Synthesis\nand Preclinical Evaluation of Dual-Specific\nProbe Targeting Glypican‑3 and Prostate-Specific Membrane Antigen\nfor Hepatocellular Carcinoma PET Imaging

Positron emission tomography (PET) is a promising modality\nfor\nearly diagnosis, accurate detection, and staging of hepatocellular\ncarcinoma (HCC). Hereby, a dual-specific probe targeting Glypican-3\n(GPC3) and prostate-specific membrane antigen (PSMA) was evaluated\nfor HCC PET imaging. The probe was prepared by conjugating TJ12P2,\na GPC3-targeting peptide previously reported by our group, to a highly\npotent PSMA inhibitor via a polyethylene glycol linker and further\ntethered to the 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)\nchelator. The resultant probe, NOTA-TJ12P2-PSMA, abbreviated as T2P,\nwas labeled with gallium-68 and fluorine-18, respectively, and evaluated\nin murine HCC models of various levels of GPC3 and PSMA expression.\nTargeting specificity was confirmed by blocking studies. The synthesized\n[<sup>68</sup>Ga]Ga-T2P and [<sup>18</sup>F]AlF-T2P were stable in\nsaline and fetal bovine serum for over 2 h, and bound to their respective\ntargets with high affinity and specificity in cell assays. PET imaging\nat 60 min postinjection (p.i.) showed that [<sup>68</sup>Ga]Ga-T2P\nexhibited higher uptake (1.75 ± 0.16%ID/g) in Huh7 models with\nhigh expression of GPC3 and PSMA than gallium-68 labeled TJ12P2 (1.25\n± 0.07%ID/g, <i>p</i> < 0.01) or gallium-68 labeled\nPSMA-617 (1.07 ± 0.06%ID/g, <i>p < 0.001</i>). The\nuptake of [<sup>68</sup>Ga]Ga-T2P in Huh7 tumors was higher than that\nin PC-3 tumors with low expression of GPC3 or PSMA (0.55 ± 0.24%ID/g, <i>p</i> < 0.01). The uptake of [<sup>18</sup>F]AlF-T2P or [<sup>68</sup>Ga]Ga-T2P in the Huh7 tumor was substantially blocked by\nTJ12P2, TJ12P2 + PSMA, or T2P, but only partially blocked by PSMA.\nAnd the PSMA and TJ12P2 monomer blocking effect was less than that\nof TJ12P2 + PSMA and T2P. [<sup>18</sup>F]AlF-T2P had higher tumor-to-muscle\nratios than [<sup>68</sup>Ga]Ga-T2P at 90 min postinjection (4.31\n± 0.10 vs 3.80 ± 0.17, <i>p < 0.05</i>) in\nHuh7 tumor models. To conclude, radiolabeled T2P exhibited a higher\nuptake and longer retention in Huh7 tumors than its monomeric counterparts.\nPET imaging via gallium-68 and fluorine-18 labeled T2P showed a similar\nimaging quality with comparable signal-to-background ratios. Our results\ndemonstrate that T2P is a promising tool for future clinical diagnosis\nof HCC.