Abstract A41: Screening of proteins related to the inhibitory receptor lymphocyte activation gene-3 (LAG-3) through BioID method
Authors: Ribeiro Priscila Rafaela/P. R. , Bonamino Martin , Chycaibam Leonardo , Pretti Marco 
Published: May 2019

Introduction: Inhibitory receptors, such as PD-1, LAG-3, TIM-3, and CTLA-4, have gained attention as potential targets for immunotherapy, once the manipulation of the negative signals mediated by these receptors may provide new therapeutic approaches for both infectious diseases, transplantation, autoimmune diseases such as for cancer. More recently, CD-4 likelymphocyte activation gene-3 (LAG-3) was described as a cell surface molecule that interacts with high affinity through its cytoplasmic domain with MHC class II molecules. Recently, in addition to these inhibitory receptors, to identify which molecules interact with these has been the new goal of this area. Once identified, such molecules can also become possible new targets to be achieved. In 2012, in order to identify interactions between proteins dependent proximity shape and biotinylation, Roux et al. (2012) developed a method called BioID, wich is based on the fusion of a protein of interest linked to a mutated biotinaligase (R118G) from Escherichia coli, which is called BirA (Choi-Rhee et al., 2004; Cronan, 2005; Roux et al., 2012.) This enzyme is able to biotinylate proteins associated with particular target protein. Once biotinylated, the proteins may be recovered by affinity (Kd = 10-14) through beads conjugated to streptavidin and subsequently identified by mass spectrometry.

Objective: To conduct a screening of proteins that interact with LAG-3 by BioID method and identify the possible signaling pathways (in silico analysis) with which these proteins are involved, validate the presence of the same by Western blot and/or flow cytometry, and relate data obtained by increasing or decreasing the immune response by T cells.

Methodology: To simulate the activation of LAG-3, we will build chimeric receptors with extracellular domain scFv anti-CD20 (chimeric receptor antigen - CAR) containing intracellular domain of Lag-3 wild type, Lag-3 Kmut (mutation K =&gt; Non KIEELE ), Lag3 EPdel (EP domain deleted), and Lag-3 Kmut EPdel (double mutant), all fused to the BirA domain. Subsequently we induce expression of these CARs cells in HEK293FT and CD4 + T lymphocyte Jurkat 20BBz +. Then we perform the identification and quantification of proteins associated to the inhibitory receptor Lag-3 by mass spectrometry, as well as perform in silico analysis of possible downstream signaling pathways in which Lag3 is involved.

Preliminary results: To date, all vectors have been cloned and to test the system, only a few attempts, only CAR antiCD20/Lag3 Wild Type-BirA was electroporated in the HEK293T cell line, presenting 75% and 90% of expression.

Western blotting was performed to verify biotinylation (incubation with Pierce™ High Sensitivity Streptavidin-HRP) and the presence of CAR (incubation with anti-Fab antibody), confirming the recruitment of molecules by the Lag-3 receptor.

Perspectives: Electroporate the remaining CARs in both HEK293T and Jurkat, double-check the presence of CAR and biotinylation pattern in all conditions, as well as proceed to an identification and quantification of proteins by mass spectrometry and validate the presence of proteins identified by flow cytometry and/or Western blot.

Citation Format: Priscila Rafaela/P. R. Ribeiro, Martin Bonamino, Leonardo Chycaibam, Marco Pretti. Screening of proteins related to the inhibitory receptor lymphocyte activation gene-3 (LAG-3) through BioID method [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A41.