Background
Despite decades of research, LAG3 remains an elusive target of great interest from an immunotherapy perspective. Similar to the PD-1/PD-L1 checkpoint, the LAG3/MHCII immune checkpoint is an inhibitory regulator of the immune system, promoting immune self-tolerance. However, both pathways can be commandeered by cancer cells to evade immune surveillance, hence the interest in immune checkpoint blockade therapies. One of the questions still surrounding LAG3 and a barrier to its development as a therapy, is a complete understanding of the function of its interaction with its ligands. While these studies are lagging behind, LAG3 has also been investigated as a target in combination with existing PD-1/PD-L1 treatment. ADAM10 is a metalloprotease responsible for the cleavage of LAG3 into its soluble form. Studies have found that responders to PD-1 immunotherapy had a lower ratio of LAG3:ADAM10, suggesting that cleavage of LAG3 is important for the anti-tumor response. To investigate, a proximity ligation assay was designed using Navinci’s sensitive and specific technology to visualise the LAG3/MHCII interaction, with additional staining of a T-cell marker, CD3, and a regulator of LAG3, ADAM10.
Materials and Methods
The LAG3/MHCII protein-protein interaction assay was generated using the in situ NaveniFlex Tissue Atto647N kit. Formalin-fixed, paraffin-embedded tissue (FFPE) sections and tissue microarrays (TMAs) were stained with optimized concentrations of antibodies specific to LAG3 and MHCII. Secondary antibodies conjugated to oligonucleotides, known as Navenibodies, were added to create an amplified signal that only occurs if the target proteins are within an interaction range of <40nm. The proximity signal was detected using a fluorescent readout. Immunofluorescent (IF) staining of CD3 and ADAM10 were added in parallel to the detection step of the proximity ligation assay.
Results
LAG3/MHCII interactions were assessed using the Naveni® proximity ligation technology in melanoma and hepatocellular carcinoma tissues and TMAs. The interaction is observed in both tissue types, with interactions occurring between lymphocytes and a healthy or tumor cell. Costaining with CD3 enables the determination of which cell type LAG3 is interacting with, and thus whether the checkpoint is being manipulated in a pro-cancer manner. Across varying levels of disease in TMAs, it is possible to visualize changes in the ADAM10 IF staining, indicating cleavage of LAG3 from the cell surface.
Conclusions
A complete picture of the mechanisms of action of LAG3 is crucial for advancing research of related immunotherapies. Possibly one of the major holdbacks is the potential for unknown impacts of LAG3 checkpoint immunotherapy based on this lack of knowledge. This study demonstrated the possibility of visualising the LAG3/MHCII interaction in situ in tissue samples, along with CD3 and ADAM10. The assay enables the assessment of LAG3 interactions and the levels of associated proteins, providing greater insight into LAG3 mechanisms. Clinically, this can be used as a prognostic biomarker assay, allowing improved patient stratification for immunotherapy recipients.
S. Bodbin: A. Employment (full or part-time); Significant; Navinci Diagnostics. J. Vennberg: A. Employment (full or part-time); Significant; Navinci Diagnostics. D. Raykova: A. Employment (full or part-time); Significant; Navinci Diagnostics. A. Zieba-Wicher: A. Employment (full or part-time); Significant; Navinci Diagnostics.