Radiofluorinated GPC3-Binding Peptides for PET Imaging of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) remains one of the most challenging diseases worldwide. Glypican-3 (GPC-3) is a cell surface proteoglycan that is overexpressed on the membrane of HCC cells. The purpose of this study was to develop a target-specific radiofluorinated peptide for positron emission tomography (PET) imaging of GPC3 expression in hepatocellular carcinoma.

New GPC3-binding peptides (GP2076 and GP2633) were radiolabeled with F-18 using Al[¹⁸F]F labeling approach, and the resulting PET probes were subsequently subject to biological evaluations. A highly hydrophilic linker was incorporated into GP2633 with an aim of reducing the probe uptake in liver and increasing tumor-to-liver (T/L) contrast. Both GP2076 and GP2633 were radiolabeled using Al[¹⁸F]F chelation approach. The binding affinity, octanol/water partition coefficient, cellular uptake and efflux, and stability of both F-18 labeled peptides were tested. Tumor targeting efficacy and biodistribution of Al[¹⁸F]F-GP2076 and Al[¹⁸F]F-GP2633 were determined by PET imaging in HCC-bearing mice. Immunohistochemistry analyses were performed to compare the findings from PET scans.

Al[¹⁸F]F-GP2076 and Al[¹⁸F]F-GP2633 were rapidly radiosynthesized within 20 min in excellent radiochemical purity (> 97 %). Al[¹⁸F]F-GP2633 was determined to be more hydrophilic than Al[¹⁸F]F-GP2076 in terms of octanol/water partition coefficient. Both Al[¹⁸F]F-GP2076 and Al[¹⁸F]F-GP2633 demonstrated good in vitro and in vivo stability and binding specificity to GPC3-positive HepG2 cells. For PET imaging, Al[¹⁸F]F-GP2633 exhibited enhanced uptake in HepG2 tumor (%ID/g 3.37 ± 0.35 vs. 2.13 ± 0.55, P = 0.031) and reduced accumulation in liver (%ID/g 1.70 ± 0.26 vs. 3.70 ± 0.98, P = 0.027) at 60 min post-injection (pi) as compared to Al[¹⁸F]F-GP2076, resulting in significantly improved tumor-to-liver (T/L) contrast (ratio 2.00 ± 0.18 vs. 0.59 ± 0.14, P = 0.0004). Higher uptake of Al[¹⁸F]F-GP2633 in GPC3-positive HepG2 tumor was observed as compared to GPC3-negative McA-RH7777 tumor (%ID/g 3.37 ± 0.35 vs. 1.64 ± 0.03, P = 0.001) at 60 min pi, confirming GPC3-specific accumulation of Al[¹⁸F]F-GP2633 in HepG2 tumor.

The results demonstrated that Al[¹⁸F]F-GP2633 is a promising probe for PET imaging of GPC3 expression in HCC. Convenient preparation, excellent GPC3 specificity in HCC, and favorable excretion profile of Al[¹⁸F]F-GP2633 warrant further investigation for clinical translation. PET imaging with a GPC3-specific probe would provide clinicians with vital diagnostic information that could have a significant impact on the management of HCC patients.