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 over, expressed in a wide range of tumour cells. The CHOEGFR-GFPI (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-KI 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 K-D 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.