中国肿瘤生物治疗杂志2013，Vol.20Issue(4)：425-431,7.DOI:10.3872/j.issn.1007-385X.2013.04.008
靶向EGFR隐蔽表位的免疫毒素的制备及其对肿瘤细胞的特异性杀伤
Preparation of immunotoxin directed to a cryptic epitope in EGFR and its antitumor activity
摘要
Abstract
Objective:To prepare recombinant immunotoxin targetting a cryptic epitope (287-302 residues) in epithelial growth factor receptor (EGFR) and to explore its biological properties.Methods:Prokaryotic expression vector pET-22b806scFv-PE38KDEL encoding anti-EGFR (287-302) 806 single-chain antibody (806scFv) fused with PE38KDEL,a truncated form of pseudomonas exotoxin A (PEA),via a flexible peptide was constructed.The immunotoxin fusion protein (806scFv-PE38KDEL) was expressed in E.coli strain BL21 (DE3) and purified.The binding capacity of the immunotoxin to EGFR was detected by ELISA and flow cytometry.The internalization of immunotoxin was showed via indirect immunofluorescent assay.The cytotoxicity effect of the immunotoxin on human glioma cell U87MG and U87MG-EGFRvⅢ,epidermis tumor cell A431,breast cancer cell MDA-MB-468 and tongue cancer CAL-27 cell lines were assessed by CCK-8 assay.Results:The recombinant immunotoxin 806scFv-PE38KDEL was constructed successfully.The induced expression product 806scFv-PE38KDEL existed in a form of inclusion body and the purity was above 95％ after purification.The protein was identified by SDS-PAGE and Western blotting.806scFv-PE38KDEL can bind to the protein of EGFRvⅢ extracellular domain and also the cancer cells with exogenous EGFRvI or with a endogenous EGFR overexpression but not to the cancer cells with a low level of endogenous EGFR.The immuno-fluorescent assay showed that the internalization of immunotoxin was mediated by 806scFv.806scFv-PE38KDEL showed the cytotocicity on targeted cells with EGFRvⅢ overexpression such as U87MGEGFRvⅢ cells with IC50 values of (5.85 ±0.03) ng/ml (P ＜ 0.01) and EGFR overexpression cancer cells such as MDAMB-468,A431 and CAL-27 cells with IC50 values of (162.80±0.06) ng/ml,(75.72 ±0.04) ng/ml,(123.70 ±0.03)ng/ml,respectively.806scFv-PE38KDEL almost completely inhibited the growth of cancer cells such as U87MG-EGFRvⅢ,MDA-MB-468,A431 and CAL-27 cells compared the control group,and the inhibitory rates significantly increased ([98.67±0.07]％ vs [2.45±2.85]％,[86.26±1.01]％ vs [0.48±1.76]％,[96.72 ± 0.16]％ vs [1.33±1.31]％,[96.29 ± 0.30] ％ vs [2.00 ± 0.60] ％ ; P ＜ 0.01) but no effect on U87 MG cells ([3.59 ± 2.09] ％ vs [0.19 ± 0.95],P ＞ 0.05).Conclusion:Recombinant immunotoxin 806scFv-PE38KDEL that targeted to EGFR (287-302) epitope prepared in this study is a candidate cancer therapeutic agent which can selectively bind and significantly inhibit the growth of the cancer cells with EGFRvⅢ or EGFR overexpression.关键词
表皮生长因子受体/隐蔽表位/免疫毒素/单链抗体/铜绿假单胞菌外毒素A/肿瘤Key words
epidermal growth factor receptor (EGFR) / cryptic epitope/ immunotoxin/ single chain variable fragment (scFv) / Pseudomonas exotoxin A (PEA) / cancer分类
医药卫生引用本文复制引用
李雅婷,石必枝,孔娟,李宗海..靶向EGFR隐蔽表位的免疫毒素的制备及其对肿瘤细胞的特异性杀伤[J].中国肿瘤生物治疗杂志,2013,20(4):425-431,7.基金项目
上海市科委生物医药重点科技攻关项目资助(No.10431903700) （No.10431903700）
"十二五"国家重大新药创制专项资助(No.2012ZX09103301-005).Project supported by the Key Science and Technology Program in Biological Pharmaceutical Medicine of Shanghai Municipal Science and Technology Commission (No.10431903700) and the National "Twelfth Five-year Plan" Key Foundation for Development of New Drugs(No.2012ZX09103301-005) （No.2012ZX09103301-005）
