64Cu-Labeled Tetrameric and Octameric RGD Peptides for Small-Animal PET of Tumor αvβ3 Integrin Expression

Integrin αvβ3 plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive imaging of αvβ3 expression and targeted radionuclide therapy. In this study, we developed 64 Cu-labeled multimeric RGD peptides, E{E[c(RGDyK)] 2 } 2 (RGD tetramer) and E(E{E[c(RGDyKj] 2 } 2 ) 2 (RGD octamer), for PET imaging of tumor integrin α v β 3 expression. Methods: Both RGD tetramer and RGD octamer were synthesized with glutamate as the linker. After conjugation with 1,4,7,10-tetra-azacyclododecane-N, N',N',N"'-tetraacetic acid (DOTA), the peptides were labeled with 64 Cu for biodistribution and small-animal PET imaging studies (U87MG human glioblastoma xenograft model and c-neu onco-mouse model). A cell adhesion assay, a cell-binding assay, receptor blocking experiments, and immunohistochemistry were also performed to evaluate the α v β 3 -binding affinity/specificity of the RGD peptide-based conjugates in vitro and in vivo. Results: RGD octamer had significantly higher integrin α v β 3 -binding affinity and specificity than RGD tetramer analog (inhibitory concentration of 50% was 10 nM for octamer vs. 35 nM for tetramer). 64 Cu-DOTA-RGD octamer had higher tumor uptake and longer tumor retention than 64 Cu-DOTA-RGD tetramer in both tumor models tested. The integrin α v β 3 specificity of both tracers was confirmed by successful receptor-blocking experiments. The high uptake and slow clearance of 64 Cu-DOTA-RGD octamer in the kidneys was attributed mainly to the integrin positivity of the kidneys, significantly higher integrin α v β 3 -binding affinity, and the larger molecular size of the octamer, as compared with the other RGD analogs. Conclusion: Polyvalency has a profound effect on the receptor-binding affinity and in vivo kinetics of radiolabeled RGD multimers. The information obtained here may guide the future development of RGD peptide-based imaging and internal radiotherapeutic agents targeting integrin α v β 3 .