Key Points hC4Nb8 is a potent cross-reactive inhibitor of CP and LP proconvertase formation. hC4Nb8 binds to a neoepitope exposed upon C4 activation and C4b generation. hC4Nb8 inhibits the CP of complement in vivo. The classical and lectin pathways of the complement system are important for the elimination of pathogens and apoptotic cells and stimulation of the adaptive immune system. Upon activation of these pathways, complement component C4 is proteolytically cleaved, and the major product C4b is deposited on the activator, enabling assembly of a C3 convertase and downstream alternative pathway amplification. Although excessive activation of the lectin and classical pathways contributes to multiple autoimmune and inflammatory diseases and overexpression of a C4 isoform has recently been linked to schizophrenia, a C4 inhibitor and structural characterization of the convertase formed by C4b is lacking. In this study, we present the nanobody hC4Nb8 that binds with picomolar affinity to human C4b and potently inhibits in vitro complement C3 deposition through the classical and lectin pathways in human serum and in mouse serum. The crystal structure of the C4b:hC4Nb8 complex and a three-dimensional reconstruction of the C4bC2 proconvertase obtained by electron microscopy together rationalize how hC4Nb8 prevents proconvertase assembly through recognition of a neoepitope exposed in C4b and reveals a unique C2 conformation compared with the alternative pathway proconvertase. On human induced pluripotent stem cell–derived neurons, the nanobody prevents C3 deposition through the classical pathway. Furthermore, hC4Nb8 inhibits the classical pathway-mediated immune complex delivery to follicular dendritic cells in vivo. The hC4Nb8 represents a novel ultrahigh-affinity inhibitor of the classical and lectin pathways of the complement cascade under both in vitro and in vivo conditions.

中文翻译：

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超高亲和力补体 C4b 特异性纳米抗体抑制经典途径前转化酶的体内组装

关键点 hC4Nb8 是一种有效的 CP 和 LP 转化酶原形成的交叉反应抑制剂。hC4Nb8 与 C4 激活和 C4b 生成时暴露的新表位结合。hC4Nb8 在体内抑制补体的 CP。补体系统的经典途径和凝集素途径对于消除病原体和凋亡细胞以及刺激适应性免疫系统很重要。这些途径激活后，补体成分 C4 被蛋白水解裂解，主要产物 C4b 沉积在激活剂上，从而能够组装 C3 转化酶和下游旁路途径扩增。尽管凝集素和经典途径的过度激活会导致多种自身免疫性和炎症性疾病，并且 C4 亚型的过度表达最近与精神分裂症有关，缺乏由 C4b 形成的转化酶的 C4 抑制剂和结构特征。在这项研究中，我们提出了纳米抗体 hC4Nb8，它以皮摩尔亲和力与人 C4b 结合，并通过人血清和小鼠血清中的经典和凝集素途径有效抑制体外补体 C3 沉积。C4b:hC4Nb8 复合物的晶体结构和通过电子显微镜获得的 C4bC2 转化原酶的三维重建共同合理化了 hC4Nb8 如何通过识别暴露在 C4b 中的新表位来阻止转化酶原组装，并与替代途径转化酶原相比揭示了独特的 C2 构象. 在人类诱导的多能干细胞衍生神经元上，纳米抗体通过经典途径阻止 C3 沉积。此外，hC4Nb8 在体内抑制经典途径介导的免疫复合物向滤泡树突细胞的传递。hC4Nb8 代表了一种在体外和体内条件下补体级联的经典和凝集素途径的新型超高亲和力抑制剂。