Department of Chemistry & Biochemistry, University of Missouri-St. Louis, St. Louis, Missouri, USA.
ExonBio, San Diego, California, USA.
J Neurochem. 2023 Jun;165(6):860-873. doi: 10.1111/jnc.15817. Epub 2023 Apr 19.
Aggregation and accumulation of amyloid-β peptide (Aβ) are a critical trigger for the onset of Alzheimer's disease (AD). While the plaques are the most outstanding Aβ pathological feature, much of the recent research emphasis has been on soluble Aβ species because of their diffusible, proinflammatory, and toxic properties. The focus on soluble aggregated Aβ species has also increased the interest in antibodies that are selective for different Aβ conformations. In the current study, we developed and characterized a new class of monoclonal antibodies (referred to as mAbSL) that are selective for Aβ protofibrils. Cloning and sequencing of the heavy and light chain variable regions for multiple antibodies identified sequence characteristics that may impart the conformational selectivity by the antibodies. Transfection of FreeStyle 293F cells with the plasmids permitted in-house expression and purification of mAbSL antibodies along with non-conformation-selective Aβ monoclonal antibodies (Aβ mAbs). Several of the purified mAbSL antibodies demonstrated significant affinity and selectivity for Aβ42 protofibrils compared with Aβ42 monomers and Aβ42 fibrils. Competition ELISA assays assessing the best overall antibody, mAbSL 113, yielded affinity constants of 7 nM for the antibody-Aβ42 protofibril interaction, while the affinity for either Aβ42 monomers or Aβ42 fibrils was roughly 80 times higher. mAbSL 113 significantly inhibited Aβ42 monomer aggregation by a unique mechanism compared with the inhibition displayed by Aβ mAb 513. Aβ42 protofibril dynamics were also markedly altered in the presence of mAbSL 113, whereby insoluble complex formation and protofibril deposition were stimulated by the antibody at low substoichiometric molar ratios. As the field contemplates the therapeutic effectiveness of Aβ conformation-selective antibodies, the findings presented here demonstrate new information on a monoclonal antibody that selectively targets Aβ protofibrils and impacts Aβ dynamics.
淀粉样蛋白-β肽（Aβ）的聚集和积累是阿尔茨海默病（AD）发病的关键触发因素。虽然斑块是最显著的 Aβ病理特征，但由于其可扩散、促炎和毒性，最近的研究重点主要集中在可溶性 Aβ 物种上。对可溶性聚集的 Aβ 物种的关注也增加了对针对不同 Aβ构象的抗体的兴趣。在目前的研究中，我们开发并表征了一类新的单克隆抗体（称为 mAbSL），它们对 Aβ原纤维具有选择性。针对多种抗体的重链和轻链可变区进行克隆和测序，确定了可能通过抗体赋予构象选择性的序列特征。用质粒转染 FreeStyle 293F 细胞，允许在内部表达和纯化 mAbSL 抗体以及非构象选择性 Aβ单克隆抗体（Aβ mAb）。几种纯化的 mAbSL 抗体与 Aβ42 单体和 Aβ42 纤维相比，对 Aβ42 原纤维表现出显著的亲和力和选择性。评估最佳整体抗体 mAbSL 113 的竞争 ELISA 测定得出该抗体与 Aβ42 原纤维相互作用的亲和力常数为 7 nM，而与 Aβ42 单体或 Aβ42 纤维的亲和力大约高 80 倍。与 Aβ mAb 513 相比，mAbSL 113 通过一种独特的机制显著抑制了 Aβ42 单体的聚集。mAbSL 113 的存在还明显改变了 Aβ42 原纤维的动力学，在低亚化学计量摩尔比下，抗体刺激不溶性复合物形成和原纤维沉积。随着该领域考虑 Aβ 构象选择性抗体的治疗效果，这里提出的发现提供了有关选择性针对 Aβ 原纤维并影响 Aβ 动力学的单克隆抗体的新信息。
