CD13 receptor as a tumor vasculature biomarker has attracted great attention in cancer research. Through phage display screening, NGR-containing peptides have been characterized as specific ligands binding to CD13 receptor. In this study, a (64)Cu-labeled dimeric NGR peptide based on sarcophagine cage was synthesized and evaluated for micropositron emission tomography (PET) imaging of CD13 expression in vivo. Macrocyclic chelating agent (sarcophagine cage) was conjugated with two azide moieties, followed by mixing with an alkyne-containing NGR peptide to rapidly provide the Sar-NGR2 peptide by click chemistry. Radiolabeling of Sar-NGR2 with (64)Cu was achieved in >90% decay-corrected yield with radiochemical purity of >99%. The cell uptake study showed that (64)Cu-Sar-NGR2 binds to CD13-positive HT-1080 cells, but not to CD13-negative MCF-7 cells. MicroPET imaging results revealed that (64)Cu-Sar-NGR2 exhibits good tumor uptake in CD13-positive HT-1080 xenografts and significantly lower tumor uptake in CD13-negative MCF-7 xenografts. The CD13-specific binding of (64)Cu-Sar-NGR2 was further verified by significant reduction of tumor uptake in HT-1080 tumor xenografts with coinjection of a nonradiolabeled NGR peptide. The biodistribution results demonstrated good tumor/muscle ratio (8.28 ± 0.37) of (64)Cu-Sar-NGR2 at 24 h pi in HT-1080 tumor xenografts, which is in agreement with the quantitative analysis of microPET imaging. In conclusion, sarcophagine cage has been successfully applied to the construction of a (64)Cu-labeled dimeric NGR-containing peptide. In vitro and in vivo studies demonstrated that (64)Cu-Sar-NGR2 is a promising PET probe for imaging CD13 expression in living mice.
Keywords: 64Cu labeling; CD13; NGR peptide; PET imaging; bivalency effect; catalyst-free click chemistry; tumor angiogenesis.