Novel conformation-specific monoclonal antibodies against amyloidogenic forms of transthyretin

<i>Introduction</i>: Transthyretin amyloidosis (ATTR amyloidosis) is caused by the misfolding and deposition of the transthyretin (TTR) protein and results in progressive multi-organ dysfunction. TTR epitopes exposed by dissociation and misfolding are targets for immunotherapeutic antibodies. We developed and characterized antibodies that selectively bound to misfolded, non-native conformations of TTR. <i>Methods</i>: Antibody clones were generated by immunizing mice with an antigenic peptide comprising a cryptotope within the TTR sequence and screened for specific binding to non-native TTR conformations, suppression of <i>in vitro</i> TTR fibrillogenesis, promotion of antibody-dependent phagocytic uptake of mis-folded TTR and specific immunolabeling of ATTR amyloidosis patient-derived tissue. <i>Results</i>: Four identified monoclonal antibodies were characterized. These antibodies selectively bound the target epitope on monomeric and non-native misfolded forms of TTR and strongly suppressed TTR fibril formation <i>in vitro</i>. These antibodies bound fluorescently tagged aggregated TTR, targeting it for phagocytic uptake by macrophage THP-1 cells, and amyloid-positive TTR deposits in heart tissue from patients with ATTR amyloidosis, but did not bind to other types of amyloid deposits or normal tissue. <i>Conclusions</i>: Conformation-specific anti-TTR antibodies selectively bind amyloidogenic but not native TTR. These novel antibodies may be therapeutically useful in preventing deposition and promoting clearance of TTR amyloid and in diagnosing TTR amyloidosis.

<i>引言</i>：运甲状腺素蛋白淀粉样变性（ATTR amyloidosis）是由运甲状腺素蛋白（transthyretin, TTR）错误折叠并沉积引发的疾病，可导致进行性多器官功能障碍。因解离与错误折叠而暴露的TTR表位，是免疫治疗抗体的潜在靶点。本研究开发并表征了可选择性结合TTR错误折叠非天然构象的抗体。
<i>方法</i>：以包含TTR序列内隐匿表位的抗原肽免疫小鼠以生成抗体克隆，并针对多项特性进行筛选：特异性结合非天然TTR构象、抑制体外（in vitro）TTR纤维形成、促进抗体依赖性吞噬摄取错误折叠TTR，以及特异性免疫标记ATTR淀粉样变性患者来源的组织样本。
<i>结果</i>：本研究共表征了4株单克隆抗体。这些抗体可选择性结合TTR单体及非天然错误折叠形式的目标表位，并在体外可强力抑制TTR纤维形成。上述抗体可结合荧光标记的聚集态TTR，介导巨噬细胞THP-1细胞对其的吞噬摄取，同时可特异性标记ATTR淀粉样变性患者心脏组织中的TTR淀粉样阳性沉积，但不会结合其他类型的淀粉样沉积或正常组织。
<i>结论</i>：构象特异性抗TTR抗体可选择性结合致淀粉样变性的TTR，而非天然状态的TTR。这些新型抗体或可用于预防TTR淀粉样蛋白沉积、促进其清除，以及用于TTR淀粉样变性的临床诊断。