Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy

Journal name:
Nature
Volume:
534,
Pages:
396–401
Date published:
DOI:
doi:10.1038/nature18300
Received
Accepted
Published online
Corrected online

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.

At a glance

Figures

  1. RNA-LPX of negative net charge deliver RNA-encoded antigens body-wide to lymphoid-resident DCs.
    Figure 1: RNA-LPX of negative net charge deliver RNA-encoded antigens body-wide to lymphoid-resident DCs.

    a, Particle size, polydispersity index (top) and zeta potential (bottom) (n = 3) of RNA-LPX constituted with DOTMA/DOPE liposomes and Luc-RNA at various charge ratios. b, Bioluminescence imaging of BALB/c mice (n = 3) after i.v. injection of Luc-LPX at various charge ratios. Pie charts show relative contribution of each organ to total signal. c, Particle size and polydispersity index of Luc-LPX either constituted freshly or stored at 4 °C followed by 24 h incubation at room temperature (top) and bioluminescence imaging of the spleens of BALB/c mice (n = 8, pooled from two experiments) after i.v. injection (bottom). d, Bioluminescence imaging after i.v. injection of Luc-LPX in CD11c-DTR mice (n = 3) depleted (depl.) of CD11c+ cells. e, Splenic localization of CD11c and Cy3 double-positive cells in BALB/c mice (n = 2) 1 h after i.v. injection of Cy3-labelled RNA-LPX. Scale bar, 100 μm. MZ, marginal zone; RP, red pulp; WP, white pulp. f, eGFP expression in splenic cell subsets of C57BL/6 mice (n = 3) 24 h after i.v. injection of eGFP-LPX by flow cytometry. g, Bioluminescence imaging of inguinal lymph nodes (LN), femur and tibia in BALB/c mice (n = 3) after i.v. injection of Luc-LPX. LDL, lower detection limit. h, Bioluminescence imaging of inguinal lymph nodes and ex vivo Luc assay of bone marrow (BM) single-cell suspensions after i.v injection of Luc-LPX in CD11c+ cell-depleted CD11c-DTR mice (n = 3). Significance was determined using unpaired two-tailed Student’s t-test. Error bars, median (c, bottom), otherwise mean ± s.d.

  2. RNA-LPX vaccines induce TLR7-triggered IFNα production, IFNAR-dependent activation of APCs and effector cells, and strong expansion of fully functional antigen-specific T cells.
    Figure 2: RNA-LPX vaccines induce TLR7-triggered IFNα production, IFNAR-dependent activation of APCs and effector cells, and strong expansion of fully functional antigen-specific T cells.

    a, b, Activation markers measured 24 h after i.v. injection of HA-LPX by in splenic immune cell subsets (n = 3 per time point) and kinetics of IFNα serum levels (n = 3 per time point) in wild-type (a) or Tlr7−/− (b) mice. c, IFNα serum levels in CD11c+ cell-depleted CD11c-DTR mice (n = 3) after i.v. injection of HA-LPX. d, Fraction of IFNα-expressing cells in splenic APC subsets after i.v. injection of HA-LPX in C57BL/6 and Ifnar1−/− mice (n = 3 per time point). e, Fraction of OVA-specific (left) and gp70-specific CD8+ T cells (right) within CD8+ T cells in blood after de novo priming of C57BL/6 mice (n = 5) and BALB/c mice (n = 5) immunized i.v. with OVA-LPX or gp70-LPX (day 0, 3, 8), respectively. f, Kinetics of OVA-specific CD8+ T cell frequencies within CD8+ T cells in blood after i.v. immunization of C57BL/6 mice (n = 5) with OVA-LPX. g, Fraction of OVA-specific CD8+ T cells within CD8+ T cells in blood of CD11c+ cell-depleted BM-chimaeric CD11c-DTR mice (n = 5) immunized i.v. with OVA-LPX (day 0, 3). Significance was determined using two-way ANOVA and Bonferroni’s multiple comparisons test (a, right, b, right, d), one-way ANOVA and Tukey’s multiple comparisons test (d), and unpaired two-tailed Student’s t-test (b, left, c, e, g). Error bars, mean ± s.d.

