Novel dual-targeted gastrin-releasing peptide receptor and fibroblast activation protein radiopharmaceuticals for molecular imaging of heterogeneous breast tumors.

OBJECTIVES: The gastrin-releasing peptide receptor-targeted radioantagonist RM2 has shown promise for breast cancer imaging, but its limited metabolic stability may hamper in vivo performance. To improve stability and pharmacokinetics while addressing tumor heterogeneity, we developed two novel dual-targeted GRPR/fibroblast activation protein (FAP) heterodimeric radioligands, htdPP-01 and htdPP-02, combining RM2 with our patented eFAP scaffold. METHODS: Both heterodimers were radiolabeled with ¹¹¹In and characterized for radiochemical yield and purity, lipophilicity, binding affinity, cell uptake/internalization, and stability. Based on in vitro performance, [¹¹¹In]In-htdPP-02 was selected for microSPECT/CT imaging in mice bearing T47-D or HT-1080.huFAP xenografts, followed by ex vivo autoradiography and FAP immunohistochemistry. RESULTS: Both heterodimers were obtained with high radiochemical yields and purities (>97%) and were hydrophilic (logD7.4: -2.51 ± 0.03 and -3.02 ± 0.04). They showed high stability in phosphate-buffered saline (>95% at 24 h) and improved murine serum stability compared with [¹¹¹In]In-RM2 (>51% vs. 12% intact at 24 h). Both retained nanomolar affinity for both targets and showed target-specific cell uptake comparable to the corresponding monomeric references. In vivo, [¹¹¹In]In-htdPP-02 displayed higher and more sustained uptake in T47-D tumors than [¹¹¹In]In-RM2 and prolonged retention in HT-1080.huFAP tumors compared with [111In]In-FAPI-46, with low background accumulation. Autoradiography confirmed target-specific binding in both models, while immunohistochemistry demonstrated extensive FAP immunoreactivity in T47-D xenografts, providing a mechanistic rationale for dual GRPR/FAP targeting. CONCLUSION: [¹¹¹In]In-htdPP-02 combines improved metabolic stability, preserved dual GRPR/FAP binding, and favorable tumor uptake and retention, supporting GRPR/FAP heterodimerization as a promising strategy for imaging heterogeneous breast tumors.