Protein Engineering vol. 16 no. 12 pp. 1107-1113, 2003
© 2003 Oxford University Press
Isolation from phage display libraries of lysine-deficient human epidermal growth factor variants for directional conjugation as targeting ligands
1vectron therapeutics AG, Rudolf-Breitscheid-Straße 24, 35037 Marburg and 2Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, Emil-Mannkopff-Straße 2, 35033 Marburg, Germany
3 To whom correspondence should be addressed. e-mail: kontermann{at}vectron-ag.com
Ligand-targeted anticancer therapeutics represent an opportunity for the selective and efficient delivery of drugs to tumours. The chemical coupling of ligands to drugs or drug carrier systems is, however, often hampered by the presence of multiple reactive groups within the ligand, for example, 
-NH2 groups in lysine side chains. In this paper, we describe the isolation by phage display of human epidermal growth factor (EGF) variants without lysine and a reduced number of arginine residues. The selection on A431 carcinoma cells also revealed that R41 is indispensable for EGF binding activity as all EGF variants contained an arginine residue at this position. One EGF variant (EGFm1) with K28Q, R45S, K48S and R53S mutations was expressed in bacteria and showed an identical binding activity as wild-type EGF. EGFm1 could be labelled with fluorescein isothiocyanate demonstrating the accessibility of the N-terminal amino group for coupling reagents. Furthermore, coupling of EGFm1 to PEGylated liposomes resulted in target cell-specific binding and internalization of the liposomes. These human EGF variants should be advantageous for the generation of anticancer therapeutics targeting the EGF receptor, which is overexpressed by a wide variety of different tumours.
Received July 8, 2003; revised October 23, 2003; accepted October 30, 2003