  3. RNA-LPX vaccines mediate rejection of advanced, aggressively growing tumours in mice.
    Figure 3: RNA-LPX vaccines mediate rejection of advanced, aggressively growing tumours in mice.

    a, Prophylactic efficacy in OVA-LPX immunized C57BL/6 (n = 6) challenged s.c. with B16-OVA melanoma and gp70-LPX immunized BALB/c mice (n = 5) challenged and rechallenged s.c. with CT26 colon carcinoma. b, B16F10-Luc growth (left) and tumour load in lungs (right) of B6 albino mice (n = 12) immunized i.v. with TRP-1-LPX, irrelevant (empty vector)-LPX or control (untreated). c, CT26-Luc growth and CT26 tumour load in lungs of BALB/c mice (n = 4–7) immunized i.v. with gp70-LPX. d, Survival of C57BL/6 mice (n = 10) with advanced s.c. TC-1-Luc tumours immunized i.v. with E6/E7-LPX or irrelevant (OVA)-LPX. e, Survival of BALB/c mice (n = 10) with i.v. CT26-Luc colon carcinoma tumours immunized i.v. with CT26-M90-LPX or irrelevant (OVA)-LPX. f, g, De novo priming in BALB/c mice (n = 3) immunized i.v. with gp70-LPX (day 0, 3, 8) and injected i.p. with anti-IFNAR1 antibody or isotype before each immunization. f, Fraction of gp70-specific CD8+ T cells within CD8+ T cells. g, Splenic CD8+ T cells upon in vitro restimulation with no (none), irrelevant (HA) or gp70 peptide. h, CT26 colon carcinoma load in lungs of BALB/c mice (n = 5) immunized i.v. with gp70-LPX and injected i.p. with anti-IFNAR1 antibody or isotype. Significance was determined using log-rank test (a, d, e), two-way ANOVA and Dunnett’s multiple comparisons test (b), and one-way ANOVA and Tukey’s multiple comparisons test (g, h). Error bars, median with interquartile range (b), otherwise mean ± s.d.

  4. Clinically administered RNA-LPX vaccines dose-dependently induce systemic INFα and de novo priming and amplification of T cells against vaccine antigens.
    Figure 4: Clinically administered RNA-LPX vaccines dose-dependently induce systemic INFα and de novo priming and amplification of T cells against vaccine antigens.

    a, Serum cytokines before (0 h) and after injection of intra-patiently escalated doses. b, c, T-cell responses against NY-ESO-1 and tyrosinase determined by restimulation with overlapping peptide mixtures or NY-ESO-1 epitopes (indicated with the amino acid position) in IFNγ ELISPOT and NY-ESO-1 specific MHC class I dextramer staining for patients 1 (b) and 3 (c). CEF, cytomegalovirus, Epstein–Barr and influenza viruses peptide pool; NM, not measured; PepMix, peptide mixture; pre-vac., pre-vaccination. d, Mechanism of action for RNA-LPX. Error bars, mean ± s.e.m.

  5. Physicochemical characteristics and biological activity of RNA-LPX constituted from different lipids at various charge ratios.
    Extended Data Fig. 1: Physicochemical characteristics and biological activity of RNA-LPX constituted from different lipids at various charge ratios.

    a, Bioluminescence imaging of Luc expression in BALB/c mice 6 h after i.v. injection of different transfection reagents and controls: PBS (n = 3), 60 μg Luc-RNA alone (n = 3), 25 μg Luc-RNA complexed with TransMessenger (Qiagen) (n = 3), 5 μg Luc-RNA complexed with Viromer RED (Lipocalyx) (n = 3). b, Cryo-TEM images of Luc-LPX constituted at a positive:negative ((+):(−)) charge ratio of 1.3:2 with DOTMA/DOPE liposomes. Scale bar, 100 nm. c, Fraction of uncomplexed RNA in Luc-LPX preparations constituted at different charge ratios with DOTMA/DOPE liposomes determined by capillary gel electrophoresis (n = 2–7). d, Particle size, polydispersity index (left) and zeta potential (right) (n = 3) of RNA-LPX constituted with Luc-RNA and differently constituted liposomes at various charge ratios. e, Bioluminescence imaging of BALB/c mice (n = 3) after i.v. injection of Luc-LPX constituted with different liposomes at various charge ratios corresponding to d. Pie charts show relative contribution of each organ to total signal. f, Relative biodistribution of Luc expression in explanted organs of BALB/c mice (n = 3) after i.v. injection of Luc-LPX constituted with DOTMA/DOPE liposomes at a charge ratio of (+):(-) of 1.3:2 or Luc-RNA alone. g, Luc expression in human immature DCs transfected with 5 μg Luc-LPX constituted freshly or stored after constitution for indicated time periods at 4 °C (left) or room temperature (right). RNA-LPX tested in duplicates (stored) or quadruplets (fresh). Each bar represents triplicates. h, Particle size (upper left) and percentage of RNA integrity (upper right) of Luc-LPX (n = 1) incubated in 50% mouse serum for indicated time periods at 37 °C. Bioluminescence imaging of Luc expression in BALB/c mice (n = 5) after i.v. injection of Luc-LPX preincubated in 50% mouse serum for 30 min at 37 °C (lower left and right). NM, not measured. Error bars, median with interquartile range (h), otherwise mean ± s.d.

