Exploring pitfalls of ⁶⁴Cu-labeled EGFR-targeting peptide GE11 as a potential PET tracer

The epidermal growth factor receptor (EGFR) represents an important molecular target for both radiotracer-based diagnostic imaging and radionuclide therapy of various cancer entities. For the delivery of radionuclides to the tumor, peptides hold great potential as a transport vehicle. With respect to EGFR, the peptide YHWYGYTPQNVI (GE11) has been reported to bind the receptor with high specificity and affinity. In the present study, GE11 with β-alanine (β-Ala-GE11) was conjugated to the chelating agent p-SCN-Bn-NOTA and radiolabeled with ⁶⁴Cu for the first radio pharmacological evaluation as a potential probe for positron emission tomography (PET)-based cancer imaging. For better water solubility, an ethylene glycol-based linker was introduced between the peptide's N terminus and the radionuclide chelator. The stability of the ⁶⁴Cu-labeled peptide conjugate and its binding to EGFR-expressing tumor cells was investigated in vitro and in vivo, and then compared with the ⁶⁴Cu-labeled EGFR-targeting antibody conjugate NOTA-cetuximab. The GE11 peptide conjugate [⁶⁴Cu]Cu-NOTA-linker-β-Ala-GE11 ([⁶⁴Cu]Cu-1) was stable in a buffer solution for at least 24 h but only 50% of the original compound was detected after 24 h of incubation in human serum. Stability could be improved by amidation of the peptide's C terminus (β-Ala-GE11-NH₂ (2)). Binding assays with both conjugates, [⁶⁴Cu]Cu-1 and [⁶⁴Cu]Cu-2, using the EGFR-expressing tumor cell lines A431 and FaDu showed no specific binding. A pilot small animal PET investigation in FaDu tumor-bearing mice revealed only low tumor uptake (standard uptake value (SUV) < 0.2) for both conjugates. The best tumor-to-muscle ratio determined was 3.75 for [⁶⁴Cu]Cu-1, at 1 h post injection. In conclusion, the GE11 conjugates in its present form are not suitable for further biological investigations, since they presumably form aggregates.