhibitors.