抑制胰岛淀粉样多肽纤维化的单区抗体的构建及其作用机制

Amyloid formation plays a critical role in a range of human diseases including Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes. Human islet amyloid polypeptide (IAPP) is a 37-residue polypeptide which is the major component of the pancreatic islet amyloid associated with type 2 diabetes. To develop highly efficient inhibitors of amyloid formation remains as a very popular yet extremely challenging research area. For our proposed work, we will develop a systematic approach to generate potent inhibitors that can effectively inhibit IAPP amyloid formation at low substoichiometric concentrations. The design of inhibitors starts from grafting small hydrophobic peptide sequences from IAPP into the complementary determining region (CDR3) of a single antibody domain (VH), yielding a new VH domain (desAb) that can bind to their cognate IAPP via homotypic interactions. In this proposed study, we will 1) design and prepare a series of desAbs by grafting IAPP fragments with different lengths and sequences, 2) systematically investigate their binding ability to the cognate IAPP, 3) study their inhibition efficacy and specificity towards IAPP amyloid formation. In addition, we will investigate how varying the number of grafted VH domain (multi domains) will impact the inhibitory and binding activity of the resulting antibodies. This proposed research project will be of significant value to both understanding the mechanism of amyloid formation and developing novel potent antibody therapeutics of type 2 diabetes.

胰岛淀粉样多肽（IAPP）可错误折叠而聚集形成淀粉样蛋白纤维，此过程和二型糖尿病紧密相关。淀粉样蛋白纤维化抑制剂的设计是非常热门但同时又极具挑战性的工作。本项目拟以单区重链多变区抗体（VH）为模板，在VH的互补决定区（CDR3）嫁接不同IAPP多肽片段，所生成的嫁接抗体（desAb）可利用同源相互作用与IAPP结合从而抑制IAPP的聚集和淀粉样蛋白纤维的生成。本项目将设计一系列嫁接不同区域和不同长度IAPP多肽的desAb，并设计具有多结合区域的抗体，系统研究此类抗体与IAPP的结合和构象特异性，以及对IAPP淀粉样蛋白纤维化和细胞毒性的抑制效果，进而得到新型高效，可以在极低化学计量浓度有效阻止淀粉样蛋白纤维生成的抗体抑制剂。本项目拟发展高效的淀粉样蛋白的抗体抑制剂，从结构方面理解抗体抑制淀粉样蛋白纤维生成的机理，并为二型糖尿病等淀粉样蛋白相关疾病药物的研发开拓新的领域。

胰岛淀粉样多肽（IAPP）的聚集过程与型糖尿病紧密相关。发展高效的抑制IAPP聚集的抑制剂极具挑战性。本项目以单区重链多变区抗体（VH）为模板，在VH的互补决定区（CDR3）嫁接不同能够与IAPP相互作用的多肽片段，所生成的嫁接抗体（desAb）通过CDR3的嫁接片段与IAPP结合从而抑制IAPP的聚集。本项目首先构建了嫁接不同区域和不同长度的IAPP多肽片段的单区抗体（desAb），发现嫁接不同IAPP多肽片段的desAb均能有效抑制IAPP聚集。另外，随着嫁接长度的增加，其与IAPP结合力和抑制IAPP聚集的效果增强。因此，嫁接能够与IAPP作用的多肽到单区抗体的CDR能够有效提高其抑制效果。基于以上研究结果，构建了嫁接能够与IAPP结合的多肽YYYY（4Y）到VH的CDR3，所生成的嫁接抗体N4Y由于限制4Y的构象并提高其稳定性，从而提升4Y与IAPP的结合力,增强其阻止IAPP聚集的能力。进一步将N4Y与高分子偶联得到复合物PDN4Y，发展了一种分级放大IAPP聚集抑制剂活性的策略，为设计高效的淀粉样蛋白聚集抑制剂提供基础；在此基础上还发展了基于限制与IAPP作用多肽构象的抗体模拟物以阻止淀粉样多肽的聚集以及降解淀粉样蛋白聚集体的策略用于淀粉样病变的研究。本项目拓展了现有淀粉样蛋白抑制剂的设计思路，为淀粉样病变药物的研发提供新策略。