Diagnosis and Treatment Response Monitoring of Liver Cancer Using Glypican-3–Targeted Single-Domain Antibody PET

PURPOSE: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death and is characterized by poor survival rates and high recurrence after surgery. Glypican-3 (GPC3) is a proteoglycan highly expressed in HCC but absent in most normal tissue, making it an attractive diagnostic and therapeutic target. In this study, we introduce a second-generation GPC3-targeted single-domain antibody probe (ssHN3) bearing a positron-emitting isotope fluorine-18 (18F), ssHN3-Al[18F]F-RESCA (Al[18F]F-ssHN3), for HCC-selective PET imaging. In addition, we show its use as a functional imaging agent following focal tumor thermal ablation. EXPERIMENTAL DESIGN: Site-specific conjugation was used to synthesize the nanobody-based PET (immunoPET) probe ssHN3-Al[18F]F-RESCA. Binding affinity was determined using biolayer interferometry, and in vivo PET/CT imaging and biodistribution studies were performed in three liver cancer models with varying GPC3 expression (HepG2 > Hep3B > Huh7). Mice inoculated with HepG2 orthotopic liver tumors were also imaged before and 1 week after thermal tumor ablation to assess response. RESULTS: Our agent exhibited high purity (>98%), nanomolar affinity for GPC3, and tumor uptake corresponding to GPC3 expression on PET/CT and biodistribution studies. In orthotopic murine models of liver cancer, Al[18F]F-ssHN3 successfully distinguished between total versus subtotal thermal ablation, accurately identifying residual, viable disease. CONCLUSIONS: We successfully designed, engineered, and tested a GPC3-targeted 18F-labeled nanobody immunoPET agent, Al[18F]F-ssHN3, demonstrating that it can be used for same-day diagnostic imaging in murine models of liver cancer. Importantly, this agent could address the limitations of current imaging methods by detecting residual disease after thermal ablation and other locoregional therapies.