Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation

Abstract

Therapeutic messenger RNA vaccines enable delivery of whole antigens, which can be advantageous over peptide vaccines. However, optimal efficacy requires both intracellular delivery, to allow antigen translation, and appropriate immune activation. Here, we developed a combinatorial library of ionizable lipid-like materials to identify mRNA delivery vehicles that facilitate mRNA delivery in vivo and provide potent and specific immune activation. Using a three-dimensional multi-component reaction system, we synthesized and evaluated the vaccine potential of over 1,000 lipid formulations. The top candidate formulations induced a robust immune response, and were able to inhibit tumor growth and prolong survival in melanoma and human papillomavirus E7 in vivo tumor models. The top-performing lipids share a common structure: an unsaturated lipid tail, a dihydroimidazole linker and cyclic amine head groups. These formulations induce antigen-presenting cell maturation via the intracellular stimulator of interferon genes (STING) pathway, rather than through Toll-like receptors, and result in limited systemic cytokine expression and enhanced anti-tumor efficacy.

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Fig. 1: Isocyanide-mediated 3-CR for high-throughput synthesis of lipidoids.
Fig. 2: In vivo and in vitro screening of lipidoids for Fluc mRNA (mLuc) delivery.
Fig. 3: Top-performing lipidoids exhibit different anti-tumor immunity.
Fig. 4: Heterocyclic amine-containing lipidoids act as mRNA vaccines with robust antigen-specific T-cell and humoral responses.
Fig. 5: Cyclic lipidoids facilitate the maturation of APCs in local lymph nodes through STING-dependent activation of type I IFN.
Fig. 6: The effect of A18 mRNA antigen vaccines on tumor growth.

Data availability

The data supporting the findings of this study are available form the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by Translate Bio (Lexington, MA) and the Juvenile Diabetes Research Foundation (grant nos. 17-2007-1063 and 3-PDF-2015-91-A-N). This work is supported in part by the Cancer Center Support (core) (grant no. P30-CA14051) from the National Institutes of Health. D.G.A. is a consultant for Translate Bio. L.M. was supported by a Misrock postdoctoral fellowship. D.D. was supported by an EPSRC E-TERM Fellowship (EP/I017801/1) and a Marie Sklodowska Curie Fellowship (IF/798348). K.S. was supported by the Ruth L. Kirschstein NRSA Postdoctoral Fellowship (no. 1F32EB025688-01A1) from the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health. L.L. and J.H. were supported by a Ming Wai Lau grant from the Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet. The authors would like to acknowledge the use of resources at the W. M. Keck Biological Imaging Facility (Whitehead Institute) and at the Microscopy, Histology, Animal Imaging & Preclinical Testing and Flow Cytometry Core Facilities (Swanson Biotechnology Center, David H. Koch Institute for Integrative Cancer Research at MIT), and acknowledge Wuxi Further Pharmaceutical Co., Ltd for synthesizing and providing lipid materials.

Author information

L.M. and D.G.A. designed experiments and analyzed data. L.L. designed the chemical structures. L.M., Y.H., D.D., Y.S., J.C., K.S., W.G. and J.H. performed experiments. J.L. and L.M. drew the schematic figures. L.M., D.D., J.C.D., Y.H. and D.G.A. wrote the manuscript. R.L. and D.G.A. supervised the study. All authors discussed the results and assisted in the preparation of the manuscript.

Correspondence to Daniel G. Anderson.

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Competing interests

L.M., Y.H., L.L. and D.G.A. have filed a patent for the development of the described lipids synthesized using the three-component reaction. R.L. receives licensing fees (to patents in which he was an inventor on) from, invested in, consults (or was on scientific advisory boards or boards of directors) for, lectured (and received a fee), or conducts sponsored research at MIT for which he was not paid for the following entities: 7th Sense, Abpro, Aleph Farms, Alkermes, Allevi, Alnylam, Artificial Cells, Arsenal Medical, BASF, Celero, Cellomics, Cellular Biomedical, Clarus, Clontech, Combined Therapeutics, Conference Forum, Curis, Domain, Eagle, Echo, Edge, Evox, Fate Therapeutics, Frequency Therapeutics, Genscript, Glycobia, Glympse, Grandhope, Greenlight, HKF Technologies, Horizon Discovery, Humacyte, Indivor, Inovio, Institute of Immunology, In Vivo Therapeutics, Ironwood Pharmaceuticals, Kallyope, Kensa, Keratinx, KSQ Therapeutics, Laderatech, Inc., Landsdowne Labs, Like Minds, Luminopia, Luye, Lyndra, Lyra, Medical Kinetics, Merck, Micelle, Moderna, Momenta, Monsanto, Mylan, Nanobiosym, Nanobiotix, Noveome, Particles for Humanity, Perosphere, Pfizer, Polaris, Portal, Pulmatrix, Puretech, Roche, Rubius, Secant, Selecta Biosciences, Setsuro, Shiseido, Sigilon, Sio2, SQZ, Stembiosys, Suono Bio, T2 Biosystems, Tara, Taris Biomedical, Tarveda, Third Rock, Tiba, Tissium, Titan Pharma, Unilever, VasoRX, Verseau Therapeutics, Vivtex, Wiki Foods and Zenomic.

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Supplementary Information

Supplementary Figs. 1–24, Tables 1, 2 and 4, and Notes 1–4.

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Supplementary Table 3

Characterization of the top-performing lipids.

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Miao, L., Li, L., Huang, Y. et al. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation. Nat Biotechnol 37, 1174–1185 (2019). https://doi.org/10.1038/s41587-019-0247-3

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