A vaccine targeting mutant IDH1 induces antitumour immunity (original) (raw)

References

1. Balss, J. et al. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol. 116, 597–602 (2008)
   Article CAS Google Scholar
2. Parsons, D. W. et al. An integrated genomic analysis of human glioblastoma multiforme. Science 321, 1807–1812 (2008)
   Article CAS ADS Google Scholar
3. Yan, H. et al. IDH1 and IDH2 mutations in gliomas. N. Engl. J. Med. 360, 765–773 (2009)
   Article CAS Google Scholar
4. Marcucci, G. et al. IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia: a cancer and leukemia group B study. J. Clin. Oncol. 28, 2348–2355 (2010)
   Article CAS Google Scholar
5. Mardis, E. R. et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N. Engl. J. Med. 361, 1058–1066 (2009)
   Article CAS Google Scholar
6. Amary, M. F. et al. IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours. J. Pathol. 224, 334–343 (2011)
   Article CAS Google Scholar
7. Dang, L. et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 462, 739–744 (2009)
   Article CAS ADS Google Scholar
8. Ward, P. S. et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 17, 225–234 (2010)
   Article CAS Google Scholar
9. Sasaki, M. et al. IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics. Nature 488, 656–659 (2012)
   Article CAS ADS Google Scholar
10. Figueroa, M. E. et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 18, 553–567 (2010)
    Article CAS Google Scholar
11. Turcan, S. et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483, 479–483 (2012)
    Article CAS ADS Google Scholar
12. Cairns, R. A. & Mak, T. W. Oncogenic isocitrate dehydrogenase mutations: mechanisms, models, and clinical opportunities. Cancer Discov 3, 730–741 (2013)
    Article CAS Google Scholar
13. Capper, D. et al. Characterization of R132H Mutation-specific IDH1 Antibody Binding in Brain Tumors. Brain Pathol. 20, 245–254 (2010)
    Article CAS Google Scholar
14. Watanabe, T., Nobusawa, S., Kleihues, P. & Ohgaki, H. IDH1 mutations are early events in the development of astrocytomas and oligodendrogliomas. Am. J. Pathol. 174, 1149–1153 (2009)
    Article CAS Google Scholar
15. Capper, D., Zentgraf, H., Balss, J., Hartmann, C. & von Deimling, A. Monoclonal antibody specific for IDH1 R132H mutation. Acta Neuropathol. 118, 599–601 (2009)
    Article CAS Google Scholar
16. Pajot, A. et al. A mouse model of human adaptive immune functions: HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice. Eur. J. Immunol. 34, 3060–3069 (2004)
    Article CAS Google Scholar
17. Hunder, N. N. et al. Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N. Engl. J. Med. 358, 2698–2703 (2008)
    Article CAS Google Scholar
18. Quezada, S. A. et al. Tumor-reactive CD4+ T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts. J. Exp. Med. 207, 637–650 (2010)
    Article CAS Google Scholar
19. Wang, F. et al. Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation. Science 340, 622–626 (2013)
    Article CAS ADS Google Scholar
20. Rohle, D. et al. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 340, 626–630 (2013)
    Article CAS ADS Google Scholar
21. Wick, W. et al. NOA-04 randomized phase III trial of sequential radiochemotherapy of anaplastic glioma with procarbazine, lomustine, and vincristine or temozolomide. J. Clin. Oncol. 27, 5874–5880 (2009)
    Article CAS Google Scholar
22. Jansen, M., Yip, S. & Louis, D. N. Molecular pathology in adult gliomas: diagnostic, prognostic, and predictive markers. Lancet Neurol. 9, 717–726 (2010)
    Article CAS Google Scholar
23. Ito, K. et al. HLA-DR4-IE chimeric class II transgenic, murine class II-deficient mice are susceptible to experimental allergic encephalomyelitis. J. Exp. Med. 183, 2635–2644 (1996)
    Article CAS Google Scholar
24. Lundegaard, C., Lund, O. & Nielsen, M. Accurate approximation method for prediction of class I MHC affinities for peptides of length 8, 10 and 11 using prediction tools trained on 9mers. Bioinformatics 24, 1397–1398 (2008)
    Article CAS Google Scholar
25. Rammensee, H., Bachmann, J., Emmerich, N. P., Bachor, O. A. & Stevanovic, S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50, 213–219 (1999)
    Article CAS Google Scholar
26. Balss, J. et al. Enzymatic assay for quantitative analysis of (D)-2-hydroxyglutarate. Acta Neuropathol. 124, 883–891 (2012)
    Article CAS Google Scholar

