A Novel CD147-Targeting Nanobody for Immuno-PET Imaging of Liver Cancer

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality, with a 5-y survival rate of approximately 20%. Many patients undergo locoregional therapy, and distinguishing viable tumor from treated necrotic tissue presents a significant clinical challenge with conventional imaging. CD147, a transmembrane glycoprotein overexpressed in approximately 60% of HCC tumors with minimal expression in normal liver, represents a promising target for tumor-selective molecular imaging to address this unmet clinical need. We aimed to develop and characterize a CD147-targeted nanobody-based PET imaging agent compatible with same-day clinical imaging workflows. Methods: We constructed a phage-display library from a llama immunized with recombinant human CD147. CD147-selective nanobodies were isolated through iterative immunopanning and characterized by enzyme-linked immunosorbent assay, biolayer interferometry, and flow cytometry using isogenic CD147-positive and CD147-knockout liver cancer cell lines. The lead candidate, denoted as DMN1, was radiolabeled with ¹⁸F via [¹⁸F]FPy pyridine-based prosthetic group conjugation. The radiolabeled nanobody was evaluated in cellular assays and further characterized in vivo using PET/CT imaging and biodistribution studies in subcutaneous and orthotopic murine HCC models. Results: DMN1 demonstrated low binding affinity to human CD147 (K _D = 0.71 ± 0.1 nM, enzyme-linked immunosorbent assay), with specificity confirmed in CD147-positive versus CD147-negative xenografts. [¹⁸F]FPy-DMN1 was synthesized with high radiochemical purity (>98%), a molar activity of 3,700-11,100 GBq/μmol, and good serum stability (87% intact in mouse serum at 3 h). In vivo, [¹⁸F]FPy-DMN1 showed robust tumor accumulation in CD147-positive (3.82 ± 0.93 %IA/g) versus CD147-negative tumor xenografts (0.21 ± 0.02 %IA/g), with excellent tumor-to-background ratios at 2 h postinjection (tumor-to-blood ratio, 18.7 ± 3.9; tumor-to-muscle ratio, 52.6 ± 22.1). In orthotopic liver tumors, [¹⁸F]FPy-DMN1 enabled clear tumor visualization with striking contrast at 3 h postinjection, whereas the control nanobody showed no tumor localization. Conclusion: [¹⁸F]FPy-DMN1 is a novel CD147-specific nanobody-based PET imaging agent that demonstrates high target specificity and favorable pharmacokinetics for same-day imaging. Its rapid blood clearance, high tumor-to-background ratios, and successful visualization of orthotopic liver tumors support further development of this agent for imaging and monitoring treatment response in patients with liver cancer.