Near-Infrared Fluorescent Macromolecular Reporters for Real-Time Imaging and Urinalysis of Cancer Immunotherapy

Real-time imaging of immunoactivation is imperative for cancer immunotherapy and drug discovery; however, most existing imaging agents possess “always-on” signals and thus have poor signal correlation with immune responses. Herein, renal-clearable near-infrared (NIR) fluorescent macromolecular reporters are synthesized to specifically detect an immunoactivation-related biomarker (granzyme B) for real-time evaluation of cancer immunotherapy. Composed of a peptide-caged NIR signaling moiety linked with a hydrophilic poly­(ethylene glycol) (PEG) passivation chain, the reporters not only specifically activate their fluorescence by granzyme B but also passively target the tumor of living mice after systemic administration. Such granzyme B induced in vivo signals of the reporters are validated to correlate well with the populations of cytotoxic T lymphocytes (CD8⁺) and T helper (CD4⁺) cells detected in tumor tissues. By virtue of their ideal renal clearance efficiency (60% injected doses at 24 h postinjection), the reporters can be used for optical urinalysis of immunoactivation simply by detecting the status of excreted reporters. This study thus proposes a molecular optical imaging approach for noninvasive evaluation of cancer immunotherapeutic efficacy in living animals.