[^99mTc]Tc-MY6349 Probe for Trop2-Targeted SPECT Imaging: From Preclinical to Pilot Clinical Study

The trophoblast cell-surface antigen 2 (Trop2) is markedly overexpressed in breast cancers, with a particularly high incidence in triple-negative breast cancer. The therapeutic relevance of Trop2 expression is underscored by the approval of an antibody-drug conjugate for triple-negative breast cancer treatment. However, there is no a predictive technique for accurate whole-body mapping of Trop2 expression in patients. In this study, we developed a novel Trop2-specific molecular probe, [^99mTc]Tc-MY6349, and evaluated its safety and feasibility for detecting Trop2 expression in breast cancer using SPECT/CT imaging. Methods: Trop2 expression in different breast cancer cell lines was assessed via immunofluorescence and flow cytometry. The Trop2-specific nanobody MY6349 was site-specifically labeled with ^99mTc via a C-terminal GGGC tag, and its binding affinity to the Trop2 receptor was tested in vitro. The in vivo tumor uptake and distribution of [^99mTc]Tc-MY6349 were examined through SPECT imaging and biodistribution studies. Furthermore, a pilot clinical study of [^99mTc]Tc-MY6349 SPECT/CT was conducted in 8 patients with breast cancer, and the results were compared with [¹⁸F]FDG PET/CT. Results: [^99mTc]Tc-MY6349 achieved a greater than 95% radiochemical purity after purification. In vitro and in vivo experiments demonstrated the binding specificity of [^99mTc]Tc-MY6349 to the Trop2 receptor. In vivo imaging and biodistribution studies revealed a significant correlation between tumor uptake and Trop2 expression levels. In the pilot clinical study, SPECT imaging with [^99mTc]Tc-MY6349 successfully detected Trop2-positive tumors 15 min after tracer injection. Delayed imaging showed reduced uptake in normal organs but sustained retention of [^99mTc]Tc-MY6349 in tumors. Importantly, [^99mTc]Tc-MY6349 SPECT/CT imaging highlighted Trop2 expression heterogeneity and visualized primary and metastatic lesions with a favorable tumor-to-background ratio in breast cancer. Conclusion: [^99mTc]Tc-MY6349 was successfully prepared and exhibited a high binding affinity and Trop2 specificity. The pilot clinical study validated the safety and feasibility of [^99mTc]Tc-MY6349 SPECT/CT for detecting Trop2 expression in vivo in patients with breast cancer. This imaging modality could complement existing methods, aiding in the guidance of Trop2-targeted therapies and advancing personalized treatment while also promoting the application of SPECT/CT nuclear medicine imaging technology.