  6. Biodistribution and cellular uptake mechanism of RNA-LPX vaccines.
    Extended Data Fig. 2: Biodistribution and cellular uptake mechanism of RNA-LPX vaccines.

    a, Uptake of Cy5-labelled RNA in splenic cell subsets of C57BL/6 mice (n = 3) 1 h after i.v. injection of 40 μg Cy5-labelled RNA-LPX. b, Localization of CD11c and Cy3 double-positive cells in the spleen of BALB/c mice (n = 2) 1 h after i.v. injection of 40 μg Cy3-labelled RNA-LPX. Nuclear staining in blue. Scale bar,100 μm. c, Half-life of RNA-LPX in circulation analysed by quantitative RT–qPCR in male and female C57BL/6 mice (n = 5 per time-point) after injection of 60 μg RNA-LPX constituted with NY-ESO-I, tyrosinase, MAGE-A3 and TPTE RNA (15 μg each). d, Localization of Cy5+ (upper left) or Thy1.1+ cells (lower left) in spleen and liver of BALB/c mice (n = 5) determined by microscopy or flow cytometry 1 h or 20 h after i.v. injection of 40 μg Cy5-labelled RNA-LPX or 40 μg 1-methyl-pseudouridine-modified Thy1.1-LPX, respectively. Nuclear staining in blue. Scale bar, 50 μm (top), 20 μm (bottom). Biodistribution of Cy5 signal in homogenized organs of BALB/c mice (n = 2) (right). Note the signal in the liver is overestimated in this analysis owing to the strong signal in the gall bladder, probably reflecting biliary secreted free dye. e, Bioluminescence imaging of lymph nodes of BALB/c mice (n = 3) 18 h after i.v. injection of 40 μg 1-methyl-pseudouridine-modified Luc-LPX. ax, axillary; ing, inguinal; mand, mandibular. f, Flow cytometry analysis of Cy5 and Thy1.1 expression in CD11c+ cells in the bone marrow of C57BL/6 mice (n = 3) 1 h or 20 h after i.v. injection of 40 μg Cy5-labelled RNA-LPX or 40 μg 1-methyl-pseudouridine-modified Thy1.1-LPX, respectively. g, h, Localization of Cy3-labelled RNA in human immature DCs after co-transfection of 1.25 μg Cy3-labelled RNA-LPX at a charge ratio of (+):(−) of 1.3:2 and 3:1 with dextran (g) or of 1.3:2 after staining for TLR7 or EEA1 (h). Nuclear staining in blue. Scale bar, 10 μm. i, Visualization and quantification of inhibited uptake of positively as well as negatively charged Cy3-labelled RNA-LPX in human immature DCs pretreated with rottlerin or cytochalasin D. Scale bar, 10 μm. j, Bioluminescence imaging of lymph nodes of BALB/c mice (n = 3) injected intranodally with 10 μM rottlerin in 10 μl PBS 15 min before i.v. injection of 80 μg Luc-LPX. k, Luminescence assay of whole blood enriched or not enriched with human immature DCs pretreated with poly I:C or not (control) before transfection with Luc-LPX at a charge ratio of 1.3:2. WB, whole blood. l, Poly-I:C-induced maturation determined by CD86 expression (left), bioluminescence imaging (middle) and eGFP expression in splenic cDC subsets (right) upon injection of BALB/c mice (n = 3) with 50 μg poly I:C i.p. 12 h before i.v. injection of 20 μg Luc-LPX or 80 μg eGFP-LPX, respectively. Significance was determined using unpaired two-tailed Student’s t-test (d, lower left, f, l, middle) and one-way ANOVA and Tukey’s multiple comparisons test (ik, l, right). Error bars, mean ± s.e.m. (k) or mean ± s.d. otherwise.