Download references

Acknowledgements

We are indebted to the patients and their relatives who agreed to participate in this study. We thank A. Gardyan, T. Lanz and W. Osen for technical advice, T. Lanz and A. Hertenstein for providing patient blood samples, S. Hundt for cloning, A. Habel and D. Krunic for technical support, W. Nicklas for pathological analysis, and J. Jung for graphics design. We acknowledge the support by the DKFZ Light Microscopy and Genomics and Proteomics Facilities. HLA-DRB1*01:01 MHC class II tetramer bound to IDH1(R132H) p123-142 was provided by NIH tetramer core facility. A2.DR1 mice were provided by Institut Pasteur. This work was supported by the Interdisciplinary Research Program of the National Center for Tumor Diseases Heidelberg (IFP III/2) to M.P. and A.V.D., the Wilhelm Sander Foundation (2012.118.1) to M.P and A.V.D., the Helmholtz Foundation (VH-NG-306) and the Andreas Zimprich Foundation to M.P. and the German Research Foundation (SFB938 TPK) to M.P. and W.W. T.S. and M.K. were supported by the Helmholtz International Graduate School, and L.B. was supported by the Heinrich F. C. Behr Foundation and the Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology MD/PhD program, University Heidelberg. F.S. was supported by a postdoctoral fellowship of the University Hospital Heidelberg.

Author information

Author notes

1. Theresa Schumacher and Lukas Bunse: These authors contributed equally to this work.

Authors and Affiliations

1. Department of Neurooncology, University Hospital Heidelberg and National Center for Tumor Diseases, 69120 Heidelberg, Germany,
   Theresa Schumacher, Lukas Bunse, Benedikt Wiestler, Oliver Menn, Matthias Osswald, Iris Oezen, Martina Ott, Melanie Keil, Katharina Rauschenbach, Wolfgang Wick & Michael Platten
2. German Cancer Consortium (DKTK) Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany,
   Theresa Schumacher, Lukas Bunse, Iris Oezen, Martina Ott, Melanie Keil, Jörg Balß, Katharina Rauschenbach & Michael Platten
3. Department of Neuropathology, University Hospital Heidelberg and National Center for Tumor Diseases, 69120 Heidelberg, Germany,
   Stefan Pusch, Felix Sahm & Andreas von Deimling
4. German Cancer Consortium (DKTK) Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany,
   Stefan Pusch, Felix Sahm, Jörg Balß & Andreas von Deimling
5. German Cancer Consortium (DKTK) Clinical Cooperation Unit Neurooncology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany,
   Benedikt Wiestler, Matthias Osswald & Wolfgang Wick
6. Department of Translational Immunology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany,
   Jasmin Quandt, Stefan B. Eichmüller & Philipp Beckhove
7. Department of Immunotherapy and –prevention Group, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany,
   Agnieszka K. Grabowska & Angelika B. Riemer
8. Ribological GmbH, 55131 Mainz, Germany,
   Isabel Vogler
9. Translational Oncology, 55131 Mainz, Germany,
   Jan Diekmann & Ugur Sahin
10. Department of Immunology, University of Tübingen, 72076 Tübingen, Germany,
    Nico Trautwein & Stefan Stevanović
11. Metabolic Centre Heidelberg, University Children’s Hospital, 69120 Heidelberg, Germany,
    Jürgen Okun
12. Center for Molecular Neurobiology, University Medical Center, Hamburg-Eppendorf, 20251 Hamburg, Germany,
    Manuel A. Friese

