Cheng Lian Sheng, Liu Ai Ping, Yang Jia Hong, Dong Yan Qiu, Li Liang Wei, Wang Jing, Wang Chao Chen, Liu Jing
School of Life Science, University of Science and Technology of China, Hefei 230027, China.
Cell Res. 2003 Feb;13(1):35-48. doi: 10.1038/sj.cr.7290149.
The c-erbB-2 proto-oncogene encodes a 185kDa protein p185, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. In order to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed the single-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichia coli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cell immunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain (ECD) of p185. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells. In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs, with a view of application in the diagnosis and treatment of human breast cancer.
c-erbB-2原癌基因编码一种185kDa的蛋白质p185，它属于表皮生长因子受体家族。该基因的扩增已被证明与某些癌症患者的不良临床预后相关。针对p185的单克隆抗体A21能特异性抑制过表达p185的肿瘤细胞增殖，因此它有望成为靶向治疗的候选药物。为了克服鼠源单克隆抗体的几个缺点，我们克隆了其VH和VL基因，并通过肽接头构建了单链Fv（scFv）。重组scFvA21在大肠杆菌中表达并通过亲和柱纯化。随后通过ELISA、Western印迹、细胞免疫组织化学和流式细胞术对其进行表征。所有这些检测均显示其对p185细胞外结构域（ECD）具有结合活性。基于scFvA21的这些特性，我们进一步构建了具有同型二聚体形式的scFv-Fc融合分子，该重组产物在哺乳动物细胞中表达。在一系列后续分析中，这种融合蛋白显示出与亲本抗体相同的抗原结合位点和活性。这些抗p185工程抗体有望进一步修饰成为肿瘤靶向药物，以期应用于人类乳腺癌的诊断和治疗。
