An immunogenic personal neoantigen vaccine for patients with melanoma (original) (raw)
Change history
14 March 2018
Please see accompanying Corrigendum (http://doi.org/10.1038/nature25145). The ‘Data availability’ statement was changed from ‘All data are available from the corresponding author upon reasonable request’ to ‘WES and RNA-seq data are deposited in dbGaP (https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs001451.v1.p1). All other data are available from the corresponding author upon reasonable request’ In addition, the following wording was added to the ‘Competing interests’ statement: ‘C.J.W. is subject to a conflict of interest management plan for the reported studies because of her competing financial interests in Neon Therapeutics. Under this plan, C.J.W. may not access identifiable human subjects’ data nor otherwise participate directly in the IRB-approved protocol reported herein. C.J.W.’s contributions to the overall program strategy and data analyses occurred on a de-identified basis.’
References
- Schumacher, T. N. & Schreiber, R. D. Neoantigens in cancer immunotherapy. Science 348, 69–74 (2015)
Article ADS CAS PubMed Google Scholar - Hacohen, N., Fritsch, E. F., Carter, T. A., Lander, E. S. & Wu, C. J. Getting personal with neoantigen-based therapeutic cancer vaccines. Cancer Immunol. Res. 1, 11–15 (2013)
Article CAS PubMed PubMed Central Google Scholar - Kenter, G. G. et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N. Engl. J. Med. 361, 1838–1847 (2009)
Article CAS PubMed Google Scholar - Caskey, M. et al. Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans. J. Exp. Med. 208, 2357–2366 (2011)
Article CAS PubMed PubMed Central Google Scholar - Robert, C. et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 372, 2521–2532 (2015)
Article CAS PubMed Google Scholar - Sykulev, Y., Joo, M., Vturina, I., Tsomides, T. J. & Eisen, H. N. Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response. Immunity 4, 565–571 (1996)
Article CAS PubMed Google Scholar - Stephen, T. L. et al. SATB1 expression governs epigenetic repression of PD-1 in tumor-reactive T cells. Immunity 46, 51–64 (2017)
Article CAS PubMed PubMed Central Google Scholar - Tran, E. et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 344, 641–645 (2014)
Article ADS CAS PubMed PubMed Central Google Scholar - Schumacher, T. et al. A vaccine targeting mutant IDH1 induces antitumour immunity. Nature 512, 324–327 (2014)
Article ADS CAS PubMed Google Scholar - 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)
Article PubMed Google Scholar - Rizvi, N. A. et al. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 348, 124–128 (2015)
Article ADS CAS PubMed PubMed Central Google Scholar - Linnemann, C. et al. High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma. Nat. Med. 21, 81–85 (2015)
Article CAS PubMed Google Scholar - Prickett, T. D. et al. Durable complete response from metastatic melanoma after transfer of autologous T cells recognizing 10 mutated tumor antigens. Cancer Immunol. Res. 4, 669–678 (2016)
Article CAS PubMed PubMed Central Google Scholar - Carreno, B. M. et al. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells. Science 348, 803–808 (2015)
Article ADS CAS PubMed PubMed Central Google Scholar - Kreiter, S. et al. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520, 692–696 (2015)
Article ADS CAS PubMed PubMed Central Google Scholar - Martin, S. D. et al. Low mutation burden in ovarian cancer may limit the utility of neoantigen-targeted vaccines. PLoS ONE 11, e0155189 (2016)
Article PubMed PubMed Central CAS Google Scholar - Stern, L. J. et al. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368, 215–221 (1994)
Article ADS CAS PubMed Google Scholar - Rossjohn, J. et al. T cell antigen receptor recognition of antigen-presenting molecules. Annu. Rev. Immunol. 33, 169–200 (2015)
Article CAS PubMed Google Scholar - Falk, K., Rötzschke, O., Stevanovic´, S., Jung, G. & Rammensee, H. G. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 351, 290–296 (1991)
Article ADS CAS PubMed Google Scholar - Mildner, A. & Jung, S. Development and function of dendritic cell subsets. Immunity 40, 642–656 (2014)
Article CAS PubMed Google Scholar - Spitzer, M. H. et al. Systemic immunity is required for effective cancer immunotherapy. Cell 168, 487–502 (2017)
Article CAS PubMed PubMed Central Google Scholar - Haabeth, O. A. et al. Idiotype-specific CD4+ T cells eradicate disseminated myeloma. Leukemia 30, 1216–1220 (2016)
Article CAS PubMed Google Scholar - Hirschhorn-Cymerman, D. et al. Induction of tumoricidal function in CD4+ T cells is associated with concomitant memory and terminally differentiated phenotype. J. Exp. Med. 209, 2113–2126 (2012)
Article CAS PubMed PubMed Central Google Scholar - Abelin, J. G. et al. Mass spectrometry profiling of HLA-associated peptidomes in mono-allelic cells enables more accurate epitope prediction. Immunity 46, 315–326 (2017)
Article CAS PubMed PubMed Central Google Scholar - Fisher, S. et al. A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Genome Biol. 12, R1 (2011)
Article PubMed PubMed Central Google Scholar - Gnirke, A. et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27, 182–189 (2009)
Article CAS PubMed PubMed Central Google Scholar - Chapman, M. A. et al. Initial genome sequencing and analysis of multiple myeloma. Nature 471, 467–472 (2011)
Article ADS CAS PubMed PubMed Central Google Scholar - Berger, M. F. et al. The genomic complexity of primary human prostate cancer. Nature 470, 214–220 (2011)
Article ADS CAS PubMed PubMed Central Google Scholar - Cibulskis, K. et al. ContEst: estimating cross-contamination of human samples in next-generation sequencing data. Bioinformatics 27, 2601–2602 (2011)
Article CAS PubMed PubMed Central Google Scholar - DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011)
Article CAS PubMed PubMed Central Google Scholar - Saunders, C. T. et al. Strelka: accurate somatic small-variant calling from sequenced tumor-normal sample pairs. Bioinformatics 28, 1811–1817 (2012)
Article CAS PubMed Google Scholar - Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011)
Article CAS PubMed PubMed Central Google Scholar - Ramos, A. H. et al. Oncotator: cancer variant annotation tool. Hum. Mutat. 36, E2423–E2429 (2015)
Article PubMed PubMed Central Google Scholar - Torres-García, W. et al. PRADA: pipeline for RNA sequencing data analysis. Bioinformatics 30, 2224–2226 (2014)
Article PubMed PubMed Central CAS Google Scholar - Hoof, I. et al. NetMHCpan, a method for MHC class I binding prediction beyond humans. Immunogenetics 61, 1–13 (2009)
Article CAS PubMed Google Scholar - Lundegaard, C., Lund, O. & Nielsen, M. Prediction of epitopes using neural network based methods. J. Immunol. Methods 374, 26–34 (2011)
Article CAS PubMed Google Scholar - Roemer, M. G. et al. Classical hodgkin lymphoma with reduced β2M/MHC Class i expression is associated with inferior outcome independent of 9p24.1 status. Cancer Immunol. Res. 4, 910–916 (2016)
Article CAS PubMed PubMed Central Google Scholar - Cai, A. et al. Mutated BCR-ABL generates immunogenic T-cell epitopes in CML patients. Clin. Cancer Res. 18, 5761–5772 (2012)
Article ADS CAS PubMed PubMed Central Google Scholar - Lu, Y. C. et al. Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions. Clinical Cancer Res. 20, 3401–3410 (2014)
Article CAS Google Scholar - Day, C. L. et al. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers. J. Clin. Invest. 112, 831–842 (2003)
Article CAS PubMed PubMed Central Google Scholar - Call, M. J. et al. In vivo enhancement of peptide display by MHC class II molecules with small molecule catalysts of peptide exchange. J. Immunol. 182, 6342–6352 (2009)
Article CAS PubMed Google Scholar - Hashimshony, T. et al. CEL-seq2: sensitive highly-multiplexed single-cell RNA-seq. Genome Biol. 17, 77 (2016)
Article PubMed PubMed Central CAS Google Scholar - Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495–502 (2015)
Article CAS PubMed PubMed Central Google Scholar - Kharchenko, P. V., Silberstein, L. & Scadden, D. T. Bayesian approach to single-cell differential expression analysis. Nat. Methods 11, 740–742 (2014)
Article CAS PubMed PubMed Central Google Scholar - Lundegaard, C. et al. NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res. 36, W509–12 (2008)
Article CAS PubMed PubMed Central Google Scholar
Acknowledgements
We thank J. Russell and the Dana-Farber Cancer Institute (DFCI) Center for Immuno-Oncology (CIO) staff; M. Copersino (Regulatory Affairs), B. Meyers, C. Harvey, and S. Bartel (Clinical Pharmacy); A. Lako (CIO), M. Bowden (Center for Molecular Oncologic Pathology); O. Sturtevant, H. Negre, S. Y. Kim, M. A. Kelley (Cell Manipulation Core Facility) and the Pasquarello Tissue Bank (all at DFCI); T. Bowman (DFHCC Specialized Histopathology Core Laboratory); the Broad Institute’s Biological Samples, Genetic Analysis, and Genome Sequencing Platforms; S. Hodi, G. Dranoff, M. Rajasagi, U. Burkhardt, S. Sarkizova, J. Fan, and P. Bachireddy for discussions; J. Petricciani and M. Krane for regulatory advice; B. McDonough (CSBio) and S. Thorne (CuriRx) for peptide development. This research was made possible by a gift from the Blavatnik Family Foundation, and was supported by grants from the Broad Institute SPARC program and the National Institutes of Health (NCI-1RO1CA155010-02 (to C.J.W.), NHLBI-5R01HL103532-03 (to C.J.W.), NCI-SPORE-2P50CA101942-11A1 (to D.B.K.); NCI-R50 RCA211482A (to S.S.)), from the Francis and Adele Kittredge Family Immuno-Oncology and Melanoma Research Fund (to P.A.O.), the Faircloth Family Research Fund (to P.A.O.), and the DFCI Center for Cancer Immunotherapy Research fellowship (to Z.H.). C.J.W. is a scholar of the Leukemia and Lymphoma Society.
Author information
Author notes
- Edward F. Fritsch
Present address: Neon Therapeutics, Inc., Cambridge, Massachusetts, 02139, USA - Patrick A. Ott and Zhuting Hu: These authors contributed equally to this work.
Authors and Affiliations
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, 02215, Massachusetts, USA
Patrick A. Ott, Zhuting Hu, Derin B. Keskin, Sachet A. Shukla, Jing Sun, David J. Bozym, Wandi Zhang, Christina Chen, Oriol Olive, Heather Daley, Elizabeth I. Buchbinder, Jerome Ritz, Edward F. Fritsch & Catherine J. Wu - Department of Medicine, Brigham and Women’s Hospital, Boston, 02215, Massachusetts, USA
Patrick A. Ott, Elizabeth I. Buchbinder, Jerome Ritz & Catherine J. Wu - Harvard Medical School, Boston, 02215, Massachusetts, USA
Patrick A. Ott, Derin B. Keskin, Elizabeth I. Buchbinder, Charles H. Yoon, Dan H. Barouch, Jon C. Aster, Gad Getz, Kai Wucherpfennig, Jerome Ritz, Eric S. Lander, Nir Hacohen & Catherine J. Wu - Broad Institute of MIT and Harvard, Cambridge, 02142, Massachusetts, USA
Derin B. Keskin, Sachet A. Shukla, Todd A. Carter, Shuqiang Li, David J. Lieb, Thomas Eisenhaure, Maegan Harden, Niall Lennon, Stacey Gabriel, Gad Getz, Eric S. Lander, Edward F. Fritsch, Nir Hacohen & Catherine J. Wu - Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, 02215, Massachusetts, USA
Adrienne Luoma & Kai Wucherpfennig - Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, 02215, Massachusetts, USA
Anita Giobbie-Hurder & Donna Neuberg - Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, 02215, Massachusetts, USA
Lauren Peter, Michael Seaman & Dan H. Barouch - Ragon Institute of MGH, MIT, and Harvard, Cambridge, 02139, Massachusetts, USA
Lauren Peter & Dan H. Barouch - Center for Immuno-Oncology (CIO), Dana-Farber Cancer Institute, Boston, 02215, Massachusetts, USA
Evisa Gjini & Scott J. Rodig - Department of Pathology, Brigham and Women’s Hospital, Boston, 02215, Massachusetts, USA
Jonathan Stevens, William J. Lane, Scott J. Rodig & Jon C. Aster - CuriRx, Inc., Wilmington, 01887, Massachusetts, USA
Indu Javeri & Kaliappanadar Nellaiappan - Oncovir, Inc., 3203 Cleveland Avenue, NW, Washington DC, 20008, USA
Andres M. Salazar - Department of Surgery, Brigham and Women’s Hospital, Boston, 02215, Massachusetts, USA
Charles H. Yoon - Department of Pathology, Massachusetts General Hospital, Boston, 02214, Massachusetts, USA
Gad Getz - Center for Cancer Research, Massachusetts General Hospital, Boston, 02214, Massachusetts, USA
Nir Hacohen
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Contributions
P.A.O. was the principal investigator and Investigational New Drug holder. C.J.W., N.H., P.A.O., and E.F.F. directed the overall study design. Z.H. designed and performed experimental and data analysis with D.B.K., D.J.B., W.Z., L.P., C.C., S.L., and D.J.L.; S.A.S., T.A.C., J.S., J.S., W.J.L., and E.F.F. analysed sequencing data and selected neoantigen targets; D.H.B. and M.S. enabled sample collection and immune monitoring; H.D. and J.R. directed vaccine preparation; A.L. and K.W. designed and generated tetramers; A.G.H. and D.N. designed and performed statistical analyses; T.E., A.M.S., I.J., and K.N. helped design the vaccine formulation; O.O. coordinated clinical research; P.A.O., E.I.B., and C.H.Y. provided patient samples; J.C.A., E.G., and S.J.R performed pathology review; M.H., N.L., S.G., and G.G. helped devise the computational pipeline; N.H., C.J.W., E.F.F., T.A.C., and E.S.L. developed the overall program strategy. P.A.O., Z.H., E.F.F., N.H., and C.J.W. wrote the manuscript; all authors discussed and interpreted results.
Corresponding author
Correspondence toCatherine J. Wu.
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Competing interests
E.F.F. is a founder and employee of Neon Therapeutics. N.H. and C.J.W. are founders of Neon Therapeutics and members of its scientific advisory board. P.A.O. has advised Neon Therapeutics. E.S.L. is a founder of Neon Therapeutics and a member of its board of directors. K.N. and I.J. are employees of CuriRx. Patent applications have been filed on aspects of the described work entitled as follows: Compositions and Methods for Personalized Neoplasia Vaccines (N.H., E.F.F., and C.J.W.), Methods for Identifying Tumour Specific Neo-Antigens (N.H. and C.J.W.), Formulations for Neoplasia Vaccines (E.F.F., K.N., and I.J.), and Combination Therapy for Neoantigen Vaccine (N.H., C.J.W., and E.F.F.). S.J.R. receives research funding from Bristol-Myers Squibb, MedImmune, and is on the scientific advisory board for Perkin Elmer. The remaining authors declare no competing financial interests. A.M.S. is a founder and employee of Oncovir, Inc. C.J.W. is subject to a conflict of interest management plan for the reported studies because of her competing financial interests in Neon Therapeutics. Under this plan, C.J.W. may not access identifiable human subjects’ data nor otherwise participate directly in the IRB-approved protocol reported herein. C.J.W.’s contributions to the overall program strategy and data analyses occurred on a de-identified basis.
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Extended data figures and tables
Extended Data Figure 1 Mutational landscape of patient melanoma and radiographic evidence of complete response after anti-PD-1 therapy for patients 2 and 6.
a, Numbers of mutations and predicted epitopes per patient tumour. Red solid lines, patients with vaccines generated and vaccinations completed (n = 6); red dashed lines, vaccines generated but vaccinations not initiated (n = 2); grey, insufficient mutation number for vaccine generation (n = 2). b, Expression (measured and normalized as transcripts per million base pairs (TPM) from the RNA-seq data) of known melanoma-associated genes in the tumour specimens for all ten patients entered into the trial (circles) compared with skin tissue (blue box, (GTEx data)) and melanoma from TCGA (red box; see Methods for analysis). The upper, middle, and lower hinge of the box are 75%, 50%, and 25% quantiles. All points above or below whiskers are the outliers. c, The overall mutational landscape of the eight patient melanoma samples for which immunizing peptides were generated (top, number of mutations per megabase; bottom, distribution of nucleotide changes) and the presence of mutations in genes previously identified as recurrent in melanoma TCGA samples (n = 290) by the MutSig2CV algorithm (middle, doi:10.7908/C1J67GCG; genes ordered on the basis of significance of recurrence reported per TCGA, Supplementary Information 2). d, Patient (Pt.) 2. Left: positron emission tomography/computed tomography (PET/CT) scans obtained 8 weeks after the last booster vaccine demonstrate new intensely fludeoxyglucose F 18 (FDG)-avid right hilar lymphadenopathy measuring ~2.1 cm (maximal standardized uptake value 20.3 (yellow arrow)). Right: PET/CT obtained 12 weeks later, after four doses of pembrolizumab at 2 mg per kg (body weight) given every 3 weeks, revealing complete interval resolution of the right hilar lymphadenopathy consistent with a complete response. Patient 6. Left panels: CT scans of the chest obtained 1 week after the last booster vaccine demonstrate multiple soft-tissue nodules along the left lateral and posterolateral chest wall (left upper panel, yellow arrow indicates a 2.1 cm × 1.8 cm soft-tissue nodule as an example) and a left lower lobe pulmonary nodule (left lower panel, yellow arrow). Right panels: CT scans obtained 16.5 weeks later, after four doses of pembrolizumab at 2 mg per kg (body weight) given every 3 weeks, revealing complete interval resolution of all lesions consistent with a complete response. On the upper panels, note that the liver is partly visualized on the pre-treatment scan (left upper panel) as a result of expirational state and not visualized on the post-treatment scan (right upper panel) as a result of inspirational state. The cross sections visualizing the soft-tissue metastases in the left chest wall versus their resolution after treatment correspond.
Extended Data Figure 2 Polyfunctional neoantigen-specific CD4+ T-cell responses are induced by vaccination in all six patients.
Frequencies of CD4+ T cells secreting cytokines in response to pools of 15- to 16-mer ASP, as measured by ICS after stimulation of PBMCs ex vivo with ASP pools. a, IFN-γ-producing CD4+ T cells detected by flow cytometry before vaccination and at week 16 after vaccination for all six vaccinated patients. Fluorescence-activated cell sorting (FACS) plots were pre-gated on CD4+ T cells. Red values, frequencies of ASP-pool-reactive IFN-γ+ cells as a proportion of all CD4+ T cells. b, Frequencies of IFN-γ-, TNF-α-, and IL-2-producing cells within CD4+ T cells in response to ASP pools tested in samples collected before vaccination (blue) and at week 16 after vaccination (red). c, Pie charts of the total CD4+ T-cell responses positive for one, two, or three cytokines. Bar graphs are shown of absolute frequencies of ASP-pool-reactive CD4+ T cells producing one, two, or three cytokines. d, Median percentage cytokine production across six patients by CD4+ T cells ex vivo against ASP peptide pools.
Extended Data Figure 3 Polyfunctional neoantigen-specific CD8+ T-cell responses are induced by vaccination in all six patients.
Frequencies of CD8+ T cells secreting cytokines in response to pools of 9- to 10-mer predicted class I EPT after a single round of pre-stimulation. PBMCs were cultured with EPT pools for 10–21 days followed by ICS. a, IFN-γ-producing CD8+ T cells detected by flow cytometry pre-vaccination and at week 16 after vaccination for all six vaccinated patients. FACS plots were pre-gated on CD8+ T cells. Percentages shown in red are frequencies of EPT-pool-reactive IFN-γ+ cells as a proportion of all CD8+ T cells. b, IFN-γ responses of CD8+ T cells against EPT pools after one round of pre-stimulation measured by ICS pre-vaccination and at 16 weeks after vaccination. c, Bar graphs show absolute frequencies of EPT-pool-reactive CD8+ T cells producing one, two, or three cytokines. Pie charts summarize the fractions of the total CD8+ T-cell responses positive for one, two, or three cytokines. d, Median percentage cytokine production across six patients by CD8+ T cells after one round of pre-stimulation against EPT peptide pools.
Extended Data Figure 4 Deconvolution of peptide pools to identify neoantigen-specific T-cell responses ex