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

Background: While replacement therapy in Haemophilia A (HA) and B (HB) has provided remarkable improvements, the development of neutralizing antibodies remains a significant complication, with rational engineering of bypassing molecules being a relevant issue. Objectives: To develop a human factor X (FX) variant containing a FIX-derived chimeric activation peptide (AP) driving FVIII or FIX deficiency bypass. Methods: Engineered variants were expressed and purified, and activation/activity properties were evaluated through functional assays, also in plasma with inhibitors from HA and HB patients. Results: After screening a panel of recombinant variants, the most promising were purified for functional characterization. Unlike wild-type (FXWT), the bypassing (BP) variants FXBP4b and FXBP4c 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 Russel Viper Venom (RVV)-X, while the intrinsic activation pathway was impaired, thus favouring their selective dose-dependent FXIa activation. In thrombin generation assays, FXBP4b-c restored the peak and the endogenous thrombin potential (ETP) in FX-deficient plasma. Remarkably, the FXBP4b outperformed FXWT in HA and HB plasma and showed a comparable or superior activity in comparison with B-domainless FVIII and FIX. Notably, FXBP4b 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 bypassing agents. Conclusions: These results support FXBP4b as a promising and versatile bypassing agent able to sustain coagulation across multiple haemophilic contexts including inhibitors presence, while maintaining favourable activation profiles.

Background 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. Objectives This study developed a human factor (F)X variant containing a FIX-derived chimeric activation peptide (AP) driving FVIII or FIX deficiency bypass. Methods 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. Results After screening a panel of recombinant variants, the most promising were purified for functional characterization. Unlike wild-type FX (FXWT), the BP variants FXBP4b and FXBP4c 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, FXBP4b-c restored the peak and the endogenous thrombin potential in FX-deficient plasma. Remarkably, the FXBP4b outperformed FXWT in HA and HB plasma and showed a comparable or superior activity in comparison with B-domainless FVIII and FIX. Notably, FXBP4b 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. Conclusion These results support FXBP4b as a promising and versatile BP agent able to sustain coagulation across multiple hemophilic contexts including inhibitors presence, while maintaining favorable activation profiles.