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Inhalable extracellular vesicle delivery of IL-12 mRNA to treat lung cancer and promote systemic immunity

Nature Nanotechnology volume 19, pages 565–575 (2024)Cite this article

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Abstract

Lung carcinoma is one of the most common cancers and has one of the lowest survival rates in the world. Cytokines such as interleukin-12 (IL-12) have demonstrated considerable potential as robust tumour suppressors. However, their applications are limited due to off-target toxicity. Here we report on a strategy involving the inhalation of IL-12 messenger RNA, encapsulated within extracellular vesicles. Inhalation and preferential uptake by cancer cells results in targeted delivery and fewer systemic side effects. The IL-12 messenger RNA generates interferon-γ production in both innate and adaptive immune-cell populations. This activation consequently incites an intense activation state in the tumour microenvironment and augments its immunogenicity. The increased immune response results in the expansion of tumour cytotoxic immune effector cells, the formation of immune memory, improved antigen presentation and tumour-specific T cell priming. The strategy is demonstrated against primary neoplastic lesions and provides profound protection against subsequent tumour rechallenge. This shows the potential for locally delivered cytokine-based immunotherapies to address orthotopic and metastatic lung tumours.

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Fig. 1: In vitro characterization of IL-12-Exo and in vivo distribution in LL/2 tumour-bearing mice.
Fig. 2: Inhaled IL-12-Exo safely stimulates local IL-12 expression in the lung TME.
Fig. 3: Local delivery of IL-12-Exo retards tumour growth in orthotopic lung tumours.
Fig. 4: IFNγ is essential for IL-12-Exo-mediated antitumour effects.
Fig. 5: CD8+ T cells are crucial for IL-12-Exo-mediated antitumour effects.
Fig. 6: The cDC1 cell, functioning as an antigen-presenting cell, plays key roles in the therapy of IL-12-Exo.

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Data availability

All data supporting the conclusions of this study are presented in the article and the Supplementary Information. The Cancer Genome Atlas database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga) and Kaplan–Meier plot database (http://kmplot.com) are used for the results of Supplementary Fig. 1.

References

  1. Wolchok, J. D. et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N. Engl. J. Med. 377, 1345–1356 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Ito, A., Kondo, S., Tada, K. & Kitano, S. Clinical development of immune checkpoint inhibitors. BioMed. Res. Int. 2015, 605478 (2015).

    PubMed  PubMed Central  Google Scholar 

  3. Anderson, K. G., Stromnes, I. M. & Greenberg, P. D. Obstacles posed by the tumor microenvironment to T cell activity: a case for synergistic therapies. Cancer Cell 31, 311–325 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Shi, Y. et al. Next-generation immunotherapies to improve anticancer immunity. Front. Pharmacol. 11, 566401 (2020).

    CAS  PubMed  Google Scholar 

  5. Mirlekar, B. & Pylayeva-Gupta, Y. IL-12 family cytokines in cancer and immunotherapy. Cancers (Basel). 13, 167 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Del Vecchio, M. et al. Interleukin-12: biological properties and clinical application. Clin. Cancer Res. 13, 4677–4685 (2007).

    PubMed  Google Scholar 

  7. Trinchieri, G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat. Rev. Immunol. 3, 133–146 (2003).

    CAS  PubMed  Google Scholar 

  8. Nguyen, K. G. et al. Localized interleukin-12 for cancer immunotherapy. Front Immunol. 11, 575597 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Moynihan, K. D. et al. Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses. Nat. Med. 22, 1402–1410 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Mace, T. A. et al. IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer. Gut 67, 320–332 (2018).

    CAS  PubMed  Google Scholar 

  11. Agarwal, Y. et al. Intratumourally injected alum-tethered cytokines elicit potent and safer local and systemic anticancer immunity. Nat. Biomed. Eng. 6, 129–143 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Jorgovanovic, D., Song, M., Wang, L. & Zhang, Y. Roles of IFN-γ in tumor progression and regression: a review. Biomark. Res. 8, 49 (2020).

    PubMed  PubMed Central  Google Scholar 

  13. Hotz, C. et al. Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across multiple preclinical tumor models. Sci. Transl. Med. 13, eabc7804 (2021).

    CAS  PubMed  Google Scholar 

  14. Li, Y. et al. Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumors and prime systemic immunity. Nat. Cancer 1, 882–893 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Liu, J. Q. et al. Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy. J. Control. Release 345, 306–313 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Liu, M. A. A comparison of plasmid DNA and mRNA as vaccine technologies. Vaccines 7, 37 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Sangro, B. et al. Phase I trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors. J. Clin. Oncol. 22, 1389–1397 (2004).

