In the diagnosis and therapy of cancer, radioactive compounds are often applied. The awareness of altered receptor expression associated with many types of cancer, has permitted the development of receptor targeting by utilising radioactively labelled peptides with selective receptor affinity. The epidermal growth factor receptor (EGFR) is involved in cellular growth and differentiation. Although EGFR is not tumour specific, it is frequently overexpressed in many types of cancers, and overexpression of EGFR is correlated with poor relapse free and survival rates. Therefore represents the EGFR a significant target for cancer diagnosis and therapy, and for the above given reasons was selected as target in this project.
The natural ligand for the EGFR, the epidermal growth factor (EGF), has strong mitogenic activity, which makes it a poor choice as an EGFR targeting drug. Therefore was GE11, a peptide analogue of EGF, selected to investigate the feasibility of an EGFR targeting drug. GE11 was synthesised by solid phase peptide synthesis, however, labelling with the fluorophore fluorescein (as free acid) was unsuccessful. GE11 was, however, successfully labelled with 5(6)carboxyfluorescein. The correct sequence and weight of the labelled GE11 was verified by mass spectrometry techniques. Purification of the product was troublesome, and signs of peptide degradation were detected. The synthesis protocol was optimised and GE11 with lysine inserted at the N-terminus was labelled with 5(6)carboxyfluorescein and obtained at a higher yield. The fluorescent drug was tested in vitro using four different variants of the Swiss 3T3 murine fibroblast cell line, NR6. NR6 lacks expression of endogenous EGFR, and has previously been stably transfected with cDNA encoding the full length human EGFR or the EGFRvIII mutant. Thus, the lines express either physiological levels of EGFR, over-expresses EGFR or expresses the truncated, cancer specific and constitutively active EGFRvIII. The expression of the receptors was partly verified by immunofluorescence, using two different antibodies, and analysed with fluorescence microscopy and fluorescence confocal laser scanning microscopy. The cell lines were incubated in medium containing the synthesised fluorescently labelled drug, and the internalisation of the cells was analysed by fluorescence microscopy. However, the targeting ability was found to be poor, as the highest intensity was found with the physiological EGFR expression cell line and the EGFRvIII expression cell line. These findings were confirmed by flow cytometry analysis. However, the flow cytometry data also revealed, unlike the fluorescence microscopy, that the lowest intensity was seen in the EGFRvIII expressing cell line.
Lysine-GE11 with the chelating agent DOTA attached to the N-terminus was also synthesised and purified, to allow the drug to be tested in vivo using a PET/CT scanner. The ability of the drug to form complexes with stable gallium and fluorescent europium were verified. However, as the affinity of the fluorescent peptide for EGFR was low and due to project time limitations, the metal DOTA-Lysine-GE11 complex was not tested in vivo.
In conclusion, it was in this project found that the affinity of lysine-GE11 for the EGFR was poor, most likely due to the added lysine amino acid. Future projects could investigate GE11 or other peptides e.g. with affinity for the cancer-specific EFGRvIII receptor, to continue the development of affinity drugs for the diagnostics and therapy of cancer.