Rational design of a novel engineered factor X with chimeric activation peptide as bypassing agent for hemophilia

While replacement therapy in hemophilia (H)A and B (HB) has provided remarkable improvements, the development of neutralizing antibodies remains a significant complication, with rational engineering of bypassing (BP) molecules being a relevant issue.

This study developed a human factor (F)X variant containing a FIX-derived chimeric activation peptide (AP) driving FVIII or FIX deficiency bypass.

Engineered variants were expressed and purified, and activation/activity properties were evaluated through functional assays, also in plasma with inhibitors from patients with HA and HB.

After screening a panel of recombinant variants, the most promising were purified for functional characterization. Unlike wild-type FX (FX^WT), the BP variants FX^BP4b and FX^BP4c at physiological concentrations effectively normalized the clotting time in HA and HB plasma. Chromogenic assays confirmed that both variants preserved activation via the extrinsic tenase complex or by Russell viper venom X, while the intrinsic activation pathway was impaired, thus favoring their selective dose-dependent FXIa activation. In thrombin generation assays, FX^BP4b-c restored the peak and the endogenous thrombin potential in FX-deficient plasma. Remarkably, the FX^BP4b outperformed FX^WT in HA and HB plasma and showed a comparable or superior activity in comparison with B-domainless FVIII and FIX. Notably, FX^BP4b significantly shortened the clotting time in plasma from patients with high-titer anti-FVIII or anti-FIX inhibitory antibodies, with efficacy levels comparable with those of well-established BP agents.

These results support FX^BP4b as a promising and versatile BP agent able to sustain coagulation across multiple hemophilic contexts including inhibitors presence, while maintaining favorable activation profiles.