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Published on: October 15, 2016
Production and functional analysis of a phage displayed scFv recombinant antibody targeting EGFR/HER2 dimerization
Mina Dabiri1, Mohsen Tehrani2, Alireza Rafiei2
1Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; Molecular and Cell Biology Research Center (MCBRC), School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; Department of Biomedical and Biological Sciences, Rowan University, 201 Mullica Hill Rd, Glassboro, NJ, 08028, United States.
Background:
Tumor cells exploit epidermal growth factor receptor (EGFR) family to develop resistance against therapeutic antibodies, such as Herceptin. Upon ligand binding, dimerization between EGFR and HER2 is one of the most important causes of treatment failure in breast cancer and other cancers expressing EGFR and HER2. The aim of this study was to develop and evaluate the function of a human recombinant single-chain variable fragment (scFv) antibody against the dimerization domain of EGFR to inhibit its interaction with other members of the epidermal growth factor receptor family, especially HER2.
Methods:
scFv against EGFR was expressed and purified. Cell-ELISA, MTT assay, inhibition of STAT3 phosphorylation, quantitative RT-PCR, and dimerization inhibition were performed on EGFR and HER2 expressing cell lines to characterize functional properties of the produced scFv. The conformational structure of the produced scFv and its binding ability to EGFR was computationally investigated.
Results:
In vitro binding analysis by cell-ELISA revealed the EGFR binding ability of the purified antibodies and confirmed by immunoblotting. ScFvs preferentially reduced the proliferation and survival of MCF7, MDA-MB-468, and SKOV3 cell lines with no effect on the VERO line. More considerably, MCF7 cells treated with the scFv antibody showed reduced STAT3 phosphorylation, decreased Bcl-2 expression, and increased Bax expression. Finally, the scFvs hindered EGFR and HER2 dimerization.
Conclusion:
The produced scFv antibody showed to be functional in a simultaneous blockade of EGFR and HER2, suggesting its potential as a promising candidate for targeted therapy against various EGFR overexpressing tumors.
Insights
A novel single-chain variable fragment (scFv) antibody targeting the epidermal growth factor receptor (EGFR) dimerization domain effectively inhibits EGFR and HER2 dimerization. This scFv shows promise for targeted therapy in EGFR-overexpressing tumors.
Area of Science:
- Oncology
- Molecular Biology
- Immunotherapy
Background:
- Tumor cells develop resistance to therapies like Herceptin by exploiting the epidermal growth factor receptor (EGFR) family.
- EGFR and HER2 dimerization is a key mechanism driving treatment failure in cancers expressing these receptors.
- Targeting EGFR-HER2 interactions is crucial for overcoming therapeutic resistance.
Purpose of the Study:
- To develop and assess a human recombinant single-chain variable fragment (scFv) antibody.
- To inhibit the dimerization domain of EGFR, specifically blocking its interaction with HER2.
- To evaluate the potential of this scFv as a targeted therapy for EGFR-overexpressing cancers.
Main Methods:
- Expression and purification of the anti-EGFR scFv antibody.
- Functional characterization using cell-ELISA, MTT assay, STAT3 phosphorylation inhibition, and quantitative RT-PCR.
- Computational investigation of scFv structure and EGFR binding affinity.
- Assessment of EGFR-HER2 dimerization inhibition.
Main Results:
- The purified scFv demonstrated binding to EGFR, confirmed by cell-ELISA and immunoblotting.
- ScFvs significantly reduced proliferation and survival in EGFR/HER2-expressing cancer cell lines (MCF7, MDA-MB-468, SKOV3).
- Treated MCF7 cells exhibited decreased STAT3 phosphorylation, reduced Bcl-2, and increased Bax expression, indicating apoptosis induction.
- The scFv successfully hindered EGFR and HER2 dimerization.
Conclusions:
- The developed scFv antibody effectively blocks EGFR and HER2 dimerization.
- This functional blockade suggests the scFv's potential as a targeted therapeutic agent.
- The scFv is a promising candidate for treating various EGFR-overexpressing tumors.
