Establishment A Cell Line Expressing EGFR Stably And Selection, Characterization Of An Internalizing EGFR ScFv

Epidermal growth factor receptor (EGFR, or ErbB1) is one of four members of the ErbB family of type-1 tyrosine kinases. EGFR is wide expressed in normal skin epithelial cells, EGFR activation drives cell proliferation, enhances cell migration and adhesion, and affects cell differentiation. But EGFR is overexpressed in a wide range of epithelial cancers, continuous deregulation of which has been believed to be associated with tumor pathogenesis, metastasis and a poor prognosis. EGFR has becoming a novel target for tumor therapy. Strategies such as monoclonal antibodies to EGFR, tyrosine kinase inhibitors to block or downregulate EGFR have been developed. Other strategies such as antibody arming with small molecule toxins and bispecific antibodies to enhancing the efficacy of antitumour antibodies have also been in a well development. In the present study, we established a cell line expressing EGFR on membrane stably and named as CHO-EGFR-GFP1. The cell line was used to study interactions between EGFR and its ligand EGF and to isolate an internalizing EGFR scFv (F4-scFv) from a large nonimmune human phagemid antibody library. There are three parts in the study.Part IEstablishment of a cell line expressing EGFR-GFPfusion proteinThe E. coli DH5α was transformed with pEGFR-EGFP plasmid by CaCl₂ method. After identified by restriction enzyme digestion, the plasmid was extracted and then transfected CHO-K1 cell by electroporation. Single clones expressing EGFR-GFP fusion protein were obtained by seeding the cells into 96-well plates with one cell per well. GFP-positive clones were detected by flow cytometry (FCM), inverted fluorescence microscopy, and western blot analysis for EGFR-GFP fusion protein expression. The growth curve of the transfected and untransfected CHO-K1 cell was compared. The transfected cell was freezed, recovered, and passaged, the expressing of the EGFR-GFP fusion protein was detected by FCM. One cell line expressing EGFR-GFP fusion protein were obtained stably and named as CHO-EGFR-GFP1, EGFR-GFP fusion protein localized primarily to the cell membrane in this cell line. Western blot analysis suggested EGFR-GFP fusion protein has a molecular weight of 166 kDa. There was no significant difference between the transfected and untranfected CHO-K1 cells on cell growth velocity. This work will be a fundament for further developing of anti-tumor durgs targeting EGFR and studying interactions between EGFR and its ligands.Part IIEffect of epidermal growth factor (EGF) on the biological characters of CHO-EGFR-GFP1 cellsInteraction between epidermal growth factor (EGF) and EGF receptor (EGFR) promotes cell growth in most cell lines, but in a number of cell lines, EGF paradoxically inhibits proliferation. In the present study, we established a cell line expressing full length human EGFR on membrane with a GFP fluorescence reporter at the C-terminal and studied the effects of EGF on cell proliferation in the transfected cell line. Our results suggested that low concentrations of EGF promoted proliferation, while high concentrations of EGF induced loss of adhesion, cell cycle arrest, apoptosis, and inhibition of proliferation. The effects of EGF on cell proliferation correlated well with the expression levels of EGFR. High concentrations of EGF induced both EGFR expression and apoptosis in a dose-dependent manner. Our study for the first time reported relationship between the effects of EGF on cell proliferation and levels of EGFR expression in one cell line expressing different levels of EGFR caused by different concentrations of EGF treatment. The study should provide considerable insight into effects of EGF on cell proliferation and tumor cell metastasis.PartIIISelection and characterization of an internalizing epidermal growth factor receptor antibodyAntibody-therapeutic agent conjugates to be delivered directly into the cytosol of tumor cells is required for many target based therapeutic strategies. For this work, a large nonimmune phage display library was used to select internalizing scFv directed against EGFR by recovering endocytosed phage from within the cells. CHO-EGFR-GFP1, a transfected cell line expressing EGFR-GFP fusion protein on membrane, and the untransfected cell line CHO-K1 were used as EGFR positive cells and negative cells respectively in the subtractive selection procedure. A novel human anti-EGFR scFv (F4-scFv) was isolated. F4-scFv bound native EGFR positive cell lines and could be internalized, but did not bind EGFR negative cell lines. F4-scFv could be used to target therapeutic agents into tumor cells and was expected to be nonimmunogenic in humans. Use of a transfected cell line expressing GFP-tagged receptors allows selection and characterization of antibodies to native receptors without the need for protein expression and purification, significantly speeding the generation of targeting antibodies.