Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers
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Katrijn Broos1,*, Marleen Keyaerts2,3,*, Quentin Lecocq1, Dries Renmans1, Tham Nguyen2,4, David Escors6, Adrian Liston7,8, Geert Raes4,5,*, Karine Breckpot1,* and Nick Devoogdt2,*
1Laboratory of Molecular and Cellular Therapy (LMCT), Vrije Universiteit Brussel, B-1090 Brussels, Belgium
2In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel, B-1090 Brussels, Belgium
3Nuclear Medicine Department, UZ Brussel, B-1090 Brussels, Belgium
4Unit of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel, B-1050 Brussels, Belgium
5Myeloid Cell Immunology Laboratory, VIB Inflammation Research Center, 9052 Ghent, Belgium
6Immunomodulation Group, Navarrabiomed-Biomedical Research Centre, 31008 Pamplona, Navarra, Spain
7Department of Microbiology and Immunology, University of Leuven (KU Leuven), 3000 Leuven, Belgium
8VIB Center for Brain and Disease Research, 3000 Leuven, Belgium
*These authors contributed equally to this work
Nick Devoogdt, email: email@example.com
Keywords: immune checkpoints, programmed death-1/programmed death-ligand 1, biomarker, nanobodies, SPECT/CT imaging
Received: January 21, 2017 Accepted: March 19, 2017 Published: March 30, 2017
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 (99mTc) 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.
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