A GRPR Antagonist Peptide-Based Pretargeting Strategy Overcoming Molecule Internalization for Enhanced Tumor Imaging

Pretargeting is an emerging strategy for tumor imaging that offers improved specificity and reduced off-target toxicity compared to conventional direct targeting approaches. However, a major limitation lies in the rapid internalization of pretargeting molecules, which reduces the availability of binding sites for effector probes and compromises imaging performance. In this study, we present a GRPR-targeted pretargeting system designed to overcome this challenge by employing the GRPR antagonist peptide RM26, which remains on the cell surface after receptor binding and thus minimizes internalization. The system is based on a bioorthogonal inverse electron-demand Diels–Alder reaction between IBA-RM26-TCO and 99mTc-HYNIC-Tz, which is completed quantitatively within 10 min under physiological conditions, with excellent stability, as evidenced by over 93% radiochemical purity after 24 h. In vitro experiments confirmed that RM26 maintained high GRPR binding affinity with minimal internalization, facilitating robust bioorthogonal reactivity. In vivo SPECT/CT imaging in GRPR-positive U-87MG xenograft mice further demonstrated more than 2-fold higher tumor uptake and an improved tumor-to-muscle ratio compared to direct targeting. Collectively, this work establishes a GRPR antagonist peptide-based pretargeting platform that not only addresses the internalization barrier but also enhances diagnostic performance, offering a safer and more effective approach for precision tumor imaging.