A FVIII-Mimetic Bispecific Antibody with an Embedded Self-Regulation Mechanism Reduces the Risk of Prothrombotic Events for the Treatment of Haemophilia a

Background Market approval of emicizumab, a Factor VIII (FVIII) mimetic bispecific antibody (bsAb) simultaneously binding coagulation Factor IX (FIX) and Factor X (FX), has significantly changed the treatment paradigm for haemophilia A (HA) patients. In contrast to FVIII, emicizumab is constitutively active and cannot be "turned off" by the natural regulation loop of the clotting cascade mediated by activated protein C (APC) and therefore remains in a permanent procoagulant state. Consequently, emicizumab is associated with a high incidence of thromboembolic events and thrombotic microangiopathies, particularly when combined with activated prothrombin complex concentrates (aPCC). The development of a next-generation FVIII-mimetic antibody equipped with a physiological self-regulation mechanism that can inactivate the bsAb when sufficient coagulation has occurred to sustain a stable clot and prevent the progression of thrombotic events may, therefore, serve as a safer therapeutic option for HA patients.

Methods A prototype of self-regulated FVIII-mimetic bsAb (SR-Ab8) was designed using a short thrombin-sensitive cleavable peptide inserted in the structure of emicizumab. FVIII-mimetic activity and inactivation were evaluated in vitro in a chromogenic assay measuring FIXa-mediated activation of FX. Thrombin Generation Assay (TGA) triggered with tissue factor was then used to characterize the prothrombotic profile of both antibodies (at 600nM) in human HA plasma in presence of aPCC (0.5U/mL). To assess the bleeding correction, FVIII knock-out (FVIIIKO) mice were injected with 3mg/kg of SR-Ab8 or emicizumab followed by a mixture of human FIX and FX. A tail clip assay was performed, and blood loss was recorded for 30min. The safety profile of SR-Ab8 was further tested in a thrombosis model where C57Bl/6 mice received a 1^st injection with SR-Ab8 or emicizumab (250µg/mice) followed by 4 injections of aPCC (2.5U/mice) every 24h to induce thrombosis. Immunofluorescent staining for vessel walls (PECAM) and platelets (αIIbβ3) was then performed on lung sections to quantify the percentage of blood vessel occlusion.

Results The kinetics of FX activation generated by SR-Ab8 or emicizumab were similar, thus indicating that the structural modification resulting from the insertion of a thrombin-sensitive peptide does not affect the basal FVIII-mimetic activity. However, when pre-exposed to purified thrombin, SR-Ab8 was progressively inactivated and eventually showed a complete loss of FVIII-mimetic activity. TGA was then used to evaluate the impact of self-regulation on thrombin generation potential. When spiked in HA plasma containing aPCC, emicizumab generated a supraphysiologic peak of thrombin with a peak height of 189% of normal plasma. In contrast, a peak height of thrombin generation equivalent to that of normal plasma was observed with SR-Ab8 (panel A). In FVIIIKO mice, SR-Ab8 and emicizumab mediated comparable reduction in total blood loss compared to untreated animals, thus demonstrating that the procoagulant biological activity had not been compromised by the addition of a thrombin-sensitive peptide. Finally, in C57Bl/6 mice that had received emicizumab followed by aPCC, immunofluorescent staining of platelets in the lungs revealed the presence of thrombi in blood vessels with an increase in the frequency of vessel occlusion (average 11.6%) when compared to animals only treated with aPCC (4.7%). Mice treated with SR-Ab8 instead of emicizumab showed a significantly lower vessel occlusion rate (6.3%) that was comparable to that of the control animals (panel B).

Conclusion The engineering of emicizumab using a carefully selected thrombin-sensitive peptide that is cleaved at a set high threshold of thrombin retains the haemostatic potential of the parental molecule whilst reducing the risk of thrombosis, as a consequence of a built-in negative feedback loop in vitro and in vivo. Therefore, this study heralds a new class of therapeutics for haemophilia A with a self-regulating off switch designed to enhance safety without compromising efficacy.

Christophe:Roche: Research Funding. Lenting:Sobi: Research Funding; Sanofi: Research Funding; Roche: Research Funding; Pfizer: Research Funding. Granger:Haleon: Current equity holder in publicly-traded company; GSK: Current equity holder in publicly-traded company, Patents & Royalties. Chester:ADC Therapeutics: Patents & Royalties; Informa PLC: Membership on an entity's Board of Directors or advisory committees; Allen & Overy LLP: Consultancy; UCL Business: Patents & Royalties; Novalgen: Consultancy, Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties. Nathwani:Biomarin: Research Funding; Genethon: Membership on an entity's Board of Directors or advisory committees.