Department of Biosciences, Paris-Lodron University of Salzburg, Salzburg, Austria.
Christian Doppler Laboratory for Innovative Tools for Biosimilar Characterization, Paris-Lodron University of Salzburg, Salzburg, Austria.
PLoS One. 2020 Nov 5;15(11):e0241560. doi: 10.1371/journal.pone.0241560. eCollection 2020.
The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides with a distinct fold, can be used to detect minor modifications or structural variations of a molecule or protein. To detect antibody molecules in a fold state occurring prior to protein precipitation, we generated DNA aptamers that were selected for extensively heat-treated rituximab. Using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX), we obtained six DNA aptamer sequences (40-mers) specific for 80°C heat-treated rituximab. In silico fold prediction and circular dichroism analysis revealed a G-quadruplex structure for one aptamer, while all others exhibited a B-DNA helix. Binding affinities ranging from 8.8-86.7 nM were determined by an enzyme-linked apta-sorbent assay (ELASA). Aptamers additionally detected structural changes in rituximab treated for 5 min at 70°C, although with lower binding activity. Notably, none of the aptamers recognized rituximab in its native state nor did they detect the antibody after it was exposed to lower temperatures or different physical stressors. Aptamers also reacted with the therapeutic antibody adalimumab incubated at 80°C suggesting similar aptamer binding motifs located on extensively heat-treated IgG1 antibodies. Within this work, we obtained the first aptamer panel, which is specific for an antibody fold state specifically present prior to protein aggregation. This study demonstrates the potential of aptamer selection for specific stress-based protein variants, which has potential impact for quality control of biopharmaceuticals.
单克隆抗 CD20 IgG1 抗体利妥昔单抗被用作 B 细胞淋巴瘤的一线治疗药物。与所有治疗性抗体一样，它是一种复杂的蛋白质，其安全性和疗效在很大程度上取决于其三维结构的完整性。适体是一种具有独特折叠的短寡核苷酸，可以用于检测分子或蛋白质的微小修饰或结构变化。为了检测在蛋白质沉淀之前发生的折叠状态的抗体分子，我们生成了针对经过充分热处理的利妥昔单抗进行选择的 DNA 适体。我们使用基于磁珠的指数富集配体系统进化（SELEX），获得了针对 80°C 热处理的利妥昔单抗的六个 DNA 适体序列（40 个核苷酸）。通过计算折叠预测和圆二色性分析，发现一个适体具有 G-四链体结构，而其他所有适体均表现出 B-DNA 螺旋。通过酶联适体吸附测定（ELASA）确定了 8.8-86.7 nM 的结合亲和力。适体还检测到在 70°C 下处理 5 分钟的利妥昔单抗的结构变化，尽管结合活性较低。值得注意的是，没有一个适体识别利妥昔单抗的天然状态，也没有在抗体暴露于较低温度或不同物理胁迫剂后检测到它。适体还与在 80°C 下孵育的治疗性抗体阿达木单抗反应，表明位于广泛热处理 IgG1 抗体上的类似适体结合基序。在这项工作中，我们获得了第一个适体面板，该面板特异性针对蛋白质聚集之前特有的抗体折叠状态。这项研究证明了适体选择用于特定基于应激的蛋白质变体的潜力，这对生物制药的质量控制具有潜在影响。
