A cell-based subtractive panning strategy for selection of conformation-specific single-chain variable-fragment (scFv) against dimerization domain of EGFR

Abstract

Background and objectiveOverexpression 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. MethodsThe 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. ResultsPCR 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. ConclusionsDirected 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.