Guangdong Provincial Key Laboratory for Biotechnology Candidate Drug Research, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, 510006, China.
Guangdong Provincial Key Laboratory for Biotechnology Candidate Drug Research, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, 510006, China.
Biochem Biophys Res Commun. 2021 Apr 9;548:78-83. doi: 10.1016/j.bbrc.2021.02.059. Epub 2021 Feb 23.
Targeting the dimer interface for the epidermal growth factor receptor (EGFR) that is highly conserved in the structure and directly involved in dimerization may solve the resistance problem that plagues anti-EGFR therapy. Heavy chain single domain antibodies have promising prospects as therapeutic antibodies. A bispecific nanobody was constructed based on previously screened humanized nanobodies that target the β-loop at the EGFR dimer interface, an anti-FcγRIIIa (CD16) of natural killer cells (NK) nanobodies and anti-human serum albumin (HSA) nanobodies. The target gene was effectively expressed and secreted while controlled by promoter GAP in Pichia pastoris X33, and the expressed product was purified with a cation exchange and nickel chelation chromatography. The bispecific nanobody specifically bound to the surfaces of EGFR-overexpressed human epidermal carcinoma A431 cells and effectively inhibited tumor cell growth both in vitro and in vivo. In the A431 cell nude mouse xenograft model, the growth inhibition effect from the bispecific nanobody was significantly increased with the assistance of peripheral blood mononuclear cells (PBMCs), which was consistent with the results obtained in vitro, suggesting that there was an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. In addition, the intraperitoneal administration of bispecific nanobodies effectively reached tumor tissues in the shoulder dorsal region, but in significantly less distributed quantities than EGFR Dimer Nb77. To conclude, a bispecific nanobody targeting the EGFR dimer interface with ADCC effect was successfully constructed.
针对表皮生长因子受体（EGFR）的二聚体界面进行靶向治疗，该受体在结构上高度保守，直接参与二聚化，可能解决抗 EGFR 治疗中存在的耐药问题。重链单域抗体作为治疗性抗体具有广阔的前景。本研究基于先前筛选出的针对 EGFR 二聚体界面β环的人源化纳米抗体，构建了一种双特异性纳米抗体，该纳米抗体靶向 EGFR 的β环，另一个靶向自然杀伤细胞（NK）FcγRIIIa（CD16）的纳米抗体和抗人血清白蛋白（HSA）的纳米抗体。目标基因在毕赤酵母 X33 中受 GAP 启动子的控制下有效表达和分泌，表达产物经阳离子交换和镍螯合层析纯化。双特异性纳米抗体特异性结合 EGFR 过表达的人表皮癌细胞 A431 表面，并在体外和体内有效抑制肿瘤细胞生长。在 A431 细胞裸鼠异种移植模型中，双特异性纳米抗体在周围血单核细胞（PBMCs）的辅助下，其生长抑制作用明显增加，与体外结果一致，表明存在抗体依赖的细胞介导的细胞毒性（ADCC）效应。此外，双特异性纳米抗体腹腔给药能有效到达肩部背部的肿瘤组织，但分布量明显少于 EGFR Dimer Nb77。总之，成功构建了一种具有 ADCC 效应的针对 EGFR 二聚体界面的双特异性纳米抗体。