Authors

1. Theresa Schumacher
   You can also search for this author inPubMed Google Scholar
2. Lukas Bunse
   You can also search for this author inPubMed Google Scholar
3. Stefan Pusch
   You can also search for this author inPubMed Google Scholar
4. Felix Sahm
   You can also search for this author inPubMed Google Scholar
5. Benedikt Wiestler
   You can also search for this author inPubMed Google Scholar
6. Jasmin Quandt
   You can also search for this author inPubMed Google Scholar
7. Oliver Menn
   You can also search for this author inPubMed Google Scholar
8. Matthias Osswald
   You can also search for this author inPubMed Google Scholar
9. Iris Oezen
   You can also search for this author inPubMed Google Scholar
10. Martina Ott
    You can also search for this author inPubMed Google Scholar
11. Melanie Keil
    You can also search for this author inPubMed Google Scholar
12. Jörg Balß
    You can also search for this author inPubMed Google Scholar
13. Katharina Rauschenbach
    You can also search for this author inPubMed Google Scholar
14. Agnieszka K. Grabowska
    You can also search for this author inPubMed Google Scholar
15. Isabel Vogler
    You can also search for this author inPubMed Google Scholar
16. Jan Diekmann
    You can also search for this author inPubMed Google Scholar
17. Nico Trautwein
    You can also search for this author inPubMed Google Scholar
18. Stefan B. Eichmüller
    You can also search for this author inPubMed Google Scholar
19. Jürgen Okun
    You can also search for this author inPubMed Google Scholar
20. Stefan Stevanović
    You can also search for this author inPubMed Google Scholar
21. Angelika B. Riemer
    You can also search for this author inPubMed Google Scholar
22. Ugur Sahin
    You can also search for this author inPubMed Google Scholar
23. Manuel A. Friese
    You can also search for this author inPubMed Google Scholar
24. Philipp Beckhove
    You can also search for this author inPubMed Google Scholar
25. Andreas von Deimling
    You can also search for this author inPubMed Google Scholar
26. Wolfgang Wick
    You can also search for this author inPubMed Google Scholar
27. Michael Platten
    You can also search for this author inPubMed Google Scholar

Contributions

T.S. and L.B. designed and performed experiments, analysed data and wrote the paper. F.S. and A.v.D. provided glioma tissue and determined IDH1 status. S.P. cloned IDH1 constructs. J.B. and J.O. performed 2-HG measurements. J.Q. and P.B. generated the A2.DR1 sarcoma cell line. K.R. and M.Ot. established stable overexpressions. B.W. performed statistical analysis. I.O. and M.K. performed animal experiments. O.M. and M.Os. provided patient blood samples. A.K.G. and A.B.R. performed epitope prediction and T2 binding assays. J.D., N.T., I.V., S.S. and U.S. analysed patient samples. S.B.E. provided DR4 mice. M.A.F. and W.W. were involved in study design and data interpretation. M.P. conceptualized the study, designed experiments, interpreted data and wrote the paper.

Corresponding author

Correspondence toMichael Platten.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 IDH1 peptide libraries are used to assess MHC binding and immunogenicity in silico, in vitro and in vivo.

a, Human wild-type IDH1 (WT) and IDH1(R132H) (RH) amino acid sequences covering the mutated residue. b, c, Peptide libraries of wild-type IDH1 and IDH1(R132H) containing peptides of 10 (b) or 15 (c) amino acids were generated. d, e, MHC peptide binding predictions for IDH1(R132H) peptides to HLA-A*0201 (A2) using NetMHC algorithm (d) and SYFPEITHI (e). f, IDH1(R132H) 10-mer peptide binding to HLA-A2 in vitro in a T2 binding assay. Fluorescence intensities are depicted after background subtraction. g, DR1-binding wild-type IDH1 15-mer epitopes were identified by Reveal Class II binding assay using the 15-mer peptide library (c). Immediate binding (yellow) and 24-h stability (red). h–j, ELISpots of IFN-γ splenocyte responses to 10-mers (h, j) and 15-mers (i) after vaccination of A2.DR1 mice with IDH1(R132H) in Montanide (j) or vehicle in CFA (h, i) (red, IDH1(R132H) (p123–142); blue, WT IDH1 (p123–142); black, IDH1(R132H) library; p123–142 (R132H versus WT), Welch _t_-test; library, ANOVA; n = 3 biological replicates). Error bars, mean + s.e.m.

Extended Data Figure 2 T-helper cell characterization after IDH1(R132H) peptide vaccination in HLA-DR transgenic mice.

a, Cytokine ELISAs of splenocyte responses to IDH1(R132H) (p123–142) or vehicle after vaccination of three A2.DR1 (top and middle panels) and three DR4 (bottom panel) mice with IDH1(R132H) (red) or vehicle (black) in CFA (top panel) or Montanide (middle and bottom panels; Welch _t_-test; n = 3 technical replicates). Error bars, mean + s.e.m. b, Representative intracellular flow cytometry of splenocyte responses to IDH1(R132H) (p123–142) or vehicle after vaccination of three A2.DR1 (left and middle panels) and three DR4 (right panel) mice with IDH1(R132H) (vacc) or vehicle (sham) using CFA (left panel) or Montanide (middle and right panels). Gated on CD4+ cells; red numbers indicate populations in per cent. c, ELISpots of IFN-γ splenocyte responses to IDH1 (p123–142) (R132H, wild type) after vaccination of DR4 mice with IDH1(R132H) (vacc) or vehicle (sham) in Montanide (Welch _t_-test; n = 3 biological replicates). Error bars, mean + s.e.m.

Extended Data Figure 3 IDH1(R132H)-specific CD4+ T cells have a TH1 phenotype and are dependent on HLA-DRA*0101 HLA-DRB1*0101 (DR1).

a, Intracellular flow cytometry of IDH1(R132H)-specific T-cell line response to IDH1(R132H) (p123–142) or vehicle. Gated on CD4+ cells; red numbers indicate populations in per cent. b, c, ELISpots of an IDH1(R132H)-specific T-cell clone (white, MOG; black, vehicle; red, IDH1(R132H); blue, wild-type IDH1; b, Welch _t_-test, error bars, mean ± s.e.m.; c, pairwise Welch _t_-test with Bonferroni correction; error bars, mean + s.e.m. n = 3 technical replicates).

Extended Data Figure 4 IDH1 peptide-coated ELISA for detection of IDH1-specific IgG in human and mouse serum.

a, Sandwich ELISA scheme. b, ELISA was established using mouse anti-IDH1(R132H) antibody as serum substitute. ELISA on indicated peptides of the 10-mer and 15-mer libraries (blue, wild-type IDH1; red, IDH1(R132H); 10-mer pool, all library peptides) and a negative control peptide (white, HIV) (left). p122–136 (R132H) was used to titrate the anti-IDH1(R132H) antibody (middle). IgG subtype-specific secondary antibodies were used with IDH1(R132H)-specific antibody on IDH1 p122–136 (red, IDH1(R132H); blue, wild-type IDH1) (right). c, Detection of IDH1(R132H)-specific IgG in IDH1(R132H)+ patient serum. IDH1 peptides of the 15-mer library and p123–142 (blue, wild type; red, R132H) were used to establish the ELISA for patient serum with MOG as negative control peptide. Serum from patients p009 and p078 was used. Scatter plot showing individual values and the mean. d, Detection of tetanus toxoid-specific IgG in serum from patients with wild-type IDH1 gliomas (blue, n = 44) and IDH1(R132H)+ gliomas (red, n = 40). IDH1(R132H)+ are grouped into IDH1(R132H)-IgG positive (responder, n = 4) and negative (non-responder, n = 36) according to Fig. 2g (Wilcoxon rank-sum test). Scatter plot showing individual values and the mean. e, IDH1(R132H)-specific IgG and IgG subtypes IgG1, IgG2, IgG3 and IgG4 were detected by p122–136 (R132H)-coated ELISA in serum from IDH1(R132H)+ glioma patients with increased relative values for IDH1(R132H)-specific IgG (pairwise Welch _t_-test with Bonferroni correction; n = 10). Scatter plot showing individual values for each patient and the mean.

Extended Data Figure 5 Generation and characterization of syngeneic IDH1(R132H)+ A2.DR1 sarcoma cells.

a, Scheme for establishment of syngeneic A2.DR1 sarcoma cell line. b, Haematoxylin and eosin staining of established A2.DR1 sarcoma cell line. c, IDH1(R132H) expression in transduced A2.DR1 sarcoma cells by immunofluorescent staining (top) and by western blot (bottom). d, 2-HG concentrations (upper panel) and IDH1(R132H) expression by western blot (lower panel) in transduced A2.DR1 syngeneic sarcoma cells in vitro (left), A2.DR1 sarcomas in vivo (IVC1) and human gliobastoma (RH) and anaplastic astrocytoma (wt) tissue (right). Red dashed lines mark the range of 2-HG concentrations found in glioma tissue. e, Growth of subcutaneous sarcomas (IDH1(R32H)+, red; wild-type IDH1+, blue) (Wilcoxon rank-sum test for median AUC; n = 5). Error bars, mean ± s.e.m. f, g, ELISA (f) and ELISpot (g, after subtraction of MOG-induced spots) of IFN-γ splenocyte responses to IDH1(R132H) (p123–142) after preventive vaccination with IDH1(R132H) (red) or vehicle (black) and injection with IDH1(R132H)+ or wild-type IDH1+ sarcomas (Welch _t_-test; n = 8 IDH1(R123H) tumours, vaccinated mice; n = 6 IDH1(R132H) tumours, sham mice; and wild-type IDH1 tumours, vaccinated mice; n = 5 wild-type IDH1 tumours, sham mice). Error bars, mean + s.e.m. h–j, IDH1/2 enzymatic activity in liver and brain (h), and ELISpot (i, after subtraction of MOG-induced spots, negative values set to zero) and ELISA (j) of IFN-γ splenocyte (spleen) and lymph node (LN) responses to IDH1 (p123-142) (RH, wild type) after therapeutic vaccination with IDH1(R132H) (red) or vehicle (black) and injection with IDH1(R132H)+ sarcomas (Welch _t_-test; n = 6, vaccinated; n = 5, sham (h); n = 7, vaccinated; n = 6, sham (i, j). Scatter plots showing individual values and the mean. All n values indicate number of biological replicates.

Extended Data Figure 6 NY-ESO-1 is a suitable antigen for DR1-dependent and TH-mediated therapeutic peptide vaccination in A2.DR1 mice.

a, Alignment of human NY-ESO-1 protein with murine CTAG2 protein. Red box, HLA-DRA*0101 DRB1*0101 (DR1)-restricted epitope human NY-ESO-1 p119–143. b, NY-ESO-1 protein detection by immunofluorescent staining and western blot in A2.DR1 sarcoma cells. c, d, ELISpot (c, after subtraction of MOG-induced spots, Wilcoxon rank-sum test, n = 7 biological replicates, error bars, mean + s.e.m.) and representative intracellular flow cytometry (d, gated on CD4+ T cells, red numbers, populations in per cent) of IFN- γ splenocyte (spleen) and lymph node (c, LN) responses to NY-ESO-1 after therapeutic vaccination with NY-ESO-1 (red) or vehicle (black) and injection with NY-ESO-1+ sarcomas.

Extended Data Figure 7 IDH1(R132H) peptide vaccination induces TH responses in A2.DR1 mice.

a, ELISpot of IFN-γ responses to IDH1 (p123–142) of CD4+ and CD8+ T cells after vaccination with IDH1(R132H) in Montanide or established T cell line (R132H, red; wild type, blue) or MOG (white), or with PMA and ionomycin (black) (Welch _t_-test; n = 3 technical replicates). Error bars, mean + s.e.m. b, c, Representative flow cytometry of blood lymphocytes from A2.DR1 mice depleted of CD4+ T cells by clone GK1.5 (b) and of B cells by clone 1D3, or isotype control treated with clone LTF-2 (b) or clone 2A3 (c), and immunized with IDH1(R132H) (vacc) or vehicle (sham). Red numbers indicate populations in per cent. d, ELISpot of IFN-γ splenocyte (spleen) and lymph node (LN) responses to IDH1 p123–142 (RH and wild type) after therapeutic vaccination with IDH1(R132H) and depletion of B cells (blue) or isotype-control treatment (red) or with vehicle (black) (Welch one-way ANOVA and pairwise Welch _t_-tests with Bonferroni correction; n = 6 biological replicates). Scatter plots showing individual values after subtraction of MOG-induced spots and the mean. e, Growth of IDH1(R132H)+ subcutaneous sarcomas after therapeutic vaccination with IDH1(R132H) and depletion of B cells (blue) or isotype-control treatment (red) or with vehicle (black) (Wilcoxon rank-sum test for median AUC with Bonferroni correction; n = 6 per group, n = 7 for vaccinated without depletion). Error bars, mean ± s.e.m.

Extended Data Table 1 Patient characteristics and analyses

Full size table

Extended Data Table 2 Clinical information of patients with IDH1 mutated gliomas

Full size table

Extended Data Table 3 Pathological analysis of organs from preventively vaccinated A2.DR1

Full size table

Supplementary information

PowerPoint slides

Rights and permissions

About this article

Cite this article

Schumacher, T., Bunse, L., Pusch, S. et al. A vaccine targeting mutant IDH1 induces antitumour immunity.Nature 512, 324–327 (2014). https://doi.org/10.1038/nature13387

Download citation

• Received: 17 July 2013
• Accepted: 17 April 2014
• Published: 25 June 2014
• Issue Date: 21 August 2014
• DOI: https://doi.org/10.1038/nature13387