The humanized platelet glycoprotein VI Fab inhibitor EMA601 protects from arterial thrombosis and ischaemic stroke in mice.

Background and Aims Glycoprotein VI (GPVI) is a platelet collagen/fibrin(ogen) receptor and an emerging pharmacological target for the treatment of thrombotic and thrombo-inflammatory diseases, notably ischaemic stroke. A first anti-human GPVI (hGPVI) antibody Fab-fragment (ACT017/glenzocimab, K _D: 4.1 nM) recently passed a clinical phase 1b/2a study in patients with acute ischaemic stroke and was found to be well tolerated, safe, and potentially beneficial. In this study, a novel humanized anti-GPVI antibody Fab-fragment (EMA601; K _D: 0.195 nM) was developed that inhibits hGPVI function with very high potency in vitro and in vivo. Methods Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6^tg/tg) mouse platelets. The in vivo effect of Emf6.1^Fab was assessed in a tail bleeding assay, an arterial thrombosis model and the transient middle cerebral artery occlusion (tMCAO) model of ischaemic stroke. Using complementary-determining region grafting, a humanized version of Emf6.1^Fab (EMA601) was generated. Emf6.1^Fab/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry. Results Emf6.1^Fab (K _D: 0.427 nM) blocked GPVI function in human and hGP6^tg/tg mouse platelets in multiple assays in vitro at concentrations ≥5 µg/mL. Emf6.1^Fab (4 mg/kg)-treated hGP6^tg/tg mice showed potent hGPVI inhibition ex vivo and were profoundly protected from arterial thrombosis as well as from cerebral infarct growth after tMCAO, whereas tail-bleeding times remained unaffected. Emf6.1^Fab binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (K _D: 0.195 nM) and fully inhibited the receptor at 0.5 µg/mL, corresponding to a >50-fold potency compared with ACT017. Conclusions EMA601 is a conceptually novel and promising anti-platelet agent to efficiently prevent or treat arterial thrombosis and thrombo-inflammatory pathologies in humans at risk. [ABSTRACT FROM AUTHOR]