Novel formulation of c-di-GMP with cytidinyl/cationic lipid reverses T cell exhaustion and activates stronger anti-tumor immunity

Rationale: Cyclic dinucleotides (cDNs) are a promising class of immunotherapeutic agent targeting stimulator of interferon genes (STING). However, enzymatic instability and transmembrane barriers limit the extensive clinical application of cDNs. Thus, a novel delivery system, composed of a neutral cytidinyl lipid DNCA and a cationic lipid CLD (Mix) that interacts with cDNs via H-bonding, pi-stacking and electrostatic interaction, is developed and optimized to overcome the above issues. Methods: The optimal composition of Mix for cDNs encapsulation was explored with RAW-Lucia ISG cells. The physicochemical properties of resulted nanoparticles were characterized. To validate the anti-tumor immunity of cDNs/Mix both in vitro and in vivo , immunogenic cell death (ICD) related markers and tumor inhibition efficacy were evaluated in cancer cells and tumor models, respectively. The mechanism by which cdG/Mix exerted the antitumor effects was explored by flow cytometric analysis and in vivo depletion. Results: Based on our developed and optimized delivery system, neutral cytidinyl lipid DNCA/cationic lipid CLD (Mix), cdG (500 nM in vitro , 1-10 μg in vivo )/Mix not only more potently stimulated production of IFNβ and related cytokines including CXCL9 and CXCL10, promoted ICD, led to NK and CD8 + T cell activation, inhibited tumor growth in both EO771 and B16F10 models and increased their survival rate (~43%), but also obviously reversed the T cell exhaustion (Tex) in tumor, meanwhile down regulated the mRNA expression of Tox and Nr4a , which are key regulators of Tex. Conclusion: cdG/Mix triggered ICD in various cancer cells and reversed the Tex systemically in tumor-burden mice, which would be a promising alternative strategy for cancer immunotherapy.