Objectives: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, with limited therapeutic options for advanced HCC. Radiopharmaceutical therapy (RPT) is emerging as a safe and effective targeted approach to treating advanced malignancies. Glypican-3 (GPC3) is a membrane-associated glycoprotein that is significantly upregulated in HCC, but not normal tissues. The differential expression of GPC3 between tumor and normal tissues makes it a promising target for RPT to treat HCC. Codrituzumab (GC33) is a full-length humanized monoclonal IgG1 specific to GPC3. We previously synthesized [²²⁵Ac]Ac–Macropa–GC33 and evaluated their therapeutic efficacy in subcutaneous xenografts with GPC3-expressing human liver cancer cell line (HepG2).^[1] Herein, we validated the therapeutic efficacy of [²²⁵Ac]Ac-Macropa-GC33 (²²⁵Ac-GC33) for radiopharmaceutical therapy as well as the performance of [⁸⁹Zr]Zr-DFO-GC33 (⁸⁹Zr-GC33) for immunoPET imaging in orthotopic HCC models.

Methods: Orthotopic hepatocellular carcinoma models were prepared by the intrahepatic injection of HepG2-Luc⁺ cells, and their tumor growth was monitored by bioluminescence imaging (BLI) with D-luciferin. The expression of GPC3 in orthotopic HCC tumors was assessed by immunohistochemistry (IHC). The chelators macropa-NCS and p-NCS-Bz-DFO were conjugated to GC33 for subsequent ²²⁵Ac and ⁸⁹Zr radiolabeling, respectively. Tumor and other organ uptakes of ²²⁵Ac-GC33 and ⁸⁹Zr-GC33 were evaluated in biodistribution studies using orthotopic HCC models at 72 h post-injection. For the RPT study in orthotopic HCC models, treatment groups (n=8 per group) included A) PBS, B) 9.25 kBq ²²⁵Ac-IgG1 (isotype control), and C) 9.25 kBq ²²⁵Ac-GC33. Treatment response and toxicity were assessed using BLI, immunoPET, and body weight and overall survival were recorded to assess the therapeutic effect and toxicity.

Results: After four weeks of intrahepatic injection of HepG2-Luc⁺ cells, it was confirmed that the orthotopic HCC tumor had reached an appropriate size (4 – 6 mm) for therapy studies. IHC results demonstrated that GPC3 was clearly expressed on the membrane of the orthotopic HCC tumor region, while normal liver tissue showed no expression. All radiolabeling procedures were successful, achieving over 95% radiolabeling efficiency and 98% radiochemical purity. Tumor uptake of ²²⁵Ac-GC33 and ⁸⁹Zr-GC33 were nearly equal (211.6 ± 7.0 %IA/g and 205.6 ± 20.3 %IA/g, respectively), and their biodistribution patterns in other organs were also very similar, showing extremely high tumor-to-organ ratios. The RPT study in orthotopic HCC models showed significant tumor growth inhibition of ²²⁵Ac-GC33 without substantial body weight reduction. Especially, complete tumor eradication was observed in some of the ²²⁵Ac-GC33 treated group (5/8). Additionally, results from BLI and PET imaging with ⁸⁹Zr-GC33 were nearly identical, but PET imaging with ⁸⁹Zr-GC33 provided a more sensitive assessment of orthotopic HCC tumor size and therapeutic efficacy compared to BLI.

Conclusions: Our study showed that GPC3-targeted ⁸⁹Zr-GC33 can selectively image orthotopic HCC tumor in ²²⁵Ac-based RPT, which can significantly inhibit the growth of orthotopic HCC tumor, resulting in complete responses in 62.5% (5/8) tumors versus 0% (0/16) in control groups. These results demonstrate that GPC3 is a promising theragnostic target for HCC, with the potential for clinical translation.

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Image/Figure Caption:

(A) Representative immunohistochemical staining in HepG2-Luc⁺ orthotopic HCC tumor. Histomorphological features were assessed using hematoxylin and eosin (H&E) staining and immunohistochemical staining for GPC3 and Ki-67. (B) Biodistribution data of ²²⁵Ac-GC33 and ⁸⁹Zr-GC33 in the major organs of HepG2-Luc⁺ orthotopic HCC tumor models at 72 h post-injection. Radioactivity uptakes in biodistribution were calculated as the percentage of the injected activity per gram of tissue (%IA/g). Data are present as the mean ± SD (n=3, respectively). (C) Comparison of tumor-to-organ ratios between ²²⁵Ac-GC33 and ⁸⁹Zr-GC33 from biodistribution data at 72 h post-injection in orthotopic HCC tumor models. (D) Quantification of BLI signal of orthotopic HCC tumor during RPT study. Data are present as the mean ± SD (n=8 per group). (E) Body weight monitoring results during RPT study. Data are present as the mean ± SD (n=8 per group). (F) At 3 weeks post-treatment, BLI and PET imaging with ⁸⁹Zr-GC33 at 72 h post-injection. (G) At 9 weeks post-treatment, BLI and PET imaging with ⁸⁹Zr-GC33 at 72 h post-injection. BLI scale in p/sec/cm²/sr. PET images display radioactivity calibrated in standardized uptake values (SUV). In BLI, mice were positioned in the supine position, while in PET/CT images, mice were positioned in the prone position. In PET/CT images, an orthotopic HCC tumor was indicated as a white triangle.

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