Discovery of novel anti-cancer microbiome and immune checkpoint protein for improving immunotherapy efficacy

Abstract

The rapid advancements in cancer immunotherapy, particularly immune checkpoint inhibitors (ICIs), have significantly transformed cancer treatment. However, their efficacy remains limited to a subset of patients due to immune evasion mechanisms and resistance. This dissertation explores two novel approaches to overcoming these challenges. First, we investigate the potential of Melanoma Cell Adhesion Molecule (MCAM) as a new immune checkpoint protein (ICP) involved in immune evasion. Through transcriptomic analysis, we identified elevated MCAM expression in non-responders to PD-1 blockade therapy. Using purified MCAM protein and MCAM- knockdown cells, produced via genome-editing based on CRISPR-Cas9, we performed T cell activity assays and employed various preclinical mouse models to determine that MCAM plays a multifaceted role in inhibiting T cell activity: (1) MCAM directly suppresses T cell proliferation and cytotoxicity by delivering inhibitory signals to T cells, leading to immune suppression within the tumor microenvironment; (2) it limits T cell infiltration into tumors, creating an immunosuppressive barrier that hinders effective immune surveillance and attack; and (3) MCAM acts independently of the PD-1/PD-L1 axis, impairing the efficacy of anti-PD-1 therapy even in patients with high PD-L1 expression, while showing synergistic effects when combined with anti-PD-1 treatment. These results suggest that MCAM is a promising target for improving ICI efficacy. Second, we investigate the role of gut microbiota in enhancing anti-tumor immunity through Lactococcus lactis subsp. lactis GEN001 (L. lac_G01), a strain isolated from healthy Korean individuals. Through multi-omics analyses and preclinical models, we identified multiple mechanisms by which L. lac_G01 exerts its anti-cancer effects: (1) L. lac_G01 treatment led to an increase in both CD8+ T cells and NK cells, but through blockade experiments, we confirmed that the anti-tumor effects were primarily mediated by CD8+ T cells, indicating that CD8+ T cells play a key role in L. lac_G01's mechanism; (2) the immune activation driven by L. lac_G01 is mediated by IL-7 and IL-15, leading to a stronger immune response against tumors; (3) metabolomics analysis identified 1-monopalmitin, an effective metabolite produced by L. lac_G01, which promotes T cell proliferation and differentiation into effector cells, further contributing to the anti-tumor effect; and (4) L. lac_G01 inhibits Monoacylglycerol lipase (MAGL), leading to reduced lipid metabolism in cancer cells, thereby limiting tumor growth. These results demonstrate the therapeutic potential of L. lac_G01 and its metabolites in modulating immune responses and metabolic pathways to enhance anti-tumor efficacy and reshape the tumor microenvironment. Together, these findings offer new strategies to overcome the limitations of current immunotherapies, providing a dual approach targeting both immune checkpoints and the microbiome to enhance cancer treatment outcomes.