Isolation from phage display libraries of lysine-deficient human epidermal growth factor variants for directional conjugation as targeting ligands

Abstract

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, epsilon-NH(2) 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.

A lysine-free mutant of epidermal growth factor as targeting moiety of a targeted toxin

“…Furthermore, EGF was utilized to deliver polyethylenimine/DNA complexes to tumor cells in vivo (Ogris et al 2003) and to target boronated acridine-loaded liposomes to EGF receptor-expressing cells (Kullberg et al 2005). Bach et al developed a phage display system to identify EGF mutants lacking lysines and some arginines (Bach et al 2003). The introduced four mutations (K28Q, R45S, K48S and R53S) exchanged not only the two lysines but also two of the three arginines to uncharged amino acids.…”

“…Based on this we expected increased affinity with our double substitution, however, the second mutation in our EGF RR (K48R) resulted in a decreased binding affinity, since both mutations together decreased the binding affinity slightly. It would be interesting to use the K48S mutation in EGF described by Bach et al in combination with K28R, since K48S seemed not to interfere with the binding properties of EGF (Bach et al 2003). This is even more likely, since Campion et al claim that no positive charge is necessary for the positions of the two lysines ).…”

In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display

“…Living cells, which express the target receptors on their surfaces, are appropriate for binding target because receptors expressed on living cells, unlike receptors in membrane fractions, assume their native structure and display only their extracellular side. In fact, living cells were used to screen DNA or RNA aptamers (29)(30)(31)(32)(33)(34)(35), as well as both peptides and antibodies, using phage display libraries (36)(37)(38)(39)(40)(41), which bind to membrane proteins expressed on the cell surface. Nevertheless, selection with live cells has not been performed with in vitro display techniques because mRNA display and ribosome display (in which a peptide is displayed on mRNA) are labile and easily degraded by ribonucleases in serum-supplemented cell culture medium.…”

Targeting the EGF Receptor for Ovarian Cancer Therapy

“…EGF receptor ligands such as mouse EGF can be conjugated through its N-terminus without affecting receptor binding ability. In contrast, human EGF has two additional amino groups due to internal lysines, and their conjugation can interfere with receptor binding [56]. For that reason mouse EGF rather than human EGF is usually employed for EGF receptor targeting.…”

“…Novel peptides that specifically bind to EGF receptor provide alternative targeting moieties. Such peptides have been identified either through screening of a virtual peptide library [57], or through screening phage display libraries [58] for peptides that specifically bind to the EGF receptor, including lysine-deficient EGF variants [56]. EGF receptor-targeting moieties are conjugated with imaging or therapeutic agents such as radionuclides, cancer chemotherapeutic agents, toxins, RNase, or photosenstizers.…”