Granzyme B Turns Nanoparticle Fluorescence "On" for Imaging Cytotoxic T Lymphocyte Activity in Vivo.

Abstract

Cytotoxic T lymphocytes (CTLs) are important immune cells, and their activation is a key step for cancer immunotherapy. Precise evaluation of CTL activity <i>in vivo</i> provides a powerful tool for monitoring cancer-immunotherapeutic outcomes, yet it faces tremendous challenges. Herein, by rationally designing a near-infrared (NIR) fluorescence probe Cys(StBu)-Ile-Glu-Phe-Asp-Lys(Cy5.5)-CBT (<b>Cy5.5-CBT</b>) and employing a reduction-instructed CBT-Cys click condensation reaction, we developed the fluorescence "dual quenched" nanoparticles <b>Cy5.5-CBT-NPs</b> for imaging of granzyme B (GraB), a biomarker tightly associated with the tumoricidal activity of CTLs. Upon GraB cleavage, <b>Cy5.5-CBT-NPs</b> disassembled, subtly turning the fluorescence signal "on". With this fluorescence "turn-on" property, <b>Cy5.5-CBT-NPs</b> enabled sensitive and real-time monitoring of GraB-mediated CTL responses against cancer cells <i>in vitro</i>. Animal experiments demonstrated that, at 16 h post injection, the fluorescence imaging signal of <b>Cy5.5-CBT-NPs</b> showed a 3.1-fold increase on the tumor sites of mice treated by an immune-activating drug <i>S</i>-(2-boronoethyl)-<i>L</i>-cysteine hydrochloride. We envision that <b>Cy5.5-CBT-NPs</b> may provide a powerful tool for noninvasive and sensitive evaluation of immunotherapeutic efficacy of cancer in the near future.