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Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Nature volume 520, pages 692–696 (2015)Cite this article

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An Erratum to this article was published on 03 June 2015

Abstract

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations1. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient’s tumour with vaccines produced ‘just in time’.

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Figure 1: Cancer‐associated mutations are frequently immunogenic and pre‐dominantly recognized by CD4+ T cells.
Figure 2: Efficient tumour control and survival benefit in B16F10 melanoma with an RNA vaccine encoding a single mutated CD4+ T‐cell epitope.
Figure 3: RNA pentatope immunization confers disease control and survival benefit in murine tumours.
Figure 4: RNA pentatope vaccines with mutations selected for in silico predicted favourable MHC class II binding and abundant expression confer potent antitumour control.

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Acknowledgements

We thank M. Holzmann, A. König, U. Schmitt, R. Roth, C. Worm and N. Krause for technical assistance; L. Ralla, J. Groß, A. Spruß, M. Erdeljan, S. Wöll and C. Rohde for immunohistochemical staining and analysis; C. Paret for sequence validation of mutations; M. Brkic for immunofluorescence staining; S. Witzel and Bodo Tillmann, S. Wurzel and Z. Yildiz for cloning of constructs; S. Kind, M. Mechler, F. Wille, B. Otte and S. Petri for RNA production as well as L. Kranz and colleagues involved in RNA formulation development. We are grateful to B. Kloke, S. Heesch, A. Kuhn, J. Buck, C. Britten and H. Haas for conceptual and technical discussions. Moreover, we would like to thank V. Bukur, J. de Graf and C. Albrecht who supported the next-generation sequencing of samples. Furthermore we like to acknowledge A. Kong for critical reading and A. Orlandini for help with graphic design. The results shown here are in part based on data generated by the TCGA Research Network http://cancergenome.nih.gov/. The study was supported by the CI3 excellence cluster program of the Federal Ministry of Education and Research (BMBF).

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Author notes

  1. Mathias Vormehr and Niels van de Roemer: These authors contributed equally to this work.

  2. Özlem Türeci and Ugur Sahin: These authors jointly supervised this work.

Authors and Affiliations

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

    Sebastian Kreiter, Mustafa Diken, Martin Löwer, Jan Diekmann, Sebastian Boegel, Barbara Schrörs, Fulvia Vascotto, John C. Castle, Arbel D. Tadmor, Özlem Türeci & Ugur Sahin

  2. Research Center for Immunotherapy (FZI), Langenbeckstrasse 1, Building 708, Mainz, 55131, Germany

    Mathias Vormehr, Niels van de Roemer, Christoph Huber & Ugur Sahin

  3. Biopharmaceutical New Technologies (BioNTech) Corporation, An der Goldgrube 12, Mainz, 55131, Germany

    Jan Diekmann & Ugur Sahin

  4. La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, 92037, California, USA

    Stephen P. Schoenberger

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Contributions

U.S. is principal investigator, conceptualized the study and experimental strategy. S.K., M.V., N.vdR., M.D., J.D., F.V. and U.S. planned and analysed experiments. M.V. and N.vdR. performed experiments. S.K., M.V., M.D., S.P.S., C.H., Ö.T. and U.S. interpreted the data and wrote the manuscript. M.L., S.B., A.D.T. and J.C.C. processed next-generation sequencing data and identified mutations. M.V. and B.S. analysed murine MHC II binding predictions. S.B. analysed TCGA data and human MHC II binding predictions.

Corresponding author

Correspondence to Ugur Sahin.

Extended data figures and tables

Extended Data Figure 1 Non synonymous cancer‐associated mutations are frequently immunogenic and pre‐dominantly recognized by CD4+ T cells.

T‐cell responses obtained by vaccinating C57BL/6 mice with antigen‐encoding RNA in the B16F10 tumour model (n = 5). Left, prevalence of non‐immunogenic, MHC-class-I- or class-II-restricted mutated epitopes. Right, detection and typing of mutation‐specific T cells (individual epitopes shown in Extended Data Table 1).

