Synthesis and Evaluation of 64Cu-Labeled Monomeric and Dimeric NGR Peptides for MicroPET Imaging of CD13 Receptor Expression

The NGR-containing peptides have been shown to bind specifically to CD13/aminopeptidase N (APN) receptor, one of the attractive tumor vasculature biomarkers. In this study, we evaluated Cu-64-labeled monomeric and dimeric NGR peptides for microPET imaging of CD13 receptor expression in vivo. Western blot analysis and immunofluorescence staining were performed to identify CD13-positive and CD13-negative cell lines. NGR-containing peptides were conjugated with 1,4,7,10-tetraazadodecane-N,N',N '',N ''-tetraacetic acid (DOTA) and labeled with Cu-64 (t(1/2) = 12.7 h) in ammonium acetate buffer. The resulting monomeric (Cu-64-DOTA-NGR1) and dimeric (Cu-64-DOTA-NGR2) peptides were then subjected to in vitro stability, cell uptake and efflux, small animal micorPET, and biodistribution studies. In vitro studies demonstrated that CD13 receptors are overexpressed in human fibrosarcoma HT-1080 cells and negative in human colon adenocarcinoma HT-29 cells. The binding affinity of Cu-64-DOTA-NGR2 to HT-1080 cells was measured to be within low nanomolar range and about 2-fold higher than that of Cu-64-DOTA-NGR1. For small animal microPET studies, Cu-64-DOTA-NGR2 displayed more favorable in vivo performance in terms of higher tumor uptake and slower tumor washout in CD13-positive HT-1080 tumor xenografts as compared to Cu-64-DOTA-NGR1. As expected, significantly lower tumor uptake and poorer tumor/normal organ contrast were observed for both Cu-64-DOTA-NGR1 and Cu-64-DOTA-NGR2 in CD13-negative HT-29 tumor xenografts in comparison with those in the HT-1080 tumor xenografts. The CD13-specific tumor activity accumulation of both Cu-64-DOTA-NGRI and Cu-64-DOTA-NGR2 was further demonstrated by significant reduction of tumor uptake in HT-1080 tumor xenografts with a coinjected blocking dose of cyclic NGR peptide [c(CNGRC)]. The biodistribution results were consistent with the quantitative analysis of microPET imaging. We concluded that both Cu-64-DOTA-NGR1 and Cu-64-DOTA-NGR2 have good and specific tumor uptake in CD13-positive HT-1080 tumor xenografts. Cu-64-DOTA-NGR2 showed higher tumor uptake and better tumor retention than Cu-64-DOTA-NGRI, presumably due to bivalency effect and increase in apparent molecular size. Cu-64-DOTA-NGR2 is a promising PET probe for noninvasive detection of CD13 receptor expression in vivo.