Cysteine cathepsin B (CTS-B) is a crucial enzyme that is overexpressed in numerous malignancies and contributes to the invasion and metastasis of cancer. Therefore, this study sets out to develop and evaluate an activity-based multimodality theranostic agent targeting CTS-B for cancer imaging and therapy. A CTS-B activity-based probe, BMX2, was synthesized and labeled efficiently with 68Ga and 90Y to produce 68Ga-BMX2 for multimodality imaging and 90Y-BMX2 for radiation therapy. The affinity and specificity of BMX2 binding with the CTS-B enzyme were determined by fluorescent western blots using recombined active human CTS-B enzyme (rh-CTS-B) and four cancer cell lines including HeLa, HepG2, MCF7, and U87MG, with CA074 as the CTS-B inhibitor for control. Confocal laser scanning microscope imaging and cell uptake measurement were also performed. Then, in vivo PET imaging and fluorescence imaging were acquired on HeLa xenografts. Finally, the therapeutic effect of 90Y-BMX2 was tested. BMX2 could be specifically activated by rh-CTS-B and stably bound to the enzyme. The binding of BMX2 with CTS-B is time-dependent and enzyme concentration-dependent. Although CTS-B expression varied between cell lines, all showed significant uptake of BMX2 and 68Ga-BMX2. In vivo optical and PET imaging showed a high tumor uptake of BMX2 and 68Ga-BMX2 and accumulation for more than 24 h. 90Y-BMX2 could significantly inhibit HeLa tumor growth. The development of 68Ga/90Y-BMX2, a radioactive and fluorescent dual modality theranostic agent, demonstrated an effective theranostic approach for PET diagnostic imaging, fluorescence imaging, and radionuclide therapy of cancers, which may have a potential for clinical translation for cancer theranostics in the future.

Purpose: Cysteine cathepsin B (CTS-B) is a crucial enzyme that is overexpressed in numerous malignancies and contributes to the invasion and metastasis of cancer. Therefore, this study set out to develop and evaluate an activity-based multi-modality theranostic agent targeting CTS-B for cancer imaging and therapy. Methods: A CTS-B activity-based probe, BMX2, was synthesized and labeled efficiently with 68Ga and 90Y to produce 68Ga-BMX2 for multi-modality imaging and 90Y-BMX2 for radiation therapy. The affinity and specificity of the BMX2 binding with the CTS-B enzyme were determined by fluorescent western blot using recombined active human CTS-B enzyme (rh-CTS-B) and four cancer cell lines including HeLa, HepG2, MCF7, and U87MG with CA074 as CTS-B inhibitor for control. Confocal laser scanning microscope imaging and cell uptake measurement was also performed. Then in vivo PET imaging and fluorescence imaging were acquired on HeLa xenografts. Finally, the therapeutic effect of 90Y-BMX2 was tested. Results: BMX2 could be specifically activated by rh-CTS-B and stably bound to the enzyme. The binding of BMX2 with CTS-B has a time-dependent and enzyme concentration-dependent manner. Although CTS-B expression varied between cell lines, all showed significant uptake of BMX2 and 68Ga-BMX2. In vivo optical and PET imaging showed high tumor uptake of BMX2 and 68Ga-BMX2 and accumulated for more than 24 hours. The 90Y-BMX2 could significantly inhibit HeLa tumor growth. Conclusion: The development of 68Ga/90Y-BMX2, a radioactive and fluorescent dual modality theranostic agent, demonstrated an effective theranostic approach for PET diagnostic imaging, fluorescence imaging, and radionuclide therapy of cancers, which may have a potential for clinical translation for cancer theranostic in the future.