Institute of Experimental Biomedicine I, Josef-Schneider-Straße 2, 97080 Würzburg, Germany.
Rudolf Virchow Center, Center for Integrative and Translational Bioimaging, University of Wuerzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany.
Eur Heart J. 2024 Nov 14;45(43):4582-4597. doi: 10.1093/eurheartj/ehae482.
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, KD: 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; KD: 0.195 nM) was developed that inhibits hGPVI function with very high potency in vitro and in vivo.
Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6tg/tg) mouse platelets. The in vivo effect of Emf6.1Fab 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 Emf6.1Fab (EMA601) was generated. Emf6.1Fab/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry.
Emf6.1Fab (KD: 0.427 nM) blocked GPVI function in human and hGP6tg/tg mouse platelets in multiple assays in vitro at concentrations ≥5 µg/mL. Emf6.1Fab (4 mg/kg)-treated hGP6tg/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. Emf6.1Fab binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (KD: 0.195 nM) and fully inhibited the receptor at 0.5 µg/mL, corresponding to a >50-fold potency compared with ACT017.
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.
糖蛋白 VI（GPVI）是血小板胶原/纤维蛋白原（原）受体，也是治疗血栓形成和血栓炎症性疾病的新兴药物靶点，尤其是缺血性中风。一种新型抗人 GPVI（hGPVI）抗体 Fab 片段（ACT017/glenzocimab，KD：4.1 nM）最近在急性缺血性中风患者中通过了临床 1b/2a 期研究，结果显示其具有良好的耐受性、安全性和潜在益处。在这项研究中，开发了一种新型人源化抗 GPVI 抗体 Fab 片段（EMA601；KD：0.195 nM），该片段在体外和体内以非常高的效价抑制 hGPVI 功能。
测试了抗 hGPVI IgG Emf6.1 的 Fab 片段在人血小板中和表达 hGPVI 的（hGP6tg/tg）小鼠血小板中对 GPVI 功能的抑制作用。在尾出血试验、动脉血栓形成模型和短暂性大脑中动脉闭塞（tMCAO）缺血性中风模型中评估了 Emf6.1Fab 的体内作用。使用互补决定区移植，生成了 Emf6.1Fab 的人源化版本（EMA601）。使用阵列格式和生物层干涉测量法映射了 Emf6.1Fab/EMA601 与 hGPVI 的相互作用，并对其动力学和定量进行了量化。
Emf6.1Fab（KD：0.427 nM）以≥5μg/ml 的浓度在多种体外测定中阻断了人源和 hGP6tg/tg 小鼠血小板中的 GPVI 功能。Emf6.1Fab（4mg/kg）治疗的 hGP6tg/tg 小鼠表现出强烈的 hGPVI 抑制作用，并能显著防止动脉血栓形成以及 tMCAO 后的脑梗死生长，而尾巴出血时间不受影响。Emf6.1Fab 结合到 GPVI 中一个迄今为止尚未描述的膜近端表位。人源化变体 EMA601 对 hGPVI 的亲和力进一步增加（KD：0.195 nM），在 0.5μg/ml 时完全抑制受体，与 ACT017 相比效力增加了>50 倍。
EMA601 是一种概念新颖、有前途的抗血小板药物，可有效预防或治疗人类高危人群的动脉血栓形成和血栓炎症性疾病。
