Noninvasive Imaging of the Immune Checkpoint LAG-3 Using Nanobodies, from Development to Pre-Clinical Use

Abstract

Immune checkpoint inhibition (ICI) is a promising cancer therapy, which has progressed rapidly from a preclinical concept to clinical implementation. Commonly considered targets in ICI are CTLA-4, PD-1/PD-L1, and LAG-3, and the list grows. As ICI is generally only beneficial for a subset of patients, there is a need to select patients that are eligible for therapy as well as to monitor therapy response. There is growing interest to do this noninvasively, by molecular imaging with target-specific tracers. To this day, noninvasive imaging has focused on CTLA-4 and PD-1/PD-L1, while there is no noninvasive tool available to accurately assess LAG-3 expression in vivo. In this proof-of-concept study, we developed nanobodies, the smallest functional fragments from camelid heavy chain-only antibodies, to noninvasively evaluate mouse LAG-3 expression using single photon emission computed tomography (SPECT)/CT imaging. The in vitro characterization of 114 nanobodies led to the selection of nine nanobodies binding to mouse LAG-3. The injection of 99mTechnetium-labeled nanobodies in healthy mice showed specific uptake in immune peripheral organs like the spleen and lymph nodes, which was not observed in LAG-3 gene knock-out mice. Moreover, nanobody uptake could be visualized using SPECT/CT and correlated to the presence of LAG-3 as assessed in flow cytometry and immunohistochemistry. SPECT/CT scans of tumor bearing mice further confirmed the diagnostic potential of the nanobodies. These findings substantiate the approach to use nanobodies as a tool to image inhibitory immune checkpoints in the tumor environment.

Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

“…However, there is an increasing demand to improve some properties of conventional antibodies that are currently being used. Nanobodies have arisen as a substitute to conventional antibodies and show great potential when used as tools in the diagnostic field [45][46][47][48]. One of the main advantages of nanobodies is that several tags can be fused in their tertiary structure by recombinant technology [9][10][11]13].…”

Nanobodies as non-invasive imaging tools

“…Anti-LAG-3 nanobodies have been developed and used for non-invasive imaging. 68 The 99m Tclabeled nanobodies showed specific uptake in secondary lymphoid organs, and no signal was detected in LAG-3 knockout mice. LAG-3 expression was confirmed by flow cytometry and immunohistochemistry analyses, and correlated well with the SPECT images.…”

“…38,103e105 An anti-human HER2 nanobody was characterized in vivo in xenograft mouse models implanted with HER2 þ and HER2-human cancer cells. Using different labeling strategies, the anti-human HER2 nanobody labeled with 18 F, 131 I and 68 Ga detected HER2 þ tumors with excellent specificity. 38e40 These studies helped set the stage for clinical translation of anti-HER2 nanobodies.…”

“…38e40 These studies helped set the stage for clinical translation of anti-HER2 nanobodies. A phase I clinical study used the 68 Ga-labeled anti-HER2 nanobody to image 20 women with primary or metastatic breast carcinoma, with a HER2 immunostaining assessment of 2 þ or 3 þ ( Figure 4A,B). 37 PET/CT images were obtained 10, 60 and 90 min after administration, and tracer accumulation in metastatic lesions was well above background.…”

“…Imaging methods that can provide a whole-body scan, such as PET and SPECT, begin to establish a non-invasive approach to the characterization of a tumor in terms of its local environment and cellular composition. PET uses isotopes that emit a positron such as 11 C (t 1/2 w20 min), 68 Ga (t 1/2 w68 min), 18 F (t 1/2 w110 min), 64 Cu (t 1/2 w12.7 h) or 89 Zr (t 1/2 w3.3 days). SPECT uses isotopes that emit gamma rays, such as 99m Tc (t 1/2 w6 h) or 111 In (t 1/2 w67.3 h).…”