  7. Systemic TLR7- and IFNAR-dependent activation of APCs and effector cells, IFNα production and strong expansion of fully functional antigen-specific T cells induced by RNA-LPX vaccines.
    Extended Data Fig. 3: Systemic TLR7- and IFNAR-dependent activation of APCs and effector cells, IFNα production and strong expansion of fully functional antigen-specific T cells induced by RNA-LPX vaccines.

    a, Localization of splenic CD11chi cells at baseline (top) and 6 h after i.v. injection of 40 μg HA-LPX (bottom) into BALB/c mice (n = 2). Nuclear staining in blue. Scale bar, 100 μm. RP, red pulp; WP, white pulp. be, Activation marker expression in splenic cell subsets and kinetics of IFNα serum levels after i.v. injection of mice (n = 3 per time point) with HA-LPX in Tlr3−/−, Tlr4−/− and Tlr9−/− mice (b), in Ifnar1−/− mice (c, d), or in BALB/c mice treated with 100 μg anti-IFNAR1 antibody or isotype i.p. 1 h before i.v. injection of HA-LPX (e). Ab, antibody. f, mRNA levels of IFNα isoforms in sorted splenic APC subsets of C57BL/6 mice (n = 3) 1 h after i.v. injection of HA-LPX determined by qRT–PCR. Data expressed as log2-fold change, as compared to control animals. g, IFNα serum levels after i.v. injection of HA-LPX in BDCA2-DTR mice (n = 3 per time point) depleted (depl) of pDCs (left) and in C57BL/6 mice (n = 3 per time point) depleted of macrophages (right). h, CFSE proliferation profile of HA-specific CD4+ T cells in lymphoid compartments of BALB/c Thy1.1+ mice (n = 3) after adoptive transfer of HA-specific Thy1.2+ HA-TCR-transgenic CD4+ T cells and subsequent immunization with HA-LPX or control (untreated). Fraction of proliferated cells indicated. tg, transgenic. i, Priming of naive HA-specific CD8+ T cells ex vivo. BALB/c (n = 3) mice were immunized with 80 μg HA-LPX, irrelevant (eGFP)-LPX or NaCl (control). Splenocytes were prepared 12 h later and co-incubated with CFSE-labelled CL4-TCR-transgenic CD8+ T cells isolated using MACS magnetic microbeads coated with CD8 antibodies at an effector:target ratio of 1:6. Four days later, proliferation profiles were analysed by flow cytometry. Numbers indicate the percentage of proliferated cells. j, Fraction of cytokine-secreting CD8+ T cells within CD8+ T cells in the spleen upon de novo priming in C57BL/6 mice (n = 5) immunized i.v. (day 0, 3, 8) with OVA-LPX after in vitro restimulation with no (none), irrelevant VSV (irrelevant) or OVA peptide and intracellular cytokine staining (top). Spleen ex vivo ELISPOT assay upon de novo priming in BALB/c mice (n = 5) immunized i.v. (day 0, 3, 8) with gp70-LPX. Stimulation with no (none), irrelevant HA (irrelevant) or gp70 peptide (lower left). gp70-specific cytotoxicity in vivo (lower right). BALB/c mice (n = 5) were immunized i.v. (day 0, 3, 8) with 40 μg gp70-LPX. Naive splenocytes were labelled with 0.5 or 5 μM CFSE and pulsed with peptide (6 μg ml−1) five days after the last immunization, and target cells (2 × 107) were adoptively transferred into immunized recipients i.v. (irrelevant HA-loaded CFSElow:gp70-loaded CFSEhigh = 1:1). Recipient splenocytes were analysed by flow cytometry 18 h after transfer, and antigen-specific lysis was determined: specific lysis (%) = (1 − (percentage of cells pulsed with gp70/percentage of cells pulsed with HA)) × 100). k, Expression of memory markers CD127 and CD62L in gp70-specific, CD44+CD8+ T cells compared to non-specific CD8+ T cells in blood (day 19) and spleen (day 67) of BALB/c mice (n = 3) after priming with gp70-LPX (day 0, 7, 14). l, Fraction of gp70-specific CD8+ T cells within total CD8+ T cells in blood, bone marrow and lymph nodes determined by MHC class I tetramer staining after de novo priming of splenectomized BALB/c mice (n = 5–7) immunized with gp70-LPX (day 0, 7) or left untreated (control). Significance was determined using unpaired two-tailed Student’s t-test (b left, c), two-way ANOVA and Bonferroni’s multiple comparisons test (b right, g) and one-way ANOVA and Tukey’s multiple comparisons test (j, l). Error bars, mean ± s.d.

