Development of novel cyclic form of fluorephore- or radiotracer-labeled c-Met peptide to detect high-c-Met expressing lung cancer

Abstract

Purpose: c-Met is a tyrosine kinase receptor for hepatocyte growth factor and have roles in induction of cancer cell growth, reduction of apoptosis, angiogenesis and scatter. c-Met overexpression in non-small cell lung cancer is clinically important, because it causes poor prognosis and leads to acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor drugs. L5-2 is a novel non-standard macrocyclic peptide which has high affinity to the external domain of the c-Met protein. The purpose of this study was to evaluate specific targeting to c-Met overexpressed lung cancer using FITC- or 68Ga-labeled macrocyclic c-Met peptide for optical or radionuclide cancer targeting study.

Methods: L5-2, macrocyclic c-Met binding peptide was synthesized by the Random non-standard Peptide Integrated Discovery (RaPID) system. The c-Met peptide was conjugated with FITC or SCN-DOTA chelator. DOTA-L5-2 c-Met peptide was labeled with 68Ga for 10 minutes (at pH 4.5) at 37 °C. Radiochemical purity was confirmed by radioTLC (0.1 M citric acid/ITLC-SG). Non-small cell lung cancer cell lines, NCI-H441 and NCI-H661 were chosen as c-Met positive and c-Met negative cell lines. Using RT-PCR assay and western blotting, total RNA and protein were prepared to measure c-Met expression level in these lung cancer cell lines. Specific binding of FITC-L5-2 c-Met peptide to cultured NCI-H441 and NCI-H661 cells was identified using high resolution confocal microscopy. In vitro binding assay was performed to analyze specific binding of 68Ga-labeled L5-2 c-Met peptide.

Results: RT-PCR and western blot analysis revealed that NCI-H441 lung cancer cell line, unlike c-Met negative NCI-H661 lung cancer cell line, highly expressed c-Met mRNA and protein. High fluorescence signals level in NCI-H441 was detected after 30 minutes of FITC-L5-2 c-Met peptide treatment. Specific binding of the FITC-L5-2 c-Met peptide to c-Met was found in NCI-H441 cell membrane. No fluorescence intensity was observed in NCI-H661 cells after treatment of FITC-L5-2 c-Met peptide. Radiochemical purity of 68Ga-labeled DOTA-L5-2 c-Met peptide was more than 90 %. In vitro radioligand binding assay exhibited that 68Ga radioactivity was gradually increased when different concentration of 68Ga-labeled DOTA-L5-2 c-Met peptide was treated in NCI-H441 cells. Approximately 3-fold higher radioactivity in NCI-H441 cells was found at 200 nM of 68Ga-labeled DOTA-L5-2 c-Met peptide compared to that in NCI-H661 cells.

Conclusion: The current in vitro fluorescence and radioligand binding study suggests that this novel non-standard macrocyclic peptide, L5-2, has a specific affinity to c-Met in lung cancer cells.. We expect that this successfully radiolabeled novel c-Met peptide will be used to detect the c-Met overexpressed lung cancer specifically in vivo.