Small-Animal PET Imaging of Human Epidermal Growth Factor Receptor Type 2 Expression with Site-Specific 18F-Labeled Protein Scaffold Molecules

Abstract

Human epidermal growth factor receptor type 2 (HER2) is a well-established tumor biomarker that is overexpressed in a wide variety of cancers and that serves as a molecular target for therapeutic intervention. HER2 also serves as a prognostic indicator of patient survival and as a predictive marker of the response to antineoplastic therapy. The development of ¹⁸F-labeled biomolecules for PET imaging of HER2 (HER2 PET) is very important because it may provide a powerful tool for the early detection of HER2-positive tumor recurrence and for the monitoring of HER2-based tumor treatment. Methods: In this study, anti-HER2 monomeric and dimeric protein scaffold molecules [Z_HER2:477 and (Z_HER2:477)₂, respectively] were radiofluorinated at a reasonable radiochemical yield (13%–18%) by use of site-specific oxime chemistry. The resulting radiofluorinated protein scaffold molecules were then evaluated as potential molecular probes for small-animal HER2 PET by use of a SKOV3 tumor–bearing mouse model. Results: The 4-¹⁸F-fluorobenzaldehyde conjugated aminooxy-protein scaffolds [¹⁸F-N-(4-fluorobenzylidene)oxime (FBO)-Z_HER2:477 and ¹⁸F-FBO-(Z_HER2:477)₂] both displayed specific HER2-binding ability in vitro. Biodistribution and small-animal PET imaging studies further revealed that ¹⁸F-FBO-Z_HER2:477 showed rapid and high SKOV3 tumor accumulation and quick clearance from normal tissues, whereas ¹⁸F-FBO-(Z_HER2:477)₂ showed poor in vivo performance (low tumor uptake and tumor-to-normal tissue ratios). The specificity of ¹⁸F-FBO-Z_HER2:477 for SKOV3 tumors was confirmed by its lower uptake on pretreatment of tumor-bearing mice with the HER2-targeting agents Z_HER2 and trastuzumab. Moreover, small-animal PET imaging studies revealed that ¹⁸F-FBO-Z_HER2:477 produced higher-quality tumor imaging than ¹⁸F-FBO-(Z_HER2:477)₂. ¹⁸F-FBO-Z_HER2:477 could clearly identify HER2-positive tumors with good contrast. Conclusion: Overall, these data demonstrate that ¹⁸F-FBO-Z_HER2:477 is a promising PET probe for imaging HER2 expression in living mice. It has a high potential for translation to clinical applications. The radiofluorination method developed can also be used as a general strategy for the site-specific labeling of other proteins with ¹⁸F. The protein scaffold molecules used here are attractive for the further development of PET probes for other molecular targets.