  8. Potent antitumour immunity and rejection of advanced aggressively growing tumours in mice conferred by RNA-LPX vaccines.
    Extended Data Fig. 4: Potent antitumour immunity and rejection of advanced aggressively growing tumours in mice conferred by RNA-LPX vaccines.

    a, B16-OVA melanoma load in lungs of C57BL/6 mice (n = 8) immunized i.v. (days 4, 7, 11) with OVA-LPX or irrelevant (eGFP)-LPX. b, Expression of activation markers measured 24 h after i.v. injection of 40 μg irrelevant (empty vector)-LPX, eGFP-LPX or OVA-LPX by flow cytometry in splenic immune cell subsets (n = 3) and IFNα serum levels (n = 3) 6 h after injection in C57BL/6 mice. c, Bioluminescence signal of tumours in different groups before immunization and on day 25 (upper left), tumour load and lung weights (upper right) and TRP-1-specific CD8+ and CD4+ T-cell responses in spleens of control (untreated), irrelevant (empty vector)-LPX and TRP-1-LPX-immunized B6 albino mice (n = 12) on day 25 detected by ELISPOT assay (bottom), depicted in Fig. 3b. d, Bioluminescence imaging of CT26-Luc carcinoma in BALB/c mice (n = 4–7) depicted in Fig. 3c (left). e, TC-1-Luc tumour growth in C57BL/6 mice (n = 10) (left), depicted in Fig. 3d, and remission of established advanced TC-1-Luc tumours in C57BL/6 mice (n = 10) immunized i.v. with 40 μg E6/E7-LPX (day 13, 20, 27) (right). f, Survival of BALB/c mice rechallenged with CT26-Luc colon carcinoma cells on day 109, depicted in Fig. 3e. Significance was determined using one-way ANOVA and Tukey’s multiple comparisons test (c), two-way ANOVA and Bonferroni’s multiple comparisons test (d), paired two-tailed Student’s t-test (f, right), unpaired two-tailed Student’s t-test (f, far right), and log-rank test (f, left). Error bars, median with interquartile range (d), mean ± s.d. otherwise.

  9. Clinical application of RNA-LPX vaccines and de novo priming and amplification of patient T-cell responses against encoded vaccine antigens.
    Extended Data Fig. 5: Clinical application of RNA-LPX vaccines and de novo priming and amplification of patient T-cell responses against encoded vaccine antigens.

    a, Vaccination scheme and monitoring for patients 1–3. b, Antigen-specific T-cell responses against NY-ESO-1 and tyrosinase determined by restimulation with overlapping peptide mixtures in IFNγ ELISPOT for patient 1. c, Antigen-specific T-cell responses against NY-ESO-1 and MAGE-A3, determined by post-IVS IFNγ ELISPOT assay at indicated days for patient 2. Values are corrected for background (no peptide). d, Antigen-specific T-cell responses against NY-ESO-I and MAGE-A3, determined by ex vivo IFNγ ELISPOT assay at indicated days for patient 3. Numbers in ELISPOT data indicate the amino acid position of each epitope. Significance was determined using unpaired two-tailed Student’s t-test. Error bars, mean ± s.e.m.

  10. Comparison of i.v. and s.c. routes for RNA-LPX administration in the context of T-cell priming and biodistribution of RNA-LPX upon s.c. administration.
    Extended Data Fig. 6: Comparison of i.v. and s.c. routes for RNA-LPX administration in the context of T-cell priming and biodistribution of RNA-LPX upon s.c. administration.

    Fraction of OVA-specific CD8+ T cells within CD8+ T cells on day 13 in blood after de novo priming of C57BL/6 mice (n = 5) immunized i.v. with OVA-LPX (day 0, 3, 8) (left). Biodistribution of Luc expression 24 h after s.c. injection of Luc-LPX in BALB/c mice (n = 3) (right). Signal can only be observed at the injection site and the draining lymph node. Significance was determined using one-way ANOVA and Tukey’s multiple comparisons test. Error bars, mean ± s.d.

Tables

  1. Findings of non-GLP pilot pharmacokinetics and pharmacodynamics study in cynomolgus monkeys
    Extended Data Table 1: Findings of non-GLP pilot pharmacokinetics and pharmacodynamics study in cynomolgus monkeys

Change history

Corrected online 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.

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Author information

  1. These authors contributed equally to this work.

    • Lena M. Kranz &
    • Mustafa Diken
  2. These authors jointly supervised this work.

