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

ABSTRACT 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 BGR _M 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.