In vivo small-animal PET/CT of EphB4 receptors using 64Cu-labeled peptide.

UNLABELLED Many solid tumors overexpress EphB4 receptor, a member of the ephrin receptor tyrosine kinase family. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed against EphB4, and improve patient outcomes. The purpose of this study was to evaluate a novel (64)Cu-labeled peptide with high receptor binding affinity for PET of EphB4 receptors. METHODS The EphB4-binding peptide TNYLFSPNGPIARAW (TNYL-RAW) was conjugated with fluorescein isothiocyanate (FITC) and DOTA. DOTA-TNYL-RAW was labeled with (64)Cu with high labeling efficiency. The binding affinity of TNYL-RAW and its derivatives to purified recombinant EphB4 was determined using surface plasmon resonance technology. In vitro binding of both FITC-TNYL-RAW and (64)Cu-DOTA-TNYL-RAW to cancer cells was assessed by fluorescent microscopy and a radioactivity count method. In vivo biodistribution and small-animal PET/CT were performed in mice bearing EphB4-expressing CT26 and PC-3M tumors as well as EphB4-negative A549 tumors. RESULTS TNYL-RAW and its derivatives displayed high binding affinity to EphB4, with equilibrium dissociation constant of 1.98-23 nM. In vitro, both FITC-TNYL-RAW and (64)Cu-DOTA-TNYL-RAW were selectively taken up by CT26 and PC-3M cells but not by A549 cells. Binding of FITC-TNYL-RAW and (64)Cu-DOTA-TNYL-RAW to CT26 and PC-3M cells could be blocked by an excess amount of TNYL-RAW. In vivo, (64)Cu-DOTA-TNYL-RAW showed significantly higher uptake in PC-3M tumors than in A549 tumors, with percentages of injected dose per gram of tumor of 0.84 ± 0.09 and 0.44 ± 0.09 at 24 h after radiotracer injection, respectively. Small-animal PET/CT clearly revealed deposition of (64)Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, uptake of (64)Cu-DOTA-TNYL-RAW in both CT26 and PC-3M tumors could be blocked by cold TNYL-RAW. CONCLUSION The expression of EphB4 receptors can be noninvasively interrogated by small-animal PET/CT using (64)Cu-DOTA-TNYL-RAW.