Near‐Infrared Bioorthogonally Activatable Fluorescence Probe for In Vivo Imaging of Immune Checkpoint in Cancer

Abstract Real‐time in vivo imaging of immune checkpoints is important for the guidance and prognosis of cancer immunotherapy. Although activatable optical probes have the advantages of high specificity and sensitivity as compared with “always‐on” probes, it is nearly impossible to adopt the reactivity‐based design approach for the detection of checkpoint proteins because they generally lack enzymatic activity. Herein, bioorthogonal‐reaction‐enabled fluorescence turn‐on detection of immune checkpoint in cancer is reported. This approach involves a bioorthogonally activatable near‐infrared fluorescence probe (BAP) and a transcyclooctene (TCO) tagged immune checkpoint antibody (αPDL1 ^TCO ). BAP is a hemicyanine fluorophore whose hydroxyl group is caged by tetrazine. Upon reaction with the TCOs of αPDL1 ^TCO , the tetrazine moiety of BAP is cleaved to release the uncaged hemicyanine with a fluorescence turn‐on response. BAP not only allows to specifically detect and track the fluctuation of PDL1 expression level in a murine colon cancer model during therapy but also shows a higher signal‐to‐background ratio than the “always‐on” fluorophore conjugated‐antibody, and a higher detection sensitivity than flow cytometric analysis of biopsied tumor tissues. It is expected that the design strategy of this bioorthogonally activatable probe can be applied for specific detection of other disease‐related protein biomarkers without enzymatic reactivity.