Thrombin-mediated AutoRegulation of FVIII-mimetic antibodies enabling dynamic control of the hemostatic response.

Emicizumab has transformed hemophilia A (HA) management by enabling effective subcutaneous prophylaxis independent of inhibitor status. However, its constitutive activity and lack of sensitivity to anticoagulant feedback contribute to excessive thrombin generation under certain conditions, particularly when combined with bypassing agents, a liability increasingly recognized as a class effect of FVIII-mimetic antibodies. We engineered an "AutoRegulated" (AR) FVIII-mimetic antibody by integrating a short thrombin cleavable peptide sensitive to the proteolytic activity of thrombin in the hinge region of both emicizumab arms. Insertion of the peptide did not impair structural integrity and biological function with AR-emicizumab demonstrating EC50 values of FVIII-mimetic activity and correction of blood loss in HA mice comparable to the parental emicizumab. However, the presence of thrombin-mediated AR enabled thrombin-responsive proteolytic inactivation upon exposure to supra-physiological thrombin levels, whereas the parental emicizumab remained unaffected. The dynamic control provided by AR efficiently reduced the procoagulant effects induced by co-administration of activated Prothrombin Complex Concentrates (aPCC). In thrombin generation assays, AR-emicizumab significantly attenuated the excessive thrombin peak observed with emicizumab. Similarly, in vivo, the presence of thrombin-mediated AR prevented platelet consumptive coagulopathy and reduced the microvascular pulmonary thrombosis induced by the parental molecule without affecting the systemic levels of the therapeutic antibody. This on-demand self-limiting mechanism aligns therapeutic activity with local hemostatic needs, may offer a promising therapeutic approach for HA patients, while establishing a generalizable strategy to improve the safety profile of next-generation hemostatic biologics.