Akbari Vajihe, Sadeghi Hamid Mir Mohammad, Jafarian-Dehkordi Abbas, Abedi Daryoush, Chou C Perry
Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran; Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada.
Protein Expr Purif. 2015 Dec;116:66-74. doi: 10.1016/j.pep.2015.07.005. Epub 2015 Jul 10.
A novel monoclonal antibody against human epidermal growth factor receptor 2 (HER2), i.e., pertuzumab (Perjeta®) developed by Genentech, has been verified to be effective in treating metastatic HER2-overexpressing breast cancer. The fact that the presence of the Fc region of the anti-HER2 is uncritical for growth inhibition of tumor cells suggests the potential biological activity of the associated antibody fragments. In the present study, we report functional expression of anti-HER2his-scFv, a single-chain variable fragment (scFv) derived from pertuzumab, in the periplasm of Escherichia coli and its purification. Biological activity of the soluble scFv produced in this manner was characterized using immunofluorescent staining, immunocytochemistry, flow cytometry and cytotoxicity assay. The effect of anti-HER2his-scFv on HER2 dimerization was also assessed by tyrosine kinase assay. It was observed that the purified scFv had a high specificity and affinity to HER2 receptors expressed on the surface of tumor cells with a selective cytotoxic effect on HER2-overexpressing SK-OV-3 cells. In addition, anti-HER2his-scFv was able to suppress phosphorylation of HER2 in the presence of heregulin. The results suggest that anti-HER2his-scFv can be a potential candidate for various therapeutic and diagnosis applications.
一种新型抗人表皮生长因子受体2（HER2）单克隆抗体，即基因泰克公司研发的帕妥珠单抗（Perjeta®），已被证实可有效治疗转移性HER2过表达乳腺癌。抗HER2的Fc区域的存在对肿瘤细胞生长抑制并非至关重要，这一事实表明相关抗体片段具有潜在生物活性。在本研究中，我们报告了抗HER2his-scFv（一种源自帕妥珠单抗的单链可变片段（scFv））在大肠杆菌周质中的功能表达及其纯化。使用免疫荧光染色、免疫细胞化学、流式细胞术和细胞毒性试验对以此方式产生的可溶性scFv的生物活性进行了表征。还通过酪氨酸激酶试验评估了抗HER2his-scFv对HER2二聚化的影响。观察到纯化的scFv对肿瘤细胞表面表达的HER2受体具有高特异性和亲和力，对HER2过表达的SK-OV-3细胞具有选择性细胞毒性作用。此外，抗HER2his-scFv在存在调理性素的情况下能够抑制HER2的磷酸化。结果表明，抗HER2his-scFv可能是各种治疗和诊断应用的潜在候选物。
