A Functional Chemiluminescent Probe for in Vivo Imaging of Natural Killer Cell Activity Against Tumours

Abstract

Natural killer (NK) cells are immune cells that can kill certain types of cancer cells. Adoptive transfer of NK cells represents a promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti‐tumour activity of NK cells in vivo. Herein, we report a new chemiluminescent probe to image in situ the granzyme B‐mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B‐specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence. The probe shows high selectivity for active granzyme B over other proteases and higher signal‐to‐noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect NK cell activity in mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.

Kees Hummelen: a creative, inspirational and unorthodox scientist from Groningen

“…14 Today, Doron Shabat in Tel Aviv is the leading scientist in using chemiluminescence in a biomedical setting. 15…”

Near-Infrared Fluorescent Probes for the Detection of Cancer-Associated Proteases

“… 70 − 73 Recently, our group developed the first chemiluminescence probe for in vivo imaging of GzmB activity released by NK cells utilizing the IEPD tetrapeptide sequence coupled to a dioxetane chemiluminescence reporter. 74 When incubated with GzmB, probe 1 underwent a 139-fold increase in chemiluminescence and was able to specifically image GzmB activity in vivo in NK cell treated tumors in mice ( Figure 3 A). Previously, Kwong et al had developed a series of GzmB reactive nanosensors for assessing acute transplant rejection in mice.…”

“…(Right mouse) Control animal without probe 1 . Adapted with permission from ref ( 74 ). Copyright Wiley-VCH 2021.…”

Development of near-infrared chemiluminescent probes for in vivo imaging

“…In addition, the CL probe 1 equipped with a granzyme-B-responsive group is first reported by Vendrell group. [60] The granzyme-B is secreted by activated T lymphocytes and NK cells for immune therapy (Figure 2-10c). This probe can detect NK cell activity in mice tumors after activation by granzyme B enzyme.…”

“…[19,158,159] After cleavage of the caged group by biomarkers, the intermediate phenolate-dioxetane derivative undergoes spontaneous decomposition and releases photons owing to the mechanism of chemically initiated electron exchange luminescence (CIEEL). Despite the ability to detect various biomarkers including enzymes, [40,60,[125][126][127] small chemical molecules and reactive oxygen species (ROS), [52,79,83,128,129,160] adamantylidene-1,2-dioxetane probes often suffer from short emission wavelengths, low aqueous chemiluminescence quantum yields (QYs), and short half-lives. Recently, Shabat et.…”