Study of PD-1/LAG-3 signaling in T cells and development of therapeutic strategies to counteract PD-1/LAG-3 mediated resistance to cancer immunotherapy

A significant number of cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. However, how LAG-3 mechanistically triggers its downstream inhibitory signalling remains largely unknown. Likewise, how LAG-3 cooperates with PD-1 to stablish a highly dysfunctional state in T cells in cancer is also unknown. A better understanding of LAG-3 and PD-1/LAG-3 signaling will uncover the reasons behind intrinsic resistance to PD-1 blockade, develop novel treatments and improve current therapies. This PhD thesis comprises the study of PD-1/LAG-3 signaling in T-cells and the development of therapeutic strategies to counteract PD-1/LAG-3-mediated resistance to cancer immunotherapy. The results of this thesis have been organized into three chapters. In chapter 1, PD-1/LAG-3 molecular co-signaling mechanisms were identified in T-cells, uncovering CBLB ubiquitin ligases as potential targets. The combination of CBL-B inhibitors with anti-PD- 1/anti-LAG-3 immunotherapies overcame PD-1/LAG-3-mediated resistance in models of lung cancer refractory to immunotherapies. In chapter 2, the consequences of PD-1/LAG-3 cosignaling over the proteome and phosphoproteome associated to the TCR signalosome were characterized. MYC was identified as an inhibited upstream regulator of PD-1/LAG-3 proteomes and phosphoproteomes in T-cells. In chapter 3, T-cell lines expressing LAG-3 mutants in its signaling domains were engineered and analyzed by quantitative highthroughput differential proteomics and phosphoproteomics. The pathways and molecules regulated by each of LAG-3 domains and their effect on the TCR signalosome both in terms of protein expression and phosphorylation activity were characterized. These results highlighted the critical role of KIEELE and EP domains to endow LAG-3 with inhibitory functions. Overall, the results will help to identify the mechanisms of intrinsic resistance to PD-1 blockade mediated by LAG-3 co-signaling, and uncover novel therapeutic targets to counteract PD- 1/LAG-3-mediated resistance.