An mRNA vaccine elicits STING-dependent antitumor immune responses.

Acta Pharmaceutica Sinica B 2023;13(3):1274e1286 Chinese Pharmaceutical Association Institute of Materia Medica, Chinese Academy of Medical Sciences Acta Pharmaceutica Sinica B w w w. e l s ev i e r. c o m / l o c a t e / a p s b w w w. s c i e n c e d i r e c t . c o m ORIGINAL ARTICLE An mRNA vaccine elicits STING-dependent antitumor immune responses Zhe Chena,b, Chaoyang Menga,c, Junhua Maia, Yongbin Liua, Hangwen Lid, Haifa Shena,e,*,# a Department of Nanomedicine, Houston Methodist Academic Institute, Houston, TX 77030, USA Xiangya Hospital of Central South University, Changsha 410000, China c Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China d Stemirna Therapeutics, Shanghai 201206, China e ImmunoQ Therapeutics, Houston, TX 77401, USA b Received 4 July 2022; received in revised form 13 September 2022; accepted 28 September 2022 KEY WORDS Cancer; Immunotherapy; mRNA; Vaccine; Dendritic cells; EDOPC; STING; MAVS Abstract Lipid-formulated RNA vaccines have been widely used for disease prevention and treatment, yet their mechanism of action and individual components contributing to such actions remain to be delineated. Here, we show that a therapeutic cancer vaccine composed of a protamine/mRNA core and a lipid shell is highly potent in promoting cytotoxic CD8þ T cell responses and mediating anti-tumor immunity. Mechanistically, both the mRNA core and lipid shell are needed to fully stimulate the expression of type I interferons and inflammatory cytokines in dendritic cells. Stimulation of interferon-b expression is exclusively dependent on STING, and antitumor activity from the mRNA vaccine is significantly compromised in mice with a defective Sting gene. Thus, the mRNA vaccine elicits STING-dependent antitumor immunity. ª 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). *Corresponding author. E-mail address: (Haifa Shen). # Current address: Stemirna Therapeutics, Shanghai 201206, China. Peer review under the responsibility of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. https://doi.org/10.1016/j.apsb.2022.11.013 2211-3835 ª 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). mRNA vaccine provokes immunogenic cancer cell killing via STING 1. Introduction Rapid development and worldwide application of mRNA vaccines for the prevention of SARS-CoV-2 infection have demonstrated the power of mRNA-based drugs in healthcare1,2. Due to their large molecular weight and negative charge, mRNA molecules need to be packaged into delivery vehicles in order to effectively enter mammalian cells3. Packaging into the nanometer-size delivery vehicles also has the benefit of protecting mRNA molecules from enzymatic degradation. Multiple delivery platforms have been developed to suit the purpose, such as lipid nanoparticle4, lipopolyplex (LPP)5, liposome-protamine-RNA (LPR)6, RNAlipoplex (RNA-LPX)7, and virus-like vaccine particle (VLVP)8. While each platform has its own unique structure and composition, most vehicles contain an ionic lipid molecule that facilitates mRNA packaging and the escape of mRNA molecules from the endosomes. With the success of the prophylactic vaccines, there is a general realization that mRNA therapeutics can be used to treat perhaps most, if not all, disease types9e12. Indeed, mRNA-based therapeutic cancer vaccines have been studied for many years13,14. Recent advances in clinical trials have also demonstrated their application potential in selected cancer patients15e17. Unlike peptide cancer vaccines that are prepared with adjuvant molecules18e20, mRNA vaccine particles can also serve as self-adjuvants21. For example, a two-component mRNA-based cancer vaccine containing free and protaminecomplexed mRNA can also activate the toll-like receptor 7 (TLR7) signaling22. However, with the increasing concern on acute innate immune toxicity from naked mRNA, most investigators and companies are using modified RNA to avoid innate recognition by the TLRs23. Consequently, the lipid components are playing an important role in enhancing adjuvant activity in the mRNAvaccine particle, preferentially by activating non-TLR signaling. A recent study on the lipid-formulated, negatively charged RNA-LPX constituted with 1,2di-octadecenyl-3-trimethylammonium (DOTMA, a cationic lipid)/ dioleoylphosphatidylethanolamine (DOPE, a helper lipid) liposome revealed activation of the interleukin 1 (IL1)-interleukin 1 receptor antagonist (IL-1ra) axis in regulating secretion of proinflammatory cytokines, and the essential role of activating the two-step inflammasome pathway in monocytes24. Interestingly, another recent investigation on the LNP-based BNT162b2 prepared with ALC-0315 (an ionized lipid), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, a helper lipid), polyethylene glycol-2000-N,N-ditetradecylacetamide (PEG2000-DTA), and cholesterol showed the key role of activating type I interferon-dependent MDA5 signaling, but not TLRs or inflammasome, in stimulating both innate and adaptive immunity of the COVID-19 vaccine25. These studies point to the possibility that delivery platforms comprised of variable lipid molecules may rely on different signal transduction pathways for vaccine activity. Thus, it is important to fully investigate the function of key molecules and their combinations in order to further improve mRNA therapeutics. In the current study, we set up experiments to dissect the functional role of individual components in a therapeutic cancer vaccine. The mRNA vaccine particle (MVP) is composed of a protamine/ mRNA core that is encapsulated in a lipid shell consisting of a cationic lipid, a helper lipid, a pegylated lipid, and cholesterol (Fig. 1A). It has been demonstrated that inclusion of charged lipid can facilitate targeted RNA delivery26, and dioleoylethylphosphatidylcholine (EDOPC) and dioleoyl-3trimethylammonium propane (DOTAP) are two of the cationic 1275 lipids that have been tested for this purpose5,27. We examined stimulation of expression of interferon-b (IFN-b), IL-1b, and tumor necrosis factor-a (TNF-a) by the mRNA core, mRNA-free vehicle, and the whole MVP, and correlated such activities to the TLR7, mitochondrial antiviral signaling (MAVS, also known as IPS-1), stimulator of IFN genes (STING), and TIR-domain-containing adapterinducing IFN-b (TRIF) signaling. Subsequently, we investigated the role of protamine in the core and cationic lipid in the shell in stimulating IFN-b an (...truncated)