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Archive > Volume 20 Issue 4 > 2013,20(4):425-431. DOI:10.3872/j.issn.1007-385X.2013.04.008 Prev Next
Basic Research
Preparation of immunotoxin directed to a cryptic epitope in EGFR and its anti-tumor activity
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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-22b-806scFv-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 immuno-fluorescent 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 EGFRvⅢ 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 U87MG-EGFRvⅢ cells with IC50 values of (5.85±0.03) ng/ml (P<0.01) and EGFR overexpression cancer cells such as MDA-MB-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 \[2.45±2.85\]%, \[8626±1.01\]% vs \[0.48±1.76\]%, \[96.72±0.16\]% vs \[1.33±1.31\]%, \[96.29±0.30\]% vs \[2.00±060\]%; P<0.01) but no effect on U87MG cells (\[3.59±2.09\]% vs \[0.19±0.95\], P>005). 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.
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Project Supported:
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）
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