Novel, dually radiolabeled peptides for simultaneous monitoring of enzymatic activity and protein targets

Objectives The goal of this study is to develop dually radiolabeled peptides for simultaneous targeting of cancer cells, through RGD binding of avb3 integrins and monitoring the enzymatic activity of cancer cells, through the cleavage of a substrate by MMP-2.

Methods A novel peptide Cyc(RGDfE)K(DOTA)PLGVRY has been designed for this purpose. The RGD moiety labeled with [64Cu] binds to the highly expressed avb3 integrins of cancer cells, thereby detecting the localization of cancer cells by PET. The matrix metalloproteinase, MMP-2, activity on the substrate PLGVR can be detected by observing the clearance of the [123I] label on the tyrosine of the peptide on the other side of the cleavage site via SPECT imaging. The peptide was successfully radiolabeled by [64Cu] and [123I]. Binding of the novel peptide to M21 and M21L cells (avb3 expressing and not expressing melanoma cells) and to pure avb3 integrins was studied. To elucidate the specificity of the binding, binding of the [64Cu] radiolabeled peptide in 1000M excess of unlabeled peptide was studied. Cleavage of the substrate by MMP-2 was also investigated.

Results The novel peptide was successfully labeled with [64Cu] and [123I] with 15 and 5 µCi/µg specific activities respectively, with >98% labeling purity. The peptide selectively bound to M21 over M21L cells by twofold. The peptide binds to avb3 integrins in the nM range (Kd = 83.4 + 13.2 nM). The specificity of Cyc(RGDfE)KPLGVRY(DOTA-[64Cu]) binding was confirmed by blocking studies. Cleavage of the peptide by activated MMP-2 was also observed via extensive HPLC analysis of the peptide fragments.

Conclusions These data demonstrate the potential of the dually radiolabeled peptide to locate protein targets and monitor enzymatic activity of cancer cells simultaneously. Animal studies are underway to determine the feasibility of in vivo imaging with this multimodality approach