Effect of a systemically administered innate immune modulator on anti-tumor activity against metastatic osteosarcoma.

e23504 Background: Osteosarcoma (OSa) is the most common malignant bone tumor with limited treatment options and poor outcomes in advanced metastatic cases. In recent decades, significant progress in OSa treatment has been limited, highlighting the urgent need for novel therapeutic approaches. Current immunotherapies, like immune checkpoint inhibitors, have shown insufficient clinical efficacy but recently, systemic activation of innate immune system with Toll-like receptor 4 (TLR4) immunostimulants has shown great promise. Unfortunately, all current TLR4 agonists are restricted to local administration due to toxicity issues limiting their capacity to address metastases and disseminated tumors. Methods: In this study, we explored the antitumor effects of an innovative chemically detoxified TLR4 agonist formulated in liposomes (HEPHA-440) in two syngeneic mouse and rat models of metastatic OSa (LM8 and OsA). HEPHA-440 was shown to have an optimized safety and solubility profile for systemic administration. We evaluated the impact of HEPHA-440 on tumor growth, lung metastases, and immune cell infiltration in wild-type and TLR4-mutant mice and performed selective immunodepletions and transcriptomic analysis in tumors. Results: HEPHA-440 showed potent antitumor effects against both localized OSa tumors, associated with increased tumor necrosis and promotion of CD8+ T cell and M1 macrophage infiltration. HEPHA-440 also significantly reduced the incidence of pulmonary metastases with up to 40% of complete regression. This was associated with a reshape of the tumor microenvironments with infiltration of CD8 + T cells and M1 macrophages in primary tumors, and CD8 + T cells infiltration with a shift of macrophages towards an M1 phenotype in lung metastases. In vitro studies confirmed that HEPHA-440 can reverse the polarization of M2 macrophages toward an M1 phenotype. The antitumor effects of HEPHA-440 were dependent on TLR4 and CD8+ T cells, and treatment increased the expression of immune checkpoint proteins (PD1, PD-L1, LAG-3 and OX40L) in OSa tumors. Furthermore, analysis of a publicly available dataset for patients with OSa revealed that higher infiltration of CD8+ T cells and M1 macrophages was correlated with better overall survival and progression-free survival. Conclusions: These findings demonstrated for the first time that a detoxified TLR4 immunostimulant can address metastatic osteosarcoma, thanks to systemic administration and capacity to reprogram tumor microenvironments through the recruitment of CD8 T-cells and M1 macrophages, and the repolarization of M2 tumor associated macrophages toward an M1 phenotype. Our data also suggest that HEPHA-440 could make OSa tumors finally responsive to Immune Checkpoint Inhibitor (ICI) treatments. This warrants further development toward clinical evaluation in cancer patients.