Noninvasive Imaging of the Immune Checkpoint LAG-3 Using Nanobodies, from Development to Pre-Clinical Use
Quentin Lecocq1, Katty Zeven2, Yannick De Vlaeminck3
1Laboratory for Molecular and Cellular Therapy (LMCT), Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. quentin.lecocq@vub.be.
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.
Insights
Researchers developed novel nanobodies for noninvasive imaging of Lymphocyte-activation gene 3 (LAG-3) expression. This breakthrough allows for potential patient selection and monitoring of immune checkpoint inhibitor therapies using SPECT/CT imaging.
Area of Science:
- Immunology
- Molecular Imaging
- Biotechnology
Background:
- Immune checkpoint inhibition (ICI) offers promising cancer therapy, targeting molecules like CTLA-4, PD-1/PD-L1, and LAG-3.
- Patient selection and therapy monitoring for ICI are crucial due to variable patient responses.
- Noninvasive molecular imaging is desired for assessing target expression, but tools for LAG-3 are lacking.
Purpose of the Study:
- To develop nanobodies for noninvasive in vivo imaging of mouse Lymphocyte-activation gene 3 (LAG-3) expression.
- To evaluate the diagnostic potential of these nanobodies using SPECT/CT imaging.
Main Methods:
- Developed and characterized 114 nanobodies against mouse LAG-3.
- Selected nine high-affinity nanobodies for further study.
- Labeled nanobodies with 99mTechnetium and performed SPECT/CT imaging in healthy and LAG-3 knockout mice, as well as tumor-bearing mice.
Main Results:
- Identified nine nanobodies that bind specifically to mouse LAG-3.
- Demonstrated specific uptake of radiolabeled nanobodies in immune organs (spleen, lymph nodes) in healthy mice, absent in knockout mice.
- Confirmed correlation between nanobody uptake visualized by SPECT/CT and LAG-3 presence via flow cytometry and immunohistochemistry.
- Showcased diagnostic potential in tumor-bearing mice.
Conclusions:
- Successfully developed nanobodies for noninvasive SPECT/CT imaging of LAG-3 expression.
- These nanobodies represent a novel tool for assessing LAG-3 in vivo.
- This approach can aid in patient selection and monitoring for immune checkpoint inhibitor therapies targeting LAG-3.
