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Abstract

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses1. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated2. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA3,4, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.

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  • 07 June 2016

    The competing financial interests statement did not display correctly online when this paper was first published; this has been corrected and the statement is now available.

References

  1. 1.

    et al. Antigen localisation regulates immune responses in a dose- and time-dependent fashion: a geographical view of immune reactivity. Immunol. Rev. 156, 199–209 (1997).

  2. 2.

    & Type I interferons in host defense. Immunity 25, 373–381 (2006).

  3. 3.

    , , & Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J. Exp. Med. 184, 465–472 (1996).

  4. 4.

    , & mRNA-based therapeutics—developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014).

  5. 5.

    & Dendritic cells and the control of immunity. Nature 392, 245–252 (1998).

  6. 6.

    , , & Dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting. Nat. Rev. Immunol. 7, 790–802 (2007).

  7. 7.

    Towards targeted delivery systems: ligand conjugation strategies for mRNA nanoparticle tumor vaccines. J. Immunol. Res. 680620 (2015).

  8. 8.

    , & Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat. Rev. Drug Discov. 13, 655–672 (2014).

  9. 9.

    et al. A cationic nanoemulsion for the delivery of next generation RNA vaccines. Mol. Ther. 22, 2118–2129 (2014).

  10. 10.

    et al. Type I IFN counteracts the induction of antigen-specific immune responses by lipid-based delivery of mRNA vaccines. Mol. Ther. 21, 251–259 (2013).

  11. 11.

    et al. RNA melanoma vaccine: induction of antitumor immunity by human glycoprotein 100 mRNA immunization. Hum. Gene Ther. 10, 2719–2724 (1999).

  12. 12.

    , , , & Vaccination with mRNAs encoding tumor-associated antigens and granulocyte-macrophage colony-stimulating factor efficiently primes CTL responses, but is insufficient to overcome tolerance to a model tumor/self antigen. Cancer Immunol. Immunother. 55, 672–683 (2006).

  13. 13.

    et al. Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA. Nanomedicine 7, 445–453 (2011).

  14. 14.

    , & Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941–951 (2015).

  15. 15.

    et al. Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung. Hum. Gene Ther. 7, 1701–1717 (1996).

  16. 16.

    et al. Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery. Nat. Biotechnol. 15, 167–173 (1997).

  17. 17.

    et al. Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation. Gene Ther. 18, 702–708 (2011).

  18. 18.

    , , & Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182, 389–400 (1995).

  19. 19.

    et al. Mature dendritic cells use endocytic receptors to capture and present antigens. Proc. Natl Acad. Sci. USA 107, 4287–4292 (2010).

  20. 20.

    , , , & & Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529–1531 (2004).

  21. 21.

    et al. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520, 692–696 (2015).

  22. 22.

    et al. Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon. Nat. Immunol. 4, 1009–1015 (2003).

  23. 23.

    et al. Interferon-α suppresses cAMP to disarm human regulatory T cells. Cancer Res. 73, 5647–5656 (2013).

  24. 24.

    , , , & Type I interferons in anticancer immunity. Nat. Rev. Immunol. 15, 405–414 (2015).

  25. 25.

    The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol. Today 13, 11–16 (1992).

  26. 26.

    & Innate immune recognition. Annu. Rev. Immunol. 20, 197–216 (2002).

  27. 27.

    , , , & Plasmacytoid dendritic cell ablation impacts early interferon responses and antiviral NK and CD8+ T cell accrual. Immunity 33, 955–966 (2010).

  28. 28.

    , , , & Type I interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia. Blood 108, 3253–3261 (2006).

  29. 29.

    et al. Functional role of type I and type II interferons in antiviral defense. Science 264, 1918–1921 (1994).

  30. 30.

    et al. In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 17, 211–220 (2002).

  31. 31.

    et al. The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product. Proc. Natl Acad. Sci. USA 93, 9730–9735 (1996).

