A Cell-Based Subtractive Panning Strategy for Selection of Conformation-Specific Single-Chain Variable-Fragment (Scfv) Against Dimerization Domain of EGFR

Abstract Background and Objective: Overexpression of the EGFR, from 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 genetically engineered VERO/EGFR cells and cancerous 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. 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 after stimulation with EGF. The dimerization inhibition test showed the capacity of the scFvs to inhibit EGFR and HER2 dimerization. Conclusions Directed HER2 targeting showed to be more effective to target the functional domain of the cell receptor for the complete blockade of the 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. The selected antibodies might then be functionally tested for antitumor effects in both in vitro and in vivo studies.