Optical imaging of protease activities in the tumor immune microenvironment

Division Of Basic Sciences - Nci

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Abstract

To measure GzmA/B proteolytic activity within the tumor microenvironment (TME), we have developed selective chemical probes that emit fluorescence only upon reaction with active Gzms (bioRxiv, 2025, in revision). We utilized a diphenyl phosphinate warhead, which enables efficient release of the quencher upon conjugation with the catalytic serine residue. For optimal imaging potential, we incorporated the near-infrared (NIR) fluorophore, sulfonated-Cy5, into Gzm-selective tetrapeptide sequences. Our activatable probes exhibit fast reaction kinetics with Gzms, high selectivity, and cell permeability with minimal background noise. Following intravenous injection, the GzmB probe was found to rapidly generate and accumulate a fluorescence signal in the tumors of living mice, which is correlated with the expression levels of GzmB and CD8 and the immunotherapy response at the early stage of treatment. Thus, our study demonstrated that Gzm-targeted optical probes have significant potential for real-time detection of immune responses. Ongoing work focuses on optimizing these probes to improve signal contrast and pharmacokinetic properties, as well as exploring additional chemical and imaging modalities.