  32. 32.

    et al. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res. 21–26 (1996).

  33. 33.

    , , & Combination of apigenin treatment with therapeutic HPV DNA vaccination generates enhanced therapeutic antitumor effects. J. Biomed. Sci. 16, 49 (2009).

  34. 34.

    et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108, 4009–4017 (2006).

  35. 35.

    et al. Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals. J. Immunol. 180, 309–318 (2008).

  36. 36.

    et al. Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Gene Ther. 17, 961–971 (2010).

  37. 37.

    et al. Enhanced antigen-specific antitumor immunity with altered peptide ligands that stabilize the MHC-peptide-TCR complex. Immunity 13, 529–538 (2000).

  38. 38.

    et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N. Engl. J. Med. 361, 1838–1847 (2009).

  39. 39.

    et al. Simultaneous ex vivo quantification of antigen-specific CD4+ and CD8+ T cell responses using in vitro transcribed RNA. Cancer Immunol. Immunother. 56, 1577–1587 (2007).

  40. 40.

    , & in Methods in Membrane Biology (ed. ) 1–68 (Springer US, 1974).

  41. 41.

    in Methods in Enzymology (Academic Press Inc, 2009).

  42. 42.

    & Single bilayer liposomes prepared without sonication. Biochim. Biophys. Acta 298, 1015–1019 (1973).

  43. 43.

    , & Complexation of siRNA and pDNA with cationic liposomes: the important aspects in lipoplex preparation. Methods Mol. Biol. 605, 461–472 (2010).

  44. 44.

    & Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages. J. Exp. Med. 170, 1635–1648 (1989).

  45. 45.

    , , & Selective inhibition by rottlerin of macropinocytosis in monocyte-derived dendritic cells. Immunology 116, 513–524 (2005).

  46. 46.

    & Defining in vivo dendritic cell functions using CD11c-DTR transgenic mice. Methods Mol. Biol. 595, 429–442 (2010).

  47. 47.

    et al. Macrophages of the splenic marginal zone are essential for trapping of blood-borne particulate antigen but dispensable for induction of specific T cell responses. J. Immunol. 171, 1148–1155 (2003).

  48. 48.

    Accurate identification of experimental pulmonary metastases. J. Natl. Cancer Inst. 36, 641–645 (1966).

  49. 49.

    et al. A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. Proc. Natl Acad. Sci. USA 94, 1914–1918 (1997).

  50. 50.

    et al. The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J. Exp. Med. 178, 489–495 (1993).

  51. 51.

    et al. Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. J. Exp. Med. 179, 921–930 (1994).

  52. 52.

    et al. Functional TCR retrieval from single antigen-specific human T cells reveals multiple novel epitopes. Cancer Immunol. Res. 2, 1230–1244 (2014).

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Acknowledgements

The authors thank M. Holzmann, R. Roth, U. Schmitt, M. Brkic, A. König, C. Worm, N. Krimmel, A.-K. Thiel, C. Bender, M. Suchan, A.-L. Popa, P. Bezerra Gomes, S. Herbert, M. Lux, D. Wintergerst, V. Bischoff, R. Krishna, Y. Hajime, J. Groß, A. Spruss, M. Erdeljan, S. Wöll, T. Bukur, H. Muramatsu and M. Baiersdörfer for technical support, NIH Tetramer Core Facility for providing gp70 MHC class I tetramer, A. Kong for critical reading, A. Kemmer-Brück, D. Schwarck, S. Bolte for clinical operations support and K. Kariko for advice. This work was supported by the technology innovation program of the Rhineland Palatinate government, the InnoTop program, the CI3 Cutting Edge Cluster Funding of the German Ministry of Technology (BMBF) and the Collaborative Research Group 1066 of Deutsche Forschungsgemeinschaft (DFG). L.M.K. was funded by the Graduate School of Immunotherapy 1043 of DFG.