    • Özlem Türeci &
    • Ugur Sahin

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

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 financial 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:

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.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Physicochemical characteristics and biological activity of RNA-LPX constituted from different lipids at various charge ratios. (415 KB)

    a, Bioluminescence imaging of Luc expression in BALB/c mice 6 h after i.v. injection of different transfection reagents and controls: PBS (n = 3), 60 μg Luc-RNA alone (n = 3), 25 μg Luc-RNA complexed with TransMessenger (Qiagen) (n = 3), 5 μg Luc-RNA complexed with Viromer RED (Lipocalyx) (n = 3). b, Cryo-TEM images of Luc-LPX constituted at a positive:negative ((+):(−)) charge ratio of 1.3:2 with DOTMA/DOPE liposomes. Scale bar, 100 nm. c, Fraction of uncomplexed RNA in Luc-LPX preparations constituted at different charge ratios with DOTMA/DOPE liposomes determined by capillary gel electrophoresis (n = 2–7). d, Particle size, polydispersity index (left) and zeta potential (right) (n = 3) of RNA-LPX constituted with Luc-RNA and differently constituted liposomes at various charge ratios. e, Bioluminescence imaging of BALB/c mice (n = 3) after i.v. injection of Luc-LPX constituted with different liposomes at various charge ratios corresponding to d. Pie charts show relative contribution of each organ to total signal. f, Relative biodistribution of Luc expression in explanted organs of BALB/c mice (n = 3) after i.v. injection of Luc-LPX constituted with DOTMA/DOPE liposomes at a charge ratio of (+):(-) of 1.3:2 or Luc-RNA alone. g, Luc expression in human immature DCs transfected with 5 μg Luc-LPX constituted freshly or stored after constitution for indicated time periods at 4 °C (left) or room temperature (right). RNA-LPX tested in duplicates (stored) or quadruplets (fresh). Each bar represents triplicates. h, Particle size (upper left) and percentage of RNA integrity (upper right) of Luc-LPX (n = 1) incubated in 50% mouse serum for indicated time periods at 37 °C. Bioluminescence imaging of Luc expression in BALB/c mice (n = 5) after i.v. injection of Luc-LPX preincubated in 50% mouse serum for 30 min at 37 °C (lower left and right). NM, not measured. Error bars, median with interquartile range (h), otherwise mean ± s.d.

  2. Extended Data Figure 2: Biodistribution and cellular uptake mechanism of RNA-LPX vaccines. (732 KB)

    a, Uptake of Cy5-labelled RNA in splenic cell subsets of C57BL/6 mice (n = 3) 1 h after i.v. injection of 40 μg Cy5-labelled RNA-LPX. b, Localization of CD11c and Cy3 double-positive cells in the spleen of BALB/c mice (n = 2) 1 h after i.v. injection of 40 μg Cy3-labelled RNA-LPX. Nuclear staining in blue. Scale bar,100 μm. c, Half-life of RNA-LPX in circulation analysed by quantitative RT–qPCR in male and female C57BL/6 mice (n = 5 per time-point) after injection of 60 μg RNA-LPX constituted with NY-ESO-I, tyrosinase, MAGE-A3 and TPTE RNA (15 μg each). d, Localization of Cy5+ (upper left) or Thy1.1+ cells (lower left) in spleen and liver of BALB/c mice (n = 5) determined by microscopy or flow cytometry 1 h or 20 h after i.v. injection of 40 μg Cy5-labelled RNA-LPX or 40 μg 1-methyl-pseudouridine-modified Thy1.1-LPX, respectively. Nuclear staining in blue. Scale bar, 50 μm (top), 20 μm (bottom). Biodistribution of Cy5 signal in homogenized organs of BALB/c mice (n = 2) (right). Note the signal in the liver is overestimated in this analysis owing to the strong signal in the gall bladder, probably reflecting biliary secreted free dye. e, Bioluminescence imaging of lymph nodes of BALB/c mice (n = 3) 18 h after i.v. injection of 40 μg 1-methyl-pseudouridine-modified Luc-LPX. ax, axillary; ing, inguinal; mand, mandibular. f, Flow cytometry analysis of Cy5 and Thy1.1 expression in CD11c+ cells in the bone marrow of C57BL/6 mice (n = 3) 1 h or 20 h after i.v. injection of 40 μg Cy5-labelled RNA-LPX or 40 μg 1-methyl-pseudouridine-modified Thy1.1-LPX, respectively. g, h, Localization of Cy3-labelled RNA in human immature DCs after co-transfection of 1.25 μg Cy3-labelled RNA-LPX at a charge ratio of (+):(−) of 1.3:2 and 3:1 with dextran (g) or of 1.3:2 after staining for TLR7 or EEA1 (h). Nuclear staining in blue. Scale bar, 10 μm. i, Visualization and quantification of inhibited uptake of positively as well as negatively charged Cy3-labelled RNA-LPX in human immature DCs pretreated with rottlerin or cytochalasin D. Scale bar, 10 μm. j, Bioluminescence imaging of lymph nodes of BALB/c mice (n = 3) injected intranodally with 10 μM rottlerin in 10 μl PBS 15 min before i.v. injection of 80 μg Luc-LPX. k, Luminescence assay of whole blood enriched or not enriched with human immature DCs pretreated with poly I:C or not (control) before transfection with Luc-LPX at a charge ratio of 1.3:2. WB, whole blood. l, Poly-I:C-induced maturation determined by CD86 expression (left), bioluminescence imaging (middle) and eGFP expression in splenic cDC subsets (right) upon injection of BALB/c mice (n = 3) with 50 μg poly I:C i.p. 12 h before i.v. injection of 20 μg Luc-LPX or 80 μg eGFP-LPX, respectively. Significance was determined using unpaired two-tailed Student’s t-test (d, lower left, f, l, middle) and one-way ANOVA and Tukey’s multiple comparisons test (ik, l, right). Error bars, mean ± s.e.m. (k) or mean ± s.d. otherwise.

