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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Castle, J. C. et al. Exploiting the mutanome for tumor vaccination. Cancer Res. 72, 1081–1091 (2012).
Castle, J. C. et al. Immunomic, genomic and transcriptomic characterization of CT26 colorectal carcinoma. BMC Genomics 15, 190 (2014).
Holtkamp, S. et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108, 4009–4017 (2006).
Kreiter, S. et al. Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals. J. Immunol. 180, 309–318 (2008).
Kuhn, A. N. 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).
Bloom, M. B. et al. Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma. J. Exp. Med. 185, 453–459 (1997).
Schumacher, T. et al. A vaccine targeting mutant IDH1 induces antitumour immunity. Nature 512, 324–327 (2014).
Tran, E. et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 344, 641–645 (2014).
Britten, C. M. et al. The regulatory landscape for actively personalized cancer immunotherapies. Nature Biotechnol. 31, 880–882 (2013).
Gerlinger, M. et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 366, 883–892 (2012).
Koebel, C. M. et al. Adaptive immunity maintains occult cancer in an equilibrium state. Nature 450, 903–907 (2007).
Matsushita, H. et al. Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting. Nature 482, 400–404 (2012).
Robbins, P. F. et al. Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nature Med. 19, 747–752 (2013).
van Rooij, N. et al. Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma. J. Clin. Oncol. 31, e439–e442 (2013).
Shen, Z. et al. Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. J. Immunol. 158, 2723–2720 (1997).
Schoenberger, S. P. et al. T-cell help for cytotoxic T lymphocytes is mediated by CD40–CD40L interactions. Nature 393, 480–483 (1998).
Linnemann, C. et al. High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma. Nature Med. 21, 81–85 (2015).
Arnold, P. Y. et al. The majority of immunogenic epitopes generate CD4+ T cells that are dependent on MHC class II-bound peptide-flanking residues. J. Immunol. 169, 739–749 (2002).
Wolfel, T. et al. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269, 1281–1284 (1995).
Wang, R. F. et al. Cloning genes encoding MHC class II-restricted antigens: mutated CDC27 as a tumor antigen. Science 284, 1351–1354 (1999).
Lu, Y. C. et al. Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression. J. Immunol. 190, 6034–6042 (2013).
Gubin, M. M. et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature 515, 577–581 (2014).
Snyder, A. et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N. Engl. J. Med. 371, 2189–2199 (2014).
Herbst, R. S. et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515, 563–567 (2014).
Tumeh, P. C. et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515, 568–571 (2014).
Rizvi, N. A. et al. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 348, 124–128 (2015).
Castle, J. C. et al. Mutated tumor alleles are expressed according to their DNA frequency. Sci. Rep. 4, 4743 (2014).
Löwer, M. et al. Confidence-based somatic mutation evaluation and prioritization. PLOS Comput. Biol. 8, e1002714 (2012).
Boegel, S. et al. A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines. OncoImmunology 3, e954893 (2014).
Kreiter, S. et al. Intranodal vaccination with naked antigen-encoding RNA elicits potent prophylactic and therapeutic antitumoral immunity. Cancer Res. 70, 9031–9040 (2010).
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).
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.).
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.
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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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