Author information

Author notes

    • Lena M. Kranz
    •  & Mustafa Diken

    These authors contributed equally to this work.

    • Özlem Türeci
    •  & Ugur Sahin

    These authors jointly supervised this work.

Affiliations

  1. TRON–Translational Oncology at the University Medical Center of the Johannes Gutenberg University gGmbH, Freiligrathstr. 12, Mainz 55131, Germany

    • Lena M. Kranz
    • , Mustafa Diken
    • , Sebastian Kreiter
    • , Fulvia Vascotto
    • , Abderraouf Selmi
    • , Richard Rae
    • , Sebastian Attig
    • , Christoph Huber
    •  & Ugur Sahin
  2. Research Center for Immunotherapy (FZI), University Medical Center at the Johannes Gutenberg University, Langenbeckstr. 1, Mainz 55131, Germany

    • Lena M. Kranz
    • , Christian Grunwitz
    • , Mathias Vormehr
    • , Abderraouf Selmi
    • , Sebastian Attig
    •  & Ugur Sahin
  3. Biopharmaceutical New Technologies (BioNTech) Corporation, An der Goldgrube 12, Mainz 55131, Germany

    • Mustafa Diken
    • , Heinrich Haas
    • , Sebastian Kreiter
    • , Kerstin C. Reuter
    • , Martin Meng
    • , Daniel Fritz
    • , Hossam Hefesha
    • , Christian Grunwitz
    • , Mathias Vormehr
    • , Yves Hüsemann
    • , Andreas N. Kuhn
    • , Janina Buck
    • , Evelyna Derhovanessian
    • , Jan Diekmann
    • , Robert A. Jabulowsky
    • , Sandra Heesch
    • , Christoph Huber
    •  & Ugur Sahin
  4. Department of Dermatology, University Medical Center of the Johannes Gutenberg University, Langenbeckstr. 1, Mainz 55131, Germany

    • Carmen Loquai
    •  & Stephan Grabbe
  5. Department of Dermatology, Heidelberg University Hospital, Im Neuenheimer Feld 440, 69120 Heidelberg, Germany

    • Jessica Hassel
  6. Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Germany, Langenbeckstr. 1, Mainz 55131, Germany

    • Peter Langguth
  7. Cluster for Individualized Immune Intervention, Kupferbergterasse 19, Mainz 55116, Germany

    • Özlem Türeci

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Contributions

U.S. was responsible for conception and experimental strategy of the study. Formulation development was performed by H.H. and P.L. Design and analysis of the experiments were done by L.M.K, M.D., H.H., S.K, M.M. and D.F. supported by K.C.R. and F.V. L.M.K, M.D., M.M., D.F., K.C.R., A.S., F.V., Y.H., Ho.He, C.G. and M.V. performed the experiments and acquired the data. C.L., J. H., J.D., A.N.K., J.B., R.J., S.H., S.G., E.D., R.R. and S.A. were involved in design, implementation or laboratory analyses of the clinical study. L.M.K., M.D., Ö.T. and U.S. interpreted the data and drafted the manuscript. C.H. supported the revision of the manuscript.

Competing interests

U.S., H.H, M.M., D.F., K.C.R, C.G., Ho.He, Y.H., M.V., A.N.K., J.B., E.D., J.D., R.J. and S.H. are employees at BioNTech AG (Mainz, Germany). M.D. and S.K. are working as consultants for BioNTech AG (Mainz, Germany). U.S., L.M.K., M.D., H.H., S.K., D.F., M.M. and K.C.R. are inventors on patents and patent applications related to this study. U.S. and C.H are stock owners, C.H. is advisor and supervisory board member and U.S. is management board member of BioNTech AG. All other authors have no potential conflict of interest.

Corresponding author

Correspondence to Ugur Sahin.

Reviewer Information: Nature thanks O. Farokhzad, C. G. Figdor, C. Melief, L. Zhang and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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https://doi.org/10.1038/nature18300

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