Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAMReport as inadecuate




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Mediators of Inflammation - Volume 2015 2015, Article ID 460264, 11 pages -

Research Article

Department of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China

Department of Neurology, Chinese Navy General Hospital, Beijing 100048, China

Department of Geriatric Gastroenterology, Chinese People’s Liberation Army General Hospital, Beijing 100853, China

Department of Immunology, Fourth Military Medical University, Xi’an 710032, China

Received 17 October 2014; Accepted 4 December 2014

Academic Editor: Lijun Xin

Copyright © 2015 Minghua Lv et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Epithelial cell adhesion molecule EpCAM is a type I transmembrane glycoprotein overexpressed in human epithelioma but with relatively low expression in normal epithelial tissues. To exploit this differential expression pattern for targeted cancer therapy, an EpCAM-targeted immunotoxin was developed and its antitumor activity was investigated in vitro. An immunotoxin scFv2A9-PE or APE was constructed by genetically fusing a truncated form PE38KDEL of Pseudomonas aeruginosa exotoxin with an anti-EpCAM single-chain variable fragment scFv. ELISA and flow cytometry were performed to verify immunotoxin scFv2A9-PE or APE antigen-binding activity with EpCAM. Cytotoxicity was measured by MTT assay. Confocal microscopy was used to observe its cellular localization. The results of ELISA and flow cytometry revealed that the immunotoxin efficiently recognized recombinant and natural EpCAM. Its antigen-binding activity was relatively lower than 2A9. MTT assay confirmed potent reduction in EpCAM-positive HHCC human hepatocellular carcinoma cell viability IC50 50 pM. Immunofluorescence revealed that the immunotoxin localized to endoplasmic reticulum 24 h later. In conclusion, we described the development of an EpCAM-targeted immunotoxin with potent activity against tumor cells, which may lay the foundation for future development of therapeutic antibody for the treatment of EpCAM-positive tumors.





Author: Minghua Lv, Feng Qiu, Tingting Li, Yuanjie Sun, Chunmei Zhang, Ping Zhu, Xiaokun Qi, Jun Wan, Kun Yang, and Kui Zhang

Source: https://www.hindawi.com/



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