Abstract
Fibroblast activation protein (FAP) is a highly expressed marker in cancer-associated fibroblasts (CAFs) across various epithelial cancers, making it an attractive target for diagnostic imaging. To address the limitations of existing FAP-targeted radiopharmaceuticals, such as poor tumor retention and off-target uptake, this study aimed to establish a comprehensive FAP nanobody library. Through systematic screening, we sought to identify a nanobody with cross-reactivity to both human and murine FAP, optimized for technetium-99m ((99m)Tc) labeling, and suitable for single-photon emission computed tomography (SPECT) imaging. A library of anti-FAP nanobodies (AFNs) was constructed and screened for binding affinity and specificity to human and murine FAP. Selected nanobodies were labeled with (99m)Tc using a site-specific radiolabeling process. In vitro assays were conducted to evaluate binding kinetics and cross-reactivity, while in vivo studies assessed pharmacokinetics, biodistribution, and imaging performance in murine tumor models. Finally, a first-in-human clinical study was performed to validate the safety and diagnostic efficacy of the lead nanobody-based radiotracer. From the nanobody library, three candidates were identified with high specificity for FAP: AFN01 (murine-specific), AFN05 (human-specific), and AFN13 (cross-reactive to both human and murine FAP). Among them, [(99m)Tc]-Tc-AFN13 demonstrated excellent binding affinity (dissociation constants: 2.16 ± 0.16 nM for murine FAP and 6.82 ± 0.54 nM for human FAP) and favorable pharmacokinetics. In vivo SPECT imaging revealed rapid tumor accumulation, prolonged retention, and minimal off-target uptake (e.g., tumor uptake of 4.41 ± 0.13% ID/cc at 30 min postinjection, declining to 2.35 ± 0.17% ID/cc at 12 h). Preliminary clinical imaging in patients confirmed the safety and specificity of [(99m)Tc]-Tc-AFN13 for FAP-expressing lesions, with no adverse events observed. In conclusion, this study successfully established a FAP nanobody library and identified [(99m)Tc]-Tc-AFN13 as a novel radiotracer with cross-reactivity to human and murine FAP. Its robust preclinical performance and promising clinical results highlight its potential for SPECT imaging in FAP-expressing cancers, paving the way for further clinical translation and theranostic applications.