    CAS  PubMed  Google Scholar 

  18. Qiu, N. et al. Tumor-associated macrophage and tumor-cell dually transfecting polyplexes for efficient interleukin-12 cancer gene therapy. Adv. Mater. 33, e2006189 (2021).

    PubMed  Google Scholar 

  19. Hewitt, S. L. et al. Intratumoral IL12 mRNA therapy promotes TH1 transformation of the tumor microenvironment. Clin. Cancer Res. 26, 6284–6298 (2020).

    CAS  PubMed  Google Scholar 

  20. Aslan, C. et al. Exosomes for mRNA delivery: a novel biotherapeutic strategy with hurdles and hope. BMC Biotechnol. 21, 20 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Popowski, K. D. et al. Inhalable dry powder mRNA vaccines based on extracellular vesicles. Matter 5, 2960–2974 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. O’Brien, K., Breyne, K., Ughetto, S., Laurent, L. C. & Breakefield, X. O. RNA delivery by extracellular vesicles in mammalian cells and its applications. Nat. Rev. Mol. Cell Biol. 21, 585–606 (2020).

    PubMed  PubMed Central  Google Scholar 

  23. Zickler, A. M. & El Andaloussi, S. Functional extracellular vesicles aplenty. Nat. Biomed. Eng. 4, 9–11 (2020).

    PubMed  Google Scholar 

  24. Cheng, K. & Kalluri, R. Guidelines for clinical translation and commercialization of extracellular vesicles and exosomes based therapeutics. Extracell. Vesicle 2, 100029 (2023).

    Google Scholar 

  25. Dinh, P. C. et al. Inhalation of lung spheroid cell secretome and exosomes promotes lung repair in pulmonary fibrosis. Nat. Commun. 11, 1064 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Wang, Z. et al. Exosomes decorated with a recombinant SARS-CoV-2 receptor-binding domain as an inhalable COVID-19 vaccine. Nat. Biomed. Eng. 6, 791–805 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Li, Z. et al. Cell-mimicking nanodecoys neutralize SARS-CoV-2 and mitigate lung injury in a non-human primate model of COVID-19. Nat. Nanotechnol. 16, 942–951 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Douguet, L. et al. A small-molecule P2RX7 activator promotes anti-tumor immune responses and sensitizes lung tumor to immunotherapy. Nat. Commun. 12, 653 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Casanova-Acebes, M. et al. Tissue-resident macrophages provide a pro-tumorigenic niche to early NSCLC cells. Nature 595, 578–584 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhu, X. et al. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. J. Extracell. Vesicles 6, 1324730 (2017).

    PubMed  PubMed Central  Google Scholar 

  31. Mizrak, A. et al. Genetically engineered microvesicles carrying suicide mRNA/protein inhibit schwannoma tumor growth. Mol. Ther. 21, 101–108 (2013).

    CAS  PubMed  Google Scholar 

  32. Kojima, R. et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat. Commun. 9, 1305 (2018).

    PubMed  PubMed Central  Google Scholar 

  33. Usman, W. M. et al. Efficient RNA drug delivery using red blood cell extracellular vesicles. Nat. Commun. 9, 2359 (2018).

    PubMed  PubMed Central  Google Scholar 

  34. Lieschke, G. J., Rao, P. K., Gately, M. K. & Mulligan, R. C. Bioactive murine and human interleukin-12 fusion proteins which retain antitumor activity in vivo. Nat. Biotechnol. 15, 35–40 (1997).

    CAS  PubMed  Google Scholar 

  35. Tsai, S. J. et al. Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity. J. Biol. Chem. 297, 101266 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Li, B. et al. Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing. Nat. Biotechnol. 41, 1410–1415 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Gao, S., Wang, L., Liu, W., Wu, Y. & Yuan, Z. The synergistic effect of homocysteine and lipopolysaccharide on the differentiation and conversion of raw264.7 macrophages. J. Inflamm. 11, 13 (2014).

    Google Scholar 

  38. Mei, X. et al. An inhaled bioadhesive hydrogel to shield non-human primates from SARS-CoV-2 infection. Nat. Mater. 22, 903–912 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Olivo Pimentel, V. et al. Releasing the brakes of tumor immunity with anti-PD-L1 and pushing its accelerator with L19-IL2 cures poorly immunogenic tumors when combined with radiotherapy. J. Immunother. Cancer 9, e001764 (2021).

    PubMed  PubMed Central  Google Scholar 

  40. Leonard, J. P. et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood 90, 2541–2548 (1997).

    CAS  PubMed  Google Scholar 

  41. Chiocca, E. A. et al. Regulatable interleukin-12 gene therapy in patients with recurrent high-grade glioma: results of a phase 1 trial. Sci. Transl. Med. 11, eaaw5680 (2019).

