Synthesis and Preclinical Evaluation of Dual-Specific Probe Targeting Glypican-3 and Prostate-Specific Membrane Antigen for Hepatocellular Carcinoma PET Imaging.

Positron emission tomography (PET) is a promising modality for early diagnosis, accurate detection, and staging of hepatocellular carcinoma (HCC). Hereby, a dual-specific probe targeting Glypican-3 (GPC3) and prostate-specific membrane antigen (PSMA) was evaluated for HCC PET imaging. The probe was prepared by conjugating TJ12P2, a GPC3-targeting peptide previously reported by our group, to a highly potent PSMA inhibitor via a polyethylene glycol linker and further tethered to the 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator. The resultant probe, NOTA-TJ12P2-PSMA, abbreviated as T2P, was labeled with gallium-68 and fluorine-18, respectively, and evaluated in murine HCC models of various levels of GPC3 and PSMA expression. Targeting specificity was confirmed by blocking studies. The synthesized [Ga]Ga-T2P and [F]AlF-T2P were stable in saline and fetal bovine serum for over 2 h, and bound to their respective targets with high affinity and specificity in cell assays. PET imaging at 60 min postinjection (p.i.) showed that [Ga]Ga-T2P exhibited higher uptake (1.75 ± 0.16%ID/g) in Huh7 models with high expression of GPC3 and PSMA than gallium-68 labeled TJ12P2 (1.25 ± 0.07%ID/g, < 0.01) or gallium-68 labeled PSMA-617 (1.07 ± 0.06%ID/g, ). The uptake of [Ga]Ga-T2P in Huh7 tumors was higher than that in PC-3 tumors with low expression of GPC3 or PSMA (0.55 ± 0.24%ID/g, < 0.01). The uptake of [F]AlF-T2P or [Ga]Ga-T2P in the Huh7 tumor was substantially blocked by TJ12P2, TJ12P2 + PSMA, or T2P, but only partially blocked by PSMA. And the PSMA and TJ12P2 monomer blocking effect was less than that of TJ12P2 + PSMA and T2P. [F]AlF-T2P had higher tumor-to-muscle ratios than [Ga]Ga-T2P at 90 min postinjection (4.31 ± 0.10 vs 3.80 ± 0.17, ) in Huh7 tumor models. To conclude, radiolabeled T2P exhibited a higher uptake and longer retention in Huh7 tumors than its monomeric counterparts. PET imaging via gallium-68 and fluorine-18 labeled T2P showed a similar imaging quality with comparable signal-to-background ratios. Our results demonstrate that T2P is a promising tool for future clinical diagnosis of HCC.