Extended Data Figure 2 Mutant epitope-specific T cells induced by RNA vaccination control tumour growth.

a, Splenocytes of mice (n = 5) vaccinated with B16‐M30 RNA were tested by ELISpot for recognition of mutated peptides as compared to the corresponding wild‐type (B16‐WT30) sequence. Right, testing of truncated variants of B16‐M30 (mean + s.e.m.). b, Mean ± s.e.m. tumour growth (left) and survival (right) of C57BL/6 mice (n = 10) inoculated s.c. with B16F10 and left untreated (control) or injected i.v. with irrelevant RNA. c, Lungs of B16F10‐Luc tumour bearing mice shown in Fig. 2b (day 27 after tumour inoculation). d, Therapeutic antitumour activity against B16F10 tumours in mice (B16‐M27, Trp2 n = 8; B16‐M30 n = 7; others n = 10) conferred by immunization with epitopes encoding immunogenic B16F10 mutations or an immunodominant wild type Trp2 epitope6. The area under the tumour growth curve at day 30 after tumour inoculation was normalized to untreated control mice and depicted as mean ± s.e.m. Red and black columns represent mutations recognized by CD8+ or CD4+ T cells, respectively. e, Spontaneous immune responses in splenocytes of irrelevant RNA treated B16F10 tumour bearing C57BL/6 mice (n = 3) were tested by ELISpot for recognition of peptides (mean + s.e.m.).

Extended Data Figure 3 Mechanism of antitumour activity of mutation specific poly‐epitope vaccines in CT26 tumour‐bearing mice.

a, BALB/c mice (n = 5) were vaccinated either with pentatope (35 µg) or the corresponding mixture of five RNA monotopes (7 µg each). T‐cell responses in peptide‐stimulated splenocytes of mice were measured ex vivo via ELISpot (medium control subtracted mean ± s.e.m.). b, c, BALB/c mice (n = 10) were inoculated i.v. with CT26 tumour cells and left untreated or injected with irrelevant, CT26‐M19 or pentatope1 or 2 RNA in absence (b) or presence of a CD8 T cell depleting antibody or a CD40L blocking antibody (c). Mean ± s.e.m. of tumour nodules per lung are shown. d, Immunofluorescence analyses of tumour‐infiltrating lymphocytes in pentatope2‐vaccinated mice. Upper panel, lung tumour tissue stained for CD4 and CD8 or CD4 and FoxP3. Scale bar, 50 µm. Lower panel left, proportion of infiltrating cells in sections of irrelevant (CD4: n = 13; CD8 = 9; FoxP3: n = 13) or pentatope (CD4: n = 17; CD8: n = 6; FoxP3: n = 10) RNA‐treated animals. Lower panel right, tumour area in sections of control (n = 22) and pentatope2‐treated (n = 20) animals (mean ± s.e.m.).

Extended Data Figure 4 Immunogenicity testing of PME pentatope‐encoded mutations.

Splenocytes of PME RNA vaccinated BALB/c mice (n = 6) were tested ex vivo for recognition of peptides representing the mutated 27mer sequences represented in PME pentatopes with or without addition of an MHC class II‐blocking antibody. Mean + s.e.m. of background (medium control) subtracted responses are shown.

Extended Data Table 1 Immunogenic B16F10 mutations
Full size table
Extended Data Table 2 Immunogenic CT26 mutations
Full size table
Extended Data Table 3 Immunogenic 4T1 mutations
Full size table
Extended Data Table 4 CT26 mutated epitopes encoded in pentatope 1+2
Full size table
Extended Data Table 5 In silico prediction of CT26 mutations with abundant expression and favourable MHC class II binding properties
Full size table

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Kreiter, S., Vormehr, M., van de Roemer, N. et al. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520, 692–696 (2015). https://doi.org/10.1038/nature14426

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