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 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.
Zhao
Xiaorong
X
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.
Dai
Wentao
W
Cao
Limin
L
Zhu
Huifen
H
Yu
Yihan
Y
Ye
Qing
Q
Wang
Min
M
Dai
Wei
W
Lei
Ping
P
Shen
Guanxin
G
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
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D066246:ErbB Receptors