Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31, Huotuo Road, Biomedical Base, Daxing District, Beijing, 102629, China.
Department of Physiology and Pathopysiology, Capital Medical University, Youanmen, Fengtai District, Beijing, 100069, China.
Sci Rep. 2020 Oct 6;10(1):16615. doi: 10.1038/s41598-020-73960-x.
Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.
中东呼吸综合征冠状病毒（MERS-CoV）是一种高毒力病原体，可引起中东呼吸综合征（MERS）。抗 MERS-CoV 抗体在预防和治疗 MERS-CoV 感染中发挥着重要作用。生物活性是治疗性抗体的关键质量属性，需要高度的准确性和精密度。评估抗病毒抗体抗病毒作用的主要方法包括使用活病毒或假病毒的中和测定，其具有高度可变性。最近的研究表明，抗病毒抗体的抗体依赖性细胞毒性（ADCC）活性与体内病毒清除效果比中和活性更一致。然而，迄今为止，尚无评估抗 MERS 抗体的 ADCC 活性的报道。在这里，我们描述了一种使用 293T/MERS 细胞作为靶细胞的稳健可靠的基于细胞的报告基因测定法的开发，该细胞稳定表达 MERS-CoV 的刺突蛋白（MERS-S），并使用工程化的 Jurkat/NFAT-luc/FcγRIIIa 作为效应细胞，该细胞稳定表达 FcγRIIIA 和 NFAT 报告基因。根据 ICH-Q2 分析方法指南，我们仔细优化了实验条件并评估了我们测定法的性能。此外，我们发现去岩藻糖基化的抗 MERS 抗体的 ADCC 活性高于其岩藻糖基化的抗体。该 ADCC 测定系统的建立为评估抗 MERS 抗体的生物活性以及通过修饰 Fc 片段的 N-糖基化来提高 ADCC 活性提供了一种新方法。
