Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM
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 develope...
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Series: | Mediators of Inflammation |
Online Access: | http://dx.doi.org/10.1155/2015/460264 |
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doaj-8cfa861d760f4fe093bb4f5321c24df42020-11-24T20:47:56ZengHindawi LimitedMediators of Inflammation0962-93511466-18612015-01-01201510.1155/2015/460264460264Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAMMinghua Lv0Feng Qiu1Tingting Li2Yuanjie Sun3Chunmei Zhang4Ping Zhu5Xiaokun Qi6Jun Wan7Kun Yang8Kui Zhang9Department of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, ChinaDepartment of Neurology, Chinese Navy General Hospital, Beijing 100048, ChinaDepartment of Geriatric Gastroenterology, Chinese People’s Liberation Army General Hospital, Beijing 100853, ChinaDepartment of Immunology, Fourth Military Medical University, Xi’an 710032, ChinaDepartment of Immunology, Fourth Military Medical University, Xi’an 710032, ChinaDepartment of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, ChinaDepartment of Neurology, Chinese Navy General Hospital, Beijing 100048, ChinaDepartment of Geriatric Gastroenterology, Chinese People’s Liberation Army General Hospital, Beijing 100853, ChinaDepartment of Immunology, Fourth Military Medical University, Xi’an 710032, ChinaDepartment of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, ChinaEpithelial 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.http://dx.doi.org/10.1155/2015/460264 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Minghua Lv Feng Qiu Tingting Li Yuanjie Sun Chunmei Zhang Ping Zhu Xiaokun Qi Jun Wan Kun Yang Kui Zhang |
spellingShingle |
Minghua Lv Feng Qiu Tingting Li Yuanjie Sun Chunmei Zhang Ping Zhu Xiaokun Qi Jun Wan Kun Yang Kui Zhang Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM Mediators of Inflammation |
author_facet |
Minghua Lv Feng Qiu Tingting Li Yuanjie Sun Chunmei Zhang Ping Zhu Xiaokun Qi Jun Wan Kun Yang Kui Zhang |
author_sort |
Minghua Lv |
title |
Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM |
title_short |
Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM |
title_full |
Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM |
title_fullStr |
Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM |
title_full_unstemmed |
Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM |
title_sort |
construction, expression, and characterization of a recombinant immunotoxin targeting epcam |
publisher |
Hindawi Limited |
series |
Mediators of Inflammation |
issn |
0962-9351 1466-1861 |
publishDate |
2015-01-01 |
description |
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. |
url |
http://dx.doi.org/10.1155/2015/460264 |
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