Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of systemic amyloidosis. The amyloidogenic gelsolin fragment AGelD187N does not play any physiological role in the body, unlike most aggregating proteins related to other protein misfolding diseases. However, no therapeutic agents that specifically and effectively target and neutralize AGelD187N exist. We used phage display technology to identify novel single-chain variable fragments that bind to different epitopes in the monomeric AGelD187N that were further maturated by variable domain shuffling and converted to antigen-binding fragment (Fab) antibodies. The generated antibody fragments had nanomolar binding affinity for full-length AGelD187N, as evaluated by biolayer interferometry. Importantly, all four Fabs selected for functional studies efficiently inhibited the amyloid formation of full-length AGelD187N as examined by thioflavin fluorescence assay and transmission electron microscopy. Two Fabs, neither of which bound to the previously proposed fibril-forming region of AGelD187N, completely blocked the amyloid formation of AGelD187N. Moreover, no small soluble aggregates, which are considered pathogenic species in protein misfolding diseases, were formed after successful inhibition of amyloid formation by the most promising aggregation inhibitor, as investigated by size-exclusion chromatography combined with multiangle light scattering. We conclude that all regions of the full-length AGelD187N are important in modulating its assembly into fibrils and that the discovered epitope-specific anti-AGelD187N antibody fragments provide a promising starting point for a disease-modifying therapy for gelsolin amyloidosis, which is currently lacking.

中文翻译：

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表位特异性抗体片段阻断 AGelD187N 的聚集，AGelD187N 是凝溶胶蛋白淀粉样变性中的一种异常肽


血浆凝溶胶蛋白异常片段 AGelD187N 的聚集是芬兰凝溶胶蛋白淀粉样变性（一种遗传性系统性淀粉样变性）病理生理学的关键事件。淀粉样蛋白生成凝溶胶蛋白片段 AGelD187N 在体内不起任何生理作用，这与大多数与其他蛋白质错误折叠疾病相关的聚集蛋白不同。然而，尚无特异性和有效靶向和中和 AGelD187N 的治疗剂。我们使用噬菌体展示技术来识别与单体 AGelD187N 中不同表位结合的新型单链可变片段，这些片段通过可变结构域改组进一步成熟并转化为抗原结合片段 （Fab） 抗体。通过生物膜干涉测定法评估，生成的抗体片段对全长 AGelD187N 具有纳摩尔结合亲和力。重要的是，通过硫黄素荧光测定和透射电子显微镜检查，选择用于功能研究的所有 4 个 Fab 都有效地抑制了全长 AGelD187N 的淀粉样蛋白形成。两个 Fab 均未与先前提出的 AGelD187N 原纤维形成区域结合，完全阻断了 AGelD187N 的淀粉样蛋白形成。此外，正如通过尺寸排阻色谱结合多角度光散射进行研究的那样，在最有前途的聚集抑制剂成功抑制淀粉样蛋白形成后，没有形成小的可溶性聚集体，这些聚集体被认为是蛋白质错误折叠疾病中的致病物种。 我们得出结论，全长 AGelD187N 的所有区域在调节其组装成原纤维方面都很重要，并且发现的表位特异性抗 AGelD187N 抗体片段为目前缺乏的凝溶胶蛋白淀粉样变性疾病修饰疗法提供了一个有希望的起点。