P8 - Non-invasive monitoring of antitumor activity of T cells via granzyme B-activated plasmonic photoacoustic nanosensors

Introduction: : Adoptive T cell therapies (ACTs) represent a promising clinical approach for treating cancer by harnessing a patient’s own T lymphocytes. As such, identifying biomarkers that can objectively assess antitumor T cell activity in conjunction with tumor anatomy has become a clinical objective, potentially enhancing the precision of ACT. A hallmark of therapeutic response following ACT is the presence of cytotoxic T cells capable of killing tumor cells through the classical granzyme-mediated pathway. Here we present a strategy to non-invasively monitor antitumor T cell activity in tumors via granzyme B (GzmB)-activated plasmonic photoacoustic (PA) nanosensors. These nanosensors consist of a gold nanosphere (GNS) core functionalized with GzmB-cleavable peptides on the surface. In the presence of GzmB, peptide cleavage induces nanosensor aggregation, resulting in strong optical responses at near-infrared (NIR) wavelengths via interparticle plasmon coupling. This enhanced optical activity leads to an increase in PA imaging signals within the NIR window. Our nanosensor platform enables real-time, functional imaging of T cell-mediated cytotoxicity both in vitro and in vivo, which may provide clinicians with an insight into therapeutic efficacy. Furthermore, owing to the modular design of this platform based on the tunability of peptide sequences, this platform may be extended to monitor diverse disease-relevant proteases, broadening its applicability across a wide range of immune and pathological contexts.

Materials and

Methods: : Gold nanospheres (GNSs) were synthesized using the Turkevich method. GzmB-cleavable peptide sequences containing a terminal cysteine residue were conjugated to the GNSs via gold-thiol bonding, yielding GzmB-activated PA nanosensors. GzmB-mediated aggregation of the nanosensors was confirmed using transmission electron microscopy (TEM). The resulting aggregation and corresponding enhancement of the nanosensors' optical response at NIR wavelengths were characterized by UV-vis-NIR spectroscopy. The optical and PA responses of the nanosensors were evaluated across different proteases to assess specificity. To investigate the nanosensors' ability to monitor T cell-mediated antitumor activity in vitro, ovalbumin (OVA)-specific OT1 T cells were co-cultured with either EL4 (OVA-negative) or EG7-OVA (OVA-positive) tumor cells. Tumor cell viability, GzmB secretion, and PA responses were quantified using flow cytometry, ELISA, and PA imaging. For in vivo studies, subcutaneous EL4 or EG7-OVA tumors were established in C57BL/6 mice, followed by ACT of OT1 T cells. To monitor antitumor T cell activity, GzmB-activated PA nanosensors were intravenously administered one day post-ACT, and Ultrasound (US)/PA imaging was performed 24 hours after particle injection.

Results, Conclusions, and Discussions:: Results and

Discussion: We developed GzmB-activated plasmonic PA nanosensors by conjugating GzmB-cleavable peptides to gold nanospheres (GNSs) via gold-thiol bonds (Fig. 1a). Upon GzmB exposure, the peptides were cleaved, destabilizing the nanosensors and subsequently inducing aggregation (Fig. 1b). This aggregation enhanced NIR optical responses due to interparticle plasmon coupling (Fig. 1c). The nanosensors responded specifically to GzmB, exhibiting minimal PA signals and contrast in the presence of other proteases including matrix metalloproteinases (MMP-7, MMP-9), caspase-3 (Cas-3), and bovine serum albumin, resulting in significantly stronger PA signals with GzmB (Fig. 1d). We next tested the nanosensors for detecting T cell-mediated antitumor activity in vitro. OT1 T cells were co-cultured with EL4 or EG7-OVA tumor cells, and tumor killing and GzmB secretion were quantified. OT1 T cells selectively targeted EG7-OVA cells, leading to increased GzmB secretion (Figs. 1e, f). Thus, the nanosensors enhanced PA signals only in the presence of both OT1 T cells and EG7-OVA cells (Fig. 1g). To assess in vivo functionality of nanosensors, we inoculated EL4 or EG7-OVA tumors to mice and administered OT1 T cells five days later (Figs. 1h, i). One day post-ACT, PA nanosensors were intravenously injected. Before nanosensor injection, PA signals were similar across tumors (Fig. 1j). After injection, EG7-OVA tumors showed significantly stronger PA signals compared to EL4 tumors (Fig. 1j). Notably, these differences in PA signal were detected two days before measurable divergence in tumor volume among groups (Figs. 1i, j).

Conclusion: Our study demonstrates a protease-activated nanosensor platform that enables non-invasive monitoring of T cell-mediated antitumor activity via US/PA imaging. The nanosensors produce specific PA responses in the presence of GzmB, distinguishing them from other proteases. This platform allows for real-time imaging of T cell cytotoxicity against tumor cells both in vitro and in vivo, while simultaneously providing tumor anatomical information through US imaging.

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