title: Selection and characterization of an internalizing epidermal-growth-factor-receptor antibody.
abstract: 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 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.
authors.affiliation: Laboratory of Molecular and Immuno-Pharmacology, Department of Immunology Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People's Republic of China.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Zhao
authors.forename: Wentao
authors.initials: W
authors.lastname: Dai
authors.forename: Limin
authors.initials: L
authors.lastname: Cao
authors.forename: Huifen
authors.initials: H
authors.lastname: Zhu
authors.forename: Yihan
authors.initials: Y
authors.lastname: Yu
authors.forename: Qing
authors.initials: Q
authors.lastname: Ye
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Dai
authors.forename: Ping
authors.initials: P
authors.lastname: Lei
authors.forename: Guanxin
authors.initials: G
authors.lastname: Shen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000276:immunology*; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D066246:ErbB Receptors