Theranostics 2022; 12(15):6723-6739. doi:10.7150/thno.71010 This issue

Research Paper

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

Xiaotong Yu1*, Jing Yu1*, Hong Dai1, Chenyun Deng1, Xudong Sun1, Sijie Long1, Zhujun Jiang1, Hongyan Jin2, Zhu Guan1, Zhenjun Yang1✉

1. State Key Laboratory of Natural & Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No.38 Xueyuan Road, Haidian District, Beijing 100191, China.
2. Department of Obstetrics and Gynecology, Peking University First Hospital, No. 8 Xishiku Street, 100034, Beijing, China.
*Authors contributed equally to this work.

This is an open access article distributed under the terms of the Creative Commons Attribution License ( See for full terms and conditions.
Yu X, Yu J, Dai H, Deng C, Sun X, Long S, Jiang Z, Jin H, Guan Z, Yang Z. Novel formulation of c-di-GMP with cytidinyl/cationic lipid reverses T cell exhaustion and activates stronger anti-tumor immunity. Theranostics 2022; 12(15):6723-6739. doi:10.7150/thno.71010. Available from

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Graphic abstract

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.

Keywords: c-di-GMP, DNCA, CLD, T cell exhaustion, STING, immunotherapy