Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers

Blockade of the inhibitory PD-1/PD-L1 immune checkpoint axis is a promising cancer treatment. Nonetheless, a significant number of patients and malignancies do not respond to this therapy. To develop a screen for response to PD-1/PD-L1 inhibition, it is critical to develop a non-invasive tool to accurately assess dynamic immune checkpoint expression. Here we evaluated non-invasive SPECT/CT imaging of PD-L1 expression, in murine tumor models with varying PD-L1 expression, using high affinity PD-L1-specific nanobodies (Nbs). We generated and characterized 37 Nbs recognizing mouse PD-L1. Among those, four Nbs C3, C7, E2 and E4 were selected and evaluated for preclinical imaging of PD-L1 in syngeneic mice. We performed SPECT/CT imaging in wild type versus PD-L1 knock-out mice, using Technetium-99m ((99m)Tc) labeled Nbs. Nb C3 and E2 showed specific antigen binding and beneficial biodistribution. Through the use of CRISPR/Cas9 PD-L1 knock-out TC-1 lung epithelial cell lines, we demonstrate that SPECT/CT imaging using Nb C3 and E2 identifies PD-L1 expressing tumors, but not PD-L1 non-expressing tumors, thereby confirming the diagnostic potential of the selected Nbs. In conclusion, these data show that Nbs C3 and E2 can be used to non-invasively image PD-L1 levels in the tumor, with the strength of the signal correlating with PD-L1 levels. These findings warrant further research into the use of Nbs as a tool to image inhibitory signals in the tumor environment.