  3. Extended Data Figure 3: Systemic TLR7- and IFNAR-dependent activation of APCs and effector cells, IFNα production and strong expansion of fully functional antigen-specific T cells induced by RNA-LPX vaccines. (444 KB)

    a, Localization of splenic CD11chi cells at baseline (top) and 6 h after i.v. injection of 40 μg HA-LPX (bottom) into BALB/c mice (n = 2). Nuclear staining in blue. Scale bar, 100 μm. RP, red pulp; WP, white pulp. be, Activation marker expression in splenic cell subsets and kinetics of IFNα serum levels after i.v. injection of mice (n = 3 per time point) with HA-LPX in Tlr3−/−, Tlr4−/− and Tlr9−/− mice (b), in Ifnar1−/− mice (c, d), or in BALB/c mice treated with 100 μg anti-IFNAR1 antibody or isotype i.p. 1 h before i.v. injection of HA-LPX (e). Ab, antibody. f, mRNA levels of IFNα isoforms in sorted splenic APC subsets of C57BL/6 mice (n = 3) 1 h after i.v. injection of HA-LPX determined by qRT–PCR. Data expressed as log2-fold change, as compared to control animals. g, IFNα serum levels after i.v. injection of HA-LPX in BDCA2-DTR mice (n = 3 per time point) depleted (depl) of pDCs (left) and in C57BL/6 mice (n = 3 per time point) depleted of macrophages (right). h, CFSE proliferation profile of HA-specific CD4+ T cells in lymphoid compartments of BALB/c Thy1.1+ mice (n = 3) after adoptive transfer of HA-specific Thy1.2+ HA-TCR-transgenic CD4+ T cells and subsequent immunization with HA-LPX or control (untreated). Fraction of proliferated cells indicated. tg, transgenic. i, Priming of naive HA-specific CD8+ T cells ex vivo. BALB/c (n = 3) mice were immunized with 80 μg HA-LPX, irrelevant (eGFP)-LPX or NaCl (control). Splenocytes were prepared 12 h later and co-incubated with CFSE-labelled CL4-TCR-transgenic CD8+ T cells isolated using MACS magnetic microbeads coated with CD8 antibodies at an effector:target ratio of 1:6. Four days later, proliferation profiles were analysed by flow cytometry. Numbers indicate the percentage of proliferated cells. j, Fraction of cytokine-secreting CD8+ T cells within CD8+ T cells in the spleen upon de novo priming in C57BL/6 mice (n = 5) immunized i.v. (day 0, 3, 8) with OVA-LPX after in vitro restimulation with no (none), irrelevant VSV (irrelevant) or OVA peptide and intracellular cytokine staining (top). Spleen ex vivo ELISPOT assay upon de novo priming in BALB/c mice (n = 5) immunized i.v. (day 0, 3, 8) with gp70-LPX. Stimulation with no (none), irrelevant HA (irrelevant) or gp70 peptide (lower left). gp70-specific cytotoxicity in vivo (lower right). BALB/c mice (n = 5) were immunized i.v. (day 0, 3, 8) with 40 μg gp70-LPX. Naive splenocytes were labelled with 0.5 or 5 μM CFSE and pulsed with peptide (6 μg ml−1) five days after the last immunization, and target cells (2 × 107) were adoptively transferred into immunized recipients i.v. (irrelevant HA-loaded CFSElow:gp70-loaded CFSEhigh = 1:1). Recipient splenocytes were analysed by flow cytometry 18 h after transfer, and antigen-specific lysis was determined: specific lysis (%) = (1 − (percentage of cells pulsed with gp70/percentage of cells pulsed with HA)) × 100). k, Expression of memory markers CD127 and CD62L in gp70-specific, CD44+CD8+ T cells compared to non-specific CD8+ T cells in blood (day 19) and spleen (day 67) of BALB/c mice (n = 3) after priming with gp70-LPX (day 0, 7, 14). l, Fraction of gp70-specific CD8+ T cells within total CD8+ T cells in blood, bone marrow and lymph nodes determined by MHC class I tetramer staining after de novo priming of splenectomized BALB/c mice (n = 5–7) immunized with gp70-LPX (day 0, 7) or left untreated (control). Significance was determined using unpaired two-tailed Student’s t-test (b left, c), two-way ANOVA and Bonferroni’s multiple comparisons test (b right, g) and one-way ANOVA and Tukey’s multiple comparisons test (j, l). Error bars, mean ± s.d.

