Zhu He, Zhao Lin, Li Zemin, Wen Biyan, Qiu Chuangnan, Liu Mengmiao, Xu Zhimin, Hu Shuzhuang, Li Huangjin
Guangdong Provincial Key Laboratory for Biotechnology Candidate Drug Research, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China.
Guangdong Provincial Key Laboratory for Biotechnology Candidate Drug Research, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China.
Protein Expr Purif. 2019 May;157:57-62. doi: 10.1016/j.pep.2019.02.003. Epub 2019 Feb 5.
Epidermal growth factor receptor (EGFR) is an effective target for the treatment of many epithelial cancers. However, EGFR inhibitors have low clinical response rates and are prone to drug resistance arising from mutations and heterodimerization of EGFR. Therefore, targeting the highly conserved dimer interface of EGFR may be an effective strategy for improving the clinical response of anti-EGFR therapies. Nanobodies have significant advantages over conventional antibodies in terms of size, solubility, stability and cost-effectiveness. To investigate the feasibility of nanobodies targeting the dimer interface of EGFR as novel anticancer drugs, four nanobodies were screened from a commercial humanized nanobody phage antibody library using the EGFR peptide from the β-hairpin loop of the dimer interface of EGFR as the antigen. A nanobody with an isoelectric point (pI) of 8.6, named EGFR dimer Nb77, was selected for further analysis of anticancer activities. EGFR dimer Nb77 was expressed in Escherichia coli Shuffle T7-B as a soluble (His)-tagged protein and purified by a CM Sepharose column and a nickel-nitrilotriacetic acid (Ni-NTA) column. Purified EGFR dimer Nb77 could specifically bind to the surface of EGFR-overexpressing A431 cells in a dose-dependent and ligand-dependent manner, and this nanobody could effectively inhibit the growth of the tumour cells, with an inhibition rate similar to that of the monoclonal antibody EGFR dimer 5G9, which also targets the dimer interface of EGFR. This work is the first to prove that nanobodies targeting the dimer interface of EGFR have promising prospects as anticancer agents.
表皮生长因子受体（EGFR）是治疗多种上皮癌的有效靶点。然而，EGFR抑制剂的临床反应率较低，且容易因EGFR的突变和异源二聚化而产生耐药性。因此，靶向EGFR高度保守的二聚体界面可能是提高抗EGFR治疗临床反应的有效策略。纳米抗体在大小、溶解性、稳定性和成本效益方面比传统抗体具有显著优势。为了研究靶向EGFR二聚体界面的纳米抗体作为新型抗癌药物的可行性，以EGFR二聚体界面β-发夹环的EGFR肽为抗原，从商业化的人源化纳米抗体噬菌体抗体库中筛选出四种纳米抗体。选择一种等电点（pI）为8.6的纳米抗体，命名为EGFR二聚体Nb77，用于进一步的抗癌活性分析。EGFR二聚体Nb77在大肠杆菌Shuffle T7-B中表达为可溶性（His）标签蛋白，并通过CM Sepharose柱和镍-亚氨基三乙酸（Ni-NTA）柱进行纯化。纯化后的EGFR二聚体Nb77能够以剂量依赖性和配体依赖性方式特异性结合EGFR过表达的A431细胞表面，并且这种纳米抗体能够有效抑制肿瘤细胞的生长，其抑制率与同样靶向EGFR二聚体界面的单克隆抗体EGFR二聚体5G9相似。这项工作首次证明靶向EGFR二聚体界面的纳米抗体作为抗癌药物具有广阔前景。
