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In vitro and in vivo anti-tumor activities of anti-EGFR single-chain variable fragment fused with recombinant gelonin toxin

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Abstract

Purpose

Epidermal growth factor receptor (EGFR) plays an important role in the growth and metastasis of many solid tumors. Strategies that target EGFR hold promising therapeutic potential for the treatment for non-small cell lung cancer (NSCLC), as EGFR is normally overexpressed in these tumors. This study was designed to determine whether an anti-EGFR immunotoxin has anti-tumor activity against NSCLC, and if so, to further investigate the possible mechanisms of cytotoxicity.

Methods

A fusion protein of anti-EGFR single-chain variable fragment (anti-EGFR scFv) and the plant toxin gelonin (rGel) was constructed, expressed in bacteria, and purified to homogeneity. Cytotoxicity of anti-EGFR scFv/rGel (E/rG) immunotoxin was assessed on A549, HCC827, and H1975 cells (EGFR-overexpressing NSCLC-derived cell lines) and A549 xenografts in nude mice.

Results

Cytotoxicity experiments using E/rG on A549, HCC827, and H1975 cells demonstrated that E/rG can specifically inhibit proliferation of these cells, whereas it did not affect the proliferation of Raji cells that do not express EGFR. Treatment for A549 xenografts in nude mice with E/rG resulted in significant suppression of tumor growth compared to controls. Immunofluorescence in frozen tissue sections confirmed that E/rG could specifically bind to tumor tissues in nude mice bearing A549 tumor xenografts, while rGel alone showed no binding activity. Furthermore, E/rG inhibited the growth of A549 cells by cytotoxic effects that blocked tumor proliferation, and the immunotoxin-induced cell death may be mediated by autophagy.

Conclusions

These results showed that E/rG might have significant potential as a novel clinical therapeutic agent against human NSCLC.

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Abbreviations

2-ME:

2-mercaptoethanol

EGFR:

Epidermal growth factor receptor

GuHCl:

Guanidine hydrochloride

scFv:

Single-chain variable fragment

rGel:

Recombinant gelonin toxin

G4S:

Glycine–glycine–glycine–glycine–serine

MVD:

Microvessel density

NSCLC:

Non-small cell lung cancer

IPTG:

Isopropyl β-d-thiogalactoside

PEA:

Pseudomonas exotoxin A

TUNEL:

TdT-mediated dUTP-X nick end labeling

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Acknowledgment

This work is supported by China National Science and Technology Programs of Significant New Drugs to Create (No.2009ZX09103-714) and the National Key Basic Research Program (973 Program) of China (No. 2010CB529900).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, No. 1 Keyuan Road 4th, Chengdu, Sichuan, 610041, People’s Republic of China

    Xikun Zhou, Ji Qiu, Zhen Wang, Nongyu Huang, Xiaolei Li, Qian Li, Yinbing Zhang, Chengjian Zhao, Can Luo, Nannan Zhang, Xiu Teng, Zhongwen Chen, Xiao Liu, Xianlian Yu, Wenling Wu, Yu-quan Wei & Jiong Li

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  1. Xikun Zhou
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  2. Ji Qiu
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  17. Jiong Li
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Correspondence to Jiong Li.

Additional information

Xikun Zhou, Ji Qiu, and Zhen Wang contributed equally to this work.

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Zhou, X., Qiu, J., Wang, Z. et al. In vitro and in vivo anti-tumor activities of anti-EGFR single-chain variable fragment fused with recombinant gelonin toxin. J Cancer Res Clin Oncol 138, 1081–1090 (2012). https://doi.org/10.1007/s00432-012-1181-7

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  • DOI: https://doi.org/10.1007/s00432-012-1181-7

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