  4. Extended Data Figure 4: Potent antitumour immunity and rejection of advanced aggressively growing tumours in mice conferred by RNA-LPX vaccines. (218 KB)

    a, B16-OVA melanoma load in lungs of C57BL/6 mice (n = 8) immunized i.v. (days 4, 7, 11) with OVA-LPX or irrelevant (eGFP)-LPX. b, Expression of activation markers measured 24 h after i.v. injection of 40 μg irrelevant (empty vector)-LPX, eGFP-LPX or OVA-LPX by flow cytometry in splenic immune cell subsets (n = 3) and IFNα serum levels (n = 3) 6 h after injection in C57BL/6 mice. c, Bioluminescence signal of tumours in different groups before immunization and on day 25 (upper left), tumour load and lung weights (upper right) and TRP-1-specific CD8+ and CD4+ T-cell responses in spleens of control (untreated), irrelevant (empty vector)-LPX and TRP-1-LPX-immunized B6 albino mice (n = 12) on day 25 detected by ELISPOT assay (bottom), depicted in Fig. 3b. d, Bioluminescence imaging of CT26-Luc carcinoma in BALB/c mice (n = 4–7) depicted in Fig. 3c (left). e, TC-1-Luc tumour growth in C57BL/6 mice (n = 10) (left), depicted in Fig. 3d, and remission of established advanced TC-1-Luc tumours in C57BL/6 mice (n = 10) immunized i.v. with 40 μg E6/E7-LPX (day 13, 20, 27) (right). f, Survival of BALB/c mice rechallenged with CT26-Luc colon carcinoma cells on day 109, depicted in Fig. 3e. Significance was determined using one-way ANOVA and Tukey’s multiple comparisons test (c), two-way ANOVA and Bonferroni’s multiple comparisons test (d), paired two-tailed Student’s t-test (f, right), unpaired two-tailed Student’s t-test (f, far right), and log-rank test (f, left). Error bars, median with interquartile range (d), mean ± s.d. otherwise.

  5. Extended Data Figure 5: Clinical application of RNA-LPX vaccines and de novo priming and amplification of patient T-cell responses against encoded vaccine antigens. (204 KB)

    a, Vaccination scheme and monitoring for patients 1–3. b, Antigen-specific T-cell responses against NY-ESO-1 and tyrosinase determined by restimulation with overlapping peptide mixtures in IFNγ ELISPOT for patient 1. c, Antigen-specific T-cell responses against NY-ESO-1 and MAGE-A3, determined by post-IVS IFNγ ELISPOT assay at indicated days for patient 2. Values are corrected for background (no peptide). d, Antigen-specific T-cell responses against NY-ESO-I and MAGE-A3, determined by ex vivo IFNγ ELISPOT assay at indicated days for patient 3. Numbers in ELISPOT data indicate the amino acid position of each epitope. Significance was determined using unpaired two-tailed Student’s t-test. Error bars, mean ± s.e.m.

  6. Extended Data Figure 6: Comparison of i.v. and s.c. routes for RNA-LPX administration in the context of T-cell priming and biodistribution of RNA-LPX upon s.c. administration. (75 KB)

    Fraction of OVA-specific CD8+ T cells within CD8+ T cells on day 13 in blood after de novo priming of C57BL/6 mice (n = 5) immunized i.v. with OVA-LPX (day 0, 3, 8) (left). Biodistribution of Luc expression 24 h after s.c. injection of Luc-LPX in BALB/c mice (n = 3) (right). Signal can only be observed at the injection site and the draining lymph node. Significance was determined using one-way ANOVA and Tukey’s multiple comparisons test. Error bars, mean ± s.d.

Extended Data Tables

  1. Extended Data Table 1: Findings of non-GLP pilot pharmacokinetics and pharmacodynamics study in cynomolgus monkeys (169 KB)

Additional data