O-10.03 Immunosensitizing effect of TLR agonist combination for the treatment of cold tumors

<h3>Background</h3> Some emerging cancer therapies aim to harness the immune system to improve tumor infiltration by immune cells and reprogram the tumor microenvironment. Converting ‘cold’ tumors into ‘hot’ tumors has become a major focus, with Toll-like receptor (TLR) agonists being explored as promising candidates. These molecules, already used in clinics (e.g., MPLA for cervical cancer, BCG for bladder cancer, and Imiquimod for superficial skin carcinomas), activate immune pathways to enhance anti-tumor immunity. However, their efficacy is limited in advanced cancers. To address this, we combined a TLR4 agonist (OM-174) and a TLR7 agonist (Resiquimod), which activate distinct signaling pathways (TRIF and MyD88 respectively), hypothesizing that their combination could elicit a more potent and synergistic anti-tumor response. <h3>Materials and Methods</h3> <i>In vivo</i>: CT26 colon carcinoma cells were subcutaneously injected into BALB/c mice. Tumor-bearing mice were treated twice a week with intravenous injections of OM-174, Resiquimod alone or in combination. Tumor growth and mouse survival were monitored for 60 days to assess treatment efficacy. Tumors were harvested 24h and 48h post-treatment for infiltrating immune cell analyses (flow cytometry, ELISA, RT-qPCR, and cytokine arrays). <i>In vitro</i>: RAW264.7 macrophages, BMDMs, BMDCs and CT26 cells were treated with OM-174, Resiquimod, or their combination. Signaling pathways, gene expression and cytokine production were analyzed by RT-qPCR, Western blot and ELISA. <h3>Results</h3> OM-174 and Resiquimod individually showed limited anti-tumor activity, but their combination resulted in significant tumor growth inhibition and even complete tumor cures with an immune memory. Mechanistic studies revealed an enhanced recruitment of neutrophils into tumors, particularly after OM-174 treatment, likely driven by the induction of CXCL1 and CXCL2 production, as shown by RT-qPCR and cytokine arrays. The combination treatment uniquely triggered robust type I interferon responses contrary to individual treatments with each agonist. Increased levels of IFN-β (ELISA) and a strong ISG signature (RT-qPCR), including increased expression of Cxcl10 (validated by ELISA) and Isg15, were observed. Neutrophils activated by type I interferons are known to exhibit anti-tumoral activity, supporting their critical role in the observed effects. Moreover, we observed that the combination treatment synergistically induces IFN-β production in macrophages and dendritic cells, which are described to be the main producers of type I interferons in the tumor microenvironment. <h3>Conclusions</h3> Our findings demonstrate that combining OM-174 (TLR4 ligand) and Resiquimod (TLR7 ligand) induces a potent and synergistic immune response, characterized by neutrophil recruitment and a robust type I interferon response ultimately leading to tumor regression. This study underscores the therapeutic potential of combining TLR agonists to overcome the limitations of single-agent therapies and offers a promising avenue for novel cancer immunotherapies, especially for cold tumors. T. Morin: None. I. Benoit-Lizon: None. C. Racoeur: None. M. Cambillard: None. E. Jacquin: None. C. Paul: None.