为了建立鉴定治疗性单克隆抗体识别蛋白质抗原表位的方法，选择程序死亡受体-1 (PD-1) 作为目的蛋白。基于丙氨酸扫描策略，建立了定点突变技术和哺乳动物细胞表达系统相结合的抗原突变体快速表达方法，确定了真核表达元件扩增和细胞转染表达的条件。共表达了150个PD-1蛋白突变体，鉴定了这些突变体与抗PD-1抗体帕博利珠单抗的结合能力。根据蛋白突变体与抗体的结合力并结合蛋白结构分析确定了帕博利珠单抗的抗原表位，与已报道的基于晶体结构的抗原表位高度一致，表明本方法操作简单、准确性高，可用于治疗性单克隆抗体的抗原表位作图。
To establish a method for identifying protein epitopes recognized by therapeutic monoclonal antibodies, the programmed death receptor-1 (PD-1) was selected as the target protein. Based on the alanine scanning strategy, a rapid expression method of antigen mutants combining site-directed mutagenesis with mammalian cell expression system was established, the conditions for eukaryotic expression element amplification and cell transfection expression were established. 150 PD-1 protein mutants were co-expressed, and the binding ability of these mutants to anti-PD-1 antibody Pembrolizumab was identified. The epitopes of Pembrolizumab were determined based on the binding ability of protein mutants to antibodies and combined with protein structure analysis, which was highly consistent with the reported crystal structure-based epitopes, indicating that this method is simple and accurate and can be used for epitope mapping of therapeutic monoclonal antibodies.
上海市青年科技启明星计划 (No. 19QB1406000) 资助。