Development and Evaluation of a Dual-Modality Theranostic Agent Targeting Cysteine Cathepsin B for Cancer Diagnosis and Therapy


Cysteine cathepsin B (CTS-B) is a protease enzyme that plays an important role in the breakdown of proteins inside cells. Its activity is associated with various diseases, including cancer, rheumatoid arthritis, and neurodegenerative diseases like Alzheimer’s. In cancer, Cathepsin B has been implicated in tumor progression and metastasis, making it a druggable target for therapeutic intervention. Its involvement in degrading the extracellular matrix contributes to the invasiveness of cancer cells. Due to its involvement in these critical biological processes and its association with various diseases, Cathepsin B is a subject of intense research. Understanding its mechanisms can lead to the development of new therapeutic strategies for treating diseases associated with its dysregulation. A new study published in Molecular Pharmaceutics and conducted by Lianbo Zhou, Feng He, Xin Xiang, Chuning Dong, Tian Xiang, Xian Li, Hong Li, Lihong Bu, Yunhua Wang, and Professor Xiaowei Ma from the Department of Nuclear Medicine at the Second Xiangya Hospital of Central South University, the authors designed a novel dual-modality activity-based theranostic probe, BMX2, specifically to target CTS-B and subsequently labeled with 68Ga for PET imaging and 90Y for radionuclide therapy. The synthesis involved conjugating a Cbz-Phe-Lys peptide with sulfo-cyanine5.5 for fluorescence imaging and 1,4,7,10-tetraazacyclododecane (DOTA) for radionuclide chelation. The radiolabeling efficiency was high, with 68Ga labeling yielding over 95% purity and 90Y labeling achieving similar purity levels. The labeled compounds demonstrated stability in human serum, suggesting their potential for in vivo applications.

The authors confirmed the binding affinity and specificity of BMX2 towards CTS-B using fluorescent western blot analysis by utilizing recombinant human CTS-B and various cancer cell lines (HeLa, HepG2, MCF7, U87MG). The experiments revealed that BMX2 binds specifically and with high affinity to CTS-B in a concentration-dependent manner. Additionally, confocal laser scanning microscopy confirmed the probe’s intracellular uptake and co-localization with CTS-B, highlighting its potential for targeted imaging and therapy. They also performed cell uptake studies to quantify the absorption of BMX2 and 68Ga-BMX2 in cancer cells. These experiments demonstrated time-dependent uptake of the probe, with significant accumulation within cells over time. The uptake was further confirmed to be CTS-B specific, as pre-incubation with a CTS-B inhibitor (CA074) significantly reduced probe absorption. The researchers conducted in vivo studies using HeLa xenografts in mice to evaluate the diagnostic potential of 68Ga -BMX2 through PET imaging and BMX2 through fluorescence imaging. The in vivo imaging results indicated high tumor uptake and retention of the probes, with clear tumor visualization over 24 hours post-injection. This was corroborated by the biodistribution studies, which confirmed the tumor-selective accumulation of the probes. The therapeutic efficacy of 90Y -BMX2 was assessed in vivo using the HeLa xenograft model. Mice treated with 90Y -BMX2 exhibited significantly slower tumor growth compared to controls, demonstrating the probe’s potential for targeted radionuclide therapy. The therapeutic effect was further validated by comparing it against a control group and a group treated with the probe in the presence of the CTS-B inhibitor, underscoring the specificity of the therapeutic action.

In conclusion, the novelty of the research work of Professor Xiaowei Ma and colleagues lies in its integrative approach to cancer theranostics, combining diagnostic imaging and targeted therapy within a single agent, thereby providing a potent tool for real-time monitoring of therapy efficacy and disease progression. The development of 68Ga/90Y-BMX2 showcases significant potential for clinical translation, emphasizing the theranostic agent’s efficacy in PET diagnostic imaging, fluorescence imaging, and radionuclide therapy of cancers. This dual modality, targeting the enzymatic activity of CTS-B, highlights a strategic pivot from conventional methods towards more targeted, effective, and personalized cancer management strategies. Moreover, the study advances the understanding of CTS-B’s role in cancer biology, offering insights into the enzyme’s variable expression across different cancer cell lines and its impact on the efficacy of the theranostic agent. The successful application of BMX2 in inhibiting tumor growth in vivo via 90Y -based therapy further validates the therapeutic potential of targeting CTS-B, opening new avenues for the development of cancer therapeutics.


Zhou L, He F, Xiang X, Dong C, Xiang T, Li X, Li H, Bu L, Wang Y, Ma X. Radioactive and Fluorescent Dual Modality Cysteine Cathepsin B Activity-Based Probe for Cancer Theranostics. Mol Pharm. 2023;20(7):3539-3548. doi: 10.1021/acs.molpharmaceut.3c00148.

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