Design, synthesis, and evaluation of ⁶⁸Ga-Labeled peptide-based LGR5-targeting radiotracers for tumor imaging

Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is critically involved in tumor initiation, progression, metastasis, and therapy resistance, thus representing a promising biomarker for tumor diagnosis and treatment. In this study, we employed computational simulations to design LGR5-targeting peptides P01 and P02, and further developed two positron emission tomography (PET) radiotracers, [⁶⁸Ga]Ga-LTP-01 and [⁶⁸Ga]Ga-LTP-02, for accurate detection of LGR5 expression. Both radiotracers have high radiochemical purity (RCP) and yield (RCY), exhibiting good stability and favorable pharmacokinetic properties. Saturation binding assays showed that [⁶⁸Ga]Ga-LTP-02 exhibited higher binding affinity for LGR5 (K_d = 74.11 ± 24.52 nM) than [⁶⁸Ga]Ga-LTP-01 (K_d = 153.40 ± 17.14 nM). Dynamic PET imaging revealed considerably higher uptake of [⁶⁸Ga]Ga-LTP-02 in LGR5-positive A549 xenografts (4.74 ± 0.44 %ID/mL) than in LGR5-negative NCI-H1299 xenografts (0.98 ± 0.08 %ID/mL). Consistently, the tumor-to-muscle ratio was markedly higher in the A549 xenograft model (4.07 ± 0.07) than in the NCI-H1299 model (0.77 ± 0.10). Moreover, biodistribution studies further confirmed that [⁶⁸Ga]Ga-LTP-02 can specifically target LGR5-positive tumors. In summary, our study synthesized two LGR5-targeting peptide-based PET radiotracers and performed their preclinical evaluation. Among them, [⁶⁸Ga]Ga-LTP-02 exhibited superior sensitivity and specificity for detecting LGR5 expression and could effectively localize LGR5-positive tumors.