Inhibiting the Interaction Between Cancer Immunotherapy Targets FGL-1 and LAG-3 Proteins Utilizing Small Interfering Peptides

Abstract

Immunotherapies are a method of cancer treatment that uses a patient’s own immune system to fight cancer cells. While immunotherapies are beneficial against numerous cancer subtypes, they are not effective within every single patient. Our study aims to inhibit the interactions between fibrinogen-like protein 1 (FGL-1), a ligand of lymphocyte-activation gene 3 (LAG-3) found on T-cells, in order to prevent inappropriate downregulation of T-cell activity. Inhibiting protein-protein interactions is typically very challenging with existing methods. Utilizing pulldowns and silver staining, we attempted to evaluate the ability of two small peptides to disrupt the protein-protein interactions between FGL-1 and LAG-3. These peptides were designed to be utilized in tandem in a multivalent inhibitor design which was validated through files from the Protein Data Bank visualized with the ChimeraX software platform. Our pulldown assay was designed to evaluate how LAG-3 and FGL-1 interact by using beads that only interact with LAG-3 and leave FGL-1 untouched. As a result, the presence of FGL-1 indicates interactions with LAG-3. We designed and validated the assay using silver stain imaging which stained FGL-1 and LAG-3 for visualization, thereby confirming the reported interaction. An additional peptide was then added to block the interaction between LAG-3 and FGL-1. This would theoretically result in absence of FGL-1 in the pulldown—however, this was not the case in our first trial. We aim to provide more accurate insights into the FGL-1/LAG-3 interaction, which is important for developing new immunotherapies. With the development of different immunotherapies, we should be able to further research and by refining our tagging methods, we enhance the reliability of our findings, contributing to the broader understanding of protein-protein interactions in immune pathways.