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Selection and characterization of an internalizing epidermal‐growth‐factor‐receptor antibody

Antibody–therapeutic agent conjugates to be delivered directly into the cytosol of tumour cells is required for many target‐based therapeutic strategies. For this work, a large non‐immune phage‐display library was used to select internalizing scFv (single chain variable fragment) directed against EGFR (epidermal growth factor receptor), a tyrosine kinase receptor that is overexpressed in a wide range of tumour cells. The CHO‐EGFR‐GFP1 (where CHO is Chinese‐hamster ovary) cell line, a transfected cell line expressing EGFR–GFP (green fluorescent protein) fusion protein on membranes, 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‐bearing cell lines and could be internalized, but did not bind EGFR‐negative cell lines. The KD value of F4‐scFv was 472 nM as determined on A431 cells. F4‐scFv could be used to target therapeutic agents into tumour cells and was expected to be non‐immunogenic 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 up the generation of targeting antibodies.