Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185c-erbB-2
Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185c-erbB-2
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DOI：
10.1038/sj.cr.7290149
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发表时间：
2003-02-01
期刊：
影响因子：
44.1
通讯作者：
Liu, J
中科院分区：
文献类型：
--
作者：
Cheng, LS;Liu, AP;Liu, J
c-erbB-2原癌基因编码一个分子量为185 kDa的p185蛋白，属于表皮生长因子受体家族。该基因的扩增已显示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特异性抑制过表达p185的肿瘤细胞的增殖，从而使其成为靶向治疗的候选者。为了克服鼠源单克隆抗体的一些缺点，我们克隆了鼠源单克隆抗体的V-H和V-L基因，并通过连接肽构建了单链抗体（scFv）。重组scFvA 21在大肠杆菌中表达，亲和层析柱纯化。随后采用ELISA、Western blot、细胞免疫组化和流式细胞仪等方法进行鉴定。所有这些试验都显示了与p185胞外区（ECD）的结合活性。基于scFvA 21的这些特性，我们进一步构建了具有同源二聚体形式的scFv-Fc融合分子，并在哺乳动物细胞中表达了重组产物。在随后的一系列分析中，该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基因工程抗体有望进一步修饰成为肿瘤靶向药物，以期应用于乳腺癌的诊断和治疗。
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 V-H and V-L 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 immunchistochemistry 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.