Montoliu-Gaya Laia, Murciano-Calles Javier, Martinez Jose C, Villegas Sandra
a Protein Folding and Stability Group, Departament de Bioquímica i Biologia Molecular , Unitat de Biociències, Universitat Autònoma de Barcelona , Spain.
b Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences , University of Granada, Campus Fuentenueva , Granada , Spain.
Amyloid. 2017 Sep;24(3):167-175. doi: 10.1080/13506129.2017.1348347. Epub 2017 Jul 12.
ScFv-h3D6 is a single-chain variable fragment derived from the monoclonal antibody bapineuzumab that prevents Aβ-induced cytotoxicity by capturing Aβ oligomers. The benefits of scFv-h3D6 treatment in Alzheimer's disease are known at the behavioural, cellular and molecular levels in the 3xTg-AD mouse model. Antibody-based therapeutics are only stable in a limited temperature range, so their benefit in vivo depends on their capability for maintaining the proper fold. Here, we have stabilized the scFv-h3D6 folding by introducing the mutation V-K64R and combining it with the previously described elongation of the V domain (C3). The stabilities of the different scFv-h3D6 constructs were calculated from urea and thermal denaturation followed by Trp-fluorescence, CD and DSC and resulted in the order C3 > K64R/C3 > V-K64R ≥ scFv-h3D6; showing that the combination of both mutations was not additive, instead they partially cancelled each other. The three mutants assayed showed a decreased aggregation tendency but maintained their capability to aggregate in the form of worm-like fibrils, basis of the protective effect of scFv-h3D6. Cytotoxicity assays showed that all the mutants recovered cell viability of Aβ-treated neuroblastoma cell cultures in a dose-dependent manner and with efficiencies that correlated with stability, therefore improving the therapeutic ability of this antibody.
单链抗体片段scFv-h3D6源自单克隆抗体巴宾纽单抗，可通过捕获Aβ寡聚体来预防Aβ诱导的细胞毒性。在3xTg-AD小鼠模型中，scFv-h3D6治疗阿尔茨海默病的益处已在行为、细胞和分子水平上得到证实。基于抗体的治疗剂仅在有限的温度范围内稳定，因此它们在体内的益处取决于其维持正确折叠的能力。在此，我们通过引入V-K64R突变并将其与先前描述的V结构域延伸（C3）相结合，稳定了scFv-h3D6的折叠。通过尿素和热变性实验，随后进行色氨酸荧光、圆二色性（CD）和差示扫描量热法（DSC），计算了不同scFv-h3D6构建体的稳定性，结果为C3＞K64R/C3＞V-K64R≥scFv-h3D6；这表明两种突变的组合不是累加性的，而是部分相互抵消。所检测的三个突变体显示出聚集倾向降低，但仍保持以蠕虫状纤维形式聚集的能力，这是scFv-h3D6发挥保护作用的基础。细胞毒性实验表明，所有突变体均以剂量依赖的方式恢复了Aβ处理的神经母细胞瘤细胞培养物的细胞活力，且效率与稳定性相关，从而提高了这种抗体的治疗能力。
