Abstract
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.
Highlights
Cancer cells express neo-antigens generated due to mutations and aberrant processing of proteins as well as cancer-germline antigens generated due to epigenetic alterations
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
Flow cytometry was performed to confirm binding to the mouse and human PD-L1 antigen when expressed on lentivirally modified B16 and HEK293T cells respectively (Supplementary Table 1)
Summary
Cancer cells express neo-antigens generated due to mutations and aberrant processing of proteins as well as cancer-germline antigens generated due to epigenetic alterations. Cancer cells co-opt specific inhibitory signaling pathways, known as immune checkpoints to evade their CD8+ T cell-mediated destruction [2] Inhibition of these inhibitory immune checkpoints has received increasing interest as a disruptive treatment for patients with solid and hematological tumors [3,4,5,6,7,8,9,10,11,12,13,14,15,16]. Several inhibitory immune checkpoints, consisting of inhibitory receptors expressed on T cells and their ligands expressed on antigen-presenting cells have been www.impactjournals.com/oncotarget described Of these the immune checkpoint consisting of programmed death-1 (PD-1, CD279) and its ligand programmed death-ligand 1 (PD-L1, B7-H1, CD274), and its blockade with monoclonal antibodies (mAbs) have received much attention [7, 11, 15,16,17,18,19]. FDA approved mAbs targeting PD-1 include Nivolumab (Bristol-Myers Squibb) and Pembrolizumab (Merck), while Atezolizumab (Roche) is the first PD-L1 targeting mAb to be FDA approved [20]
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