SYNTHESIS, CHARACTERIZATION, AND RADIOLABELING OF MODIFIED EGFR-TARGETING PEPTIDES: POTENTIAL THERANOSTIC AGENTS?

Introduction/Justification

Cancer remains one of the leading causes of mortality worldwide. Consequently, efforts to overcome the limitations of conventional therapies have increasingly focused on molecularly targeted treatments, with particular emphasis on peptides due to their anti-tumorigenic properties and high affinity for receptors overexpressed in tumors. Peptides designed to inhibit intracellular signaling pathways play a key role in molecularly targeted therapies, often focusing on receptors such as the Epidermal Growth Factor receptor (EGFr), which is overexpressed in many solid tumors. As targeting biomolecules, these peptides can also serve as carriers for radionuclides, enabling both molecular imaging and targeted radionuclide therapy.

Objectives

This study aimed to develop modified peptides with high affinity for EGFr, thereby enabling their potential application as theranostic molecules.

Materials and Methods

Anti-EGFr peptides were modified by incorporating two different spacers—hexa-aminocaproic acid (C6) or dodeca-aminocaproic acid (C12)—and by adding the chelating agent DOTA. These peptides were synthesized using the Fmoc/tBu strategy for peptide synthesis. Cleavage from the resin was performed using a reagent mixture with a high concentration of trifluoroacetic acid (reagent K). Subsequently, the peptides underwent characterization and purification through high-performance liquid chromatography (HPLC) and mass spectrometry. A preliminary radiolabeling assay of DOTA-C6-anti-EGFR was conducted using cyclotron-produced yttrium-86 (⁸⁶Y) in a NaOAc buffer (pH 5.5). The radiochemical reaction was carried out at 95°C for 30 min, followed by purification through a Sep-Pak C18 cartridge to determine the radiolabeling yield.

Results

The peptides DOTA-C6-anti-EGFr and DOTA-C12-anti-EGFr were successfully synthesized, with yields of 33.8% and 3.3%. HPLC and mass spectrometry analyses confirmed the efficiency of the synthesis, cleavage, and purification processes, as evidenced by the molecular masses corresponding to the expected peptides. Preliminary radiolabeling data for DOTA-C6-anti-EGFr with ⁸⁶Y demonstrated a radiochemical yield of approximately 96.5%.

Conclusion

The modified peptides targeting EGFr were successfully synthesized, characterized, and purified. The significantly lower yield obtained for the C12 spacer suggests that peptides incorporating the C6 spacer are more viable for further development. Moreover, the high radiochemical yield of DOTA-C6-anti-EGFR highlights its potential for future radiochemical and theranostic applications, warranting further investigation.