Bioorthogonal Fluorogenic Reporters for Noninvasive Imaging and Urinalysis of Immunotherapeutic Response in Renal Cell Carcinoma.

Real-time monitoring of kidney-infiltrating cytotoxic T lymphocytes (CTLs) is crucial for evaluating immunotherapy in renal cell carcinoma (RCC). However, existing imaging probes often exhibit "always-on" signals and poor renal clearance, limiting their ability to detect renal immune responses. Herein, we report renal-clearable bio-orthogonal near-infrared (NIR) fluorogenic probes (BGRs) that specifically detect granzyme B (GzmB), a biomarker of CTL activation, for dynamic evaluation of RCC immunotherapy. BGRs are built on a nitrile-substituted hemicyanine scaffold with a biothiol-responsive cysteine tail that is dually locked with a GzmB-responsive peptide and conjugated to renal clearable (2-hydroxypropyl)-β-cyclodextrin (HPβCD). Following injection in RCC mouse models under immunotherapy, non-fluorescent BGRM accumulates in the renal tumor, where dual cleavage by elevated glutathione (GSH) and GzmB releases HPβCD and exposes an aminothiol group, triggering nitrile-aminothiol biorthogonal click reaction that activates NIR fluorescence and drives probe self-assembly for enhanced imaging performance. BGRs not only differentiate immunotherapeutic responses in living mice but also enable sensitive optical urinalysis of GzmB in clinical specimens from RCC patients (n = 21), allowing precise stratification of immune activation before and after treatment. This work thus establishes a generalizable strategy for translational optical reporters that addresses the unmet clinical need for dynamic monitoring of immunotherapies in RCC and other urological cancers.