    PubMed  PubMed Central  Google Scholar 

  42. Liu, Y. et al. Armored inducible expression of IL-12 enhances antitumor activity of glypican-3-targeted chimeric antigen receptor-engineered T cells in hepatocellular carcinoma. J. Immunol. 203, 198–207 (2019).

    CAS  PubMed  Google Scholar 

  43. Zhu, M. L., Nagavalli, A. & Su, M. A. Aire deficiency promotes TRP-1-specific immune rejection of melanoma. Cancer Res. 73, 2104–2116 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Lizotte, P. H. et al. In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer. Nat. Nanotechnol. 11, 295–303 (2015).

    PubMed  PubMed Central  Google Scholar 

  45. Gollob, J. A. et al. Phase I trial of twice-weekly intravenous interleukin 12 in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain IFN-gamma induction is associated with clinical response. Clin. Cancer Res. 6, 1678–1692 (2000).

    CAS  PubMed  Google Scholar 

  46. Smyth, M. J., Taniguchi, M. & Street, S. E. The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent. J. Immunol. 165, 2665–2670 (2000).

    CAS  PubMed  Google Scholar 

  47. Xue, D. et al. A tumor-specific pro-IL-12 activates preexisting cytotoxic T cells to control established tumors. Sci. Immunol. 7, eabi6899 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Karin, N. Chemokines in the landscape of cancer immunotherapy: how they and their receptors can be used to turn cold tumors into hot ones? Cancers 13, 6317 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Jones, D. S. 2nd et al. Cell surface-tethered IL-12 repolarizes the tumor immune microenvironment to enhance the efficacy of adoptive T cell therapy. Sci. Adv. 8, eabi8075 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Rubinstein, M. P. et al. Ex vivo interleukin-12-priming during CD8(+) T cell activation dramatically improves adoptive T cell transfer antitumor efficacy in a lymphodepleted host. J. Am. Coll. Surg. 214, 700–707 (2012).

    PubMed  PubMed Central  Google Scholar 

  51. Müller, J. M. et al. In vivo induction of interferon gamma expression in grey horses with metastatic melanoma resulting from direct injection of plasmid DNA coding for equine interleukin 12. Schweiz Arch. Tierheilkd. 153, 509–513 (2011).

    PubMed  Google Scholar 

  52. Goldszmid, R. S. et al. NK cell-derived interferon-γ orchestrates cellular dynamics and the differentiation of monocytes into dendritic cells at the site of infection. Immunity 36, 1047–1059 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Kerkar, S. P. et al. Collapse of the tumor stroma is triggered by IL-12 induction of Fas. Mol. Ther. 21, 1369–1377 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Geall, A. J. et al. Nonviral delivery of self-amplifying RNA vaccines. Proc. Natl Acad. Sci. USA 109, 14604–14609 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work is supported by grants from the National Institutes of Health (NIH) of the United States (nos. HL123920, HL137093, HL144002, HL146153, HL147357 and HL149940 to K.C.). This research was funded in part through the NIH/NCI Cancer Center Support grant no. P30CA013696. We extend our gratitude to A. Beg from the Moffitt Cancer Center, Tampa, FL, United States, for the invaluable gift of the LL/2-OVA cell line.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Columbia University, New York, NY, USA

    Mengrui Liu, Shiqi Hu, Na Yan & Ke Cheng

  2. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA

    Mengrui Liu, Shiqi Hu, Na Yan & Ke Cheng

  3. Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill/Raleigh, NC, USA

    Kristen D. Popowski & Ke Cheng

  4. Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC, USA

    Kristen D. Popowski & Ke Cheng

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Contributions

M.L. and K.C. conceived and designed the research. M.L. and S.H. established animal models. M.L., S.H., N.Y. and K.D.P. performed the experiments assessments. M.L. and N.Y. analysed the data. M.L. and K.C. wrote the article. All authors read and approved the final article. All authors provided the corresponding author with written permission to be named in the article.

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Correspondence to Ke Cheng.

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K.C. is a cofounder and equity holder of Xsome Biotech Inc. Xsome provided no funding to this research. The remaining authors declare no competing interests.

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Nature Nanotechnology thanks Minhyung Lee, Raghu Kalluri and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Table 1, Figs. 1–18, IL-12 gene sequence and information.

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Liu, M., Hu, S., Yan, N. et al. Inhalable extracellular vesicle delivery of IL-12 mRNA to treat lung cancer and promote systemic immunity. Nat. Nanotechnol. 19, 565–575 (2024). https://doi.org/10.1038/s41565-023-01580-3

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