68Ga-NOTA-FAPI-IF7: A Heterobivalent PET Probe for Dual-Targeted Imaging of Fibroblast Activation Protein and Annexin A1 in Tumor Microenvironment

Abstract Fibroblast activation protein (FAP)-targeted PET tracers enable high-contrast tumor imaging but suffer from rapid clearance and non-specific uptake in inflammatory lesions. The heptapeptide IF7 binds annexin A1 (ANXA1) on tumor vascular endothelium, a target directly accessible from the bloodstream. To overcome these limitations, we developed a heterobivalent PET probe, NOTA-FAPI-IF7, based on a rationally designed dual-targeting strategy that simultaneously engages FAP in cancer-associated fibroblasts and ANXA1 on tumor vasculature. Radiolabeling with 68Ga achieved a radiochemical yield of 92.3 ± 3.6% (decay-corrected, n = 5) with excellent stability (radiochemical purity >97% at all time points). In vitro assays confirmed dual-target binding (IC50 = 27.9 nM, KD = 3.77 nM). Micro-PET imaging demonstrated rapid tumor visualization as early as 10 min post-injection (6.02 ± 0.07% ID/g), with uptake significantly reduced by blocking with excess unlabeled FAPI, IF7, or NOTA-FAPI-IF7. The probe exhibited favorable hydrophilicity (log P = −1.20) and renal clearance with low hepatobiliary uptake. This bispecific strategy, distinct from monospecific and homomultimeric approaches, offers a promising avenue for precision oncologic PET imaging.