AND-gated protease-activated nanosensors for programmable detection of anti-tumour immunity

The forward design of biosensors that implement Boolean logic to improve detection precision primarily relies on programming genetic components to control transcriptional responses. However, cell- and gene-free nanomaterials programmed with logical functions may present lower barriers for clinical translation. Here we report the design of activity-based nanosensors that implement AND-gate logic without genetic parts via bi-labile cyclic peptides. These actuate by releasing a reporter if and only if cleaved by a specific pair of proteases. AND-gated nanosensors that detect the concomitant activity of the granzyme B protease secreted by CD8 T cells and matrix metalloproteinases overexpressed by cancer cells identify the unique condition of cytotoxic T cell killing of tumour cells. In preclinical mouse models, AND-gated nanosensors discriminate tumours that are responsive to immune checkpoint blockade therapy from B2m^-/- tumours that are resistant to it, minimize signals from tissues without co-localized protease expression including the lungs during acute influenza infection, and release a reporter locally in tissue or distally in the urine for facile detection.