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A cell-based subtractive panning strategy for selection of conformation-specific single-chain variable-fragment
Reza Valadan1, Mina Dabiri1, Mohsen Tehrani1
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.
Background And Objective:
Overexpression of EGFR, a member of the ErbB receptor family, has been observed in several cancers and causes resistance to therapeutic antibodies, such as Herceptin. In this study, we produced a recombinant single-chain variable fragment (scFv) antibody against the EGFR dimerization domain.
Methods:
The recombinant scFv was generated using a cell-based subtractive panning strategy. Subtractive panning was performed on a genetically engineered, VERO/EGFR, cells as well as a triple-negative breast cancer, MDA-MB-468, cells. Phage cell-ELISA was used to monitor the binding of the selected scFvs to the dimerization domain of EGFR. Inhibition of EGFR and HER2 dimerization by the produced scFvs were finally evaluated using the dimerization inhibition test and the expression of apoptosis-related genes were measured using the quantitative RT-PCR.
Results:
PCR fingerprinting results showed a uniform digestion pattern following the third round of panning that confirmed the success of subtractive panning. Moreover, cell-ELISA validated the reactivity of the produced scFvs to EGFR following stimulation with EGF. Dimerization inhibition test showed the capacity of the scFvs to inhibit EGFR and HER2 dimerization. Investigation of apoptosis-related genes showed that treatment with the scFv antibody caused increased Bax and decreased Bcl2 expression.
Conclusions:
Directed HER2 targeting was shown to be effective enough to block the functional domain of the cell receptor and its intracellular signaling pathway. The subtractive panning strategy used in this study could control the process of directed selection of specific antibodies against the dimerization domain of EGFR. Selected antibodies might then be functionally tested for antitumor effects in both in vitro and in vivo studies.
Insights
Researchers developed a new single-chain variable fragment (scFv) antibody targeting the EGFR dimerization domain. This antibody inhibits EGFR and HER2 dimerization, potentially overcoming cancer therapy resistance.
Area of Science:
- Molecular Biology
- Immunology
- Oncology
Background:
- Epidermal Growth Factor Receptor (EGFR) overexpression is linked to various cancers and therapeutic resistance.
- EGFR is a key member of the ErbB receptor family, implicated in cancer progression.
Purpose of the Study:
- To produce a recombinant single-chain variable fragment (scFv) antibody targeting the EGFR dimerization domain.
- To evaluate the antibody's ability to inhibit EGFR and HER2 dimerization and induce apoptosis.
Main Methods:
- Cell-based subtractive panning strategy using VERO/EGFR and MDA-MB-468 cells.
- Phage cell-ELISA for binding validation and dimerization inhibition tests.
- Quantitative RT-PCR to measure apoptosis-related gene expression.
Main Results:
- Successful selection of scFv antibodies against the EGFR dimerization domain confirmed by PCR fingerprinting and cell-ELISA.
- The produced scFvs inhibited both EGFR and HER2 dimerization.
- Treatment with scFv antibodies increased Bax and decreased Bcl2 gene expression, indicating apoptosis induction.
Conclusions:
- Targeting HER2 effectively blocks EGFR signaling pathways, offering a potential therapeutic strategy.
- The subtractive panning method enables directed selection of specific antibodies against EGFR's dimerization domain.
- Further in vitro and in vivo studies are warranted to assess the antitumor effects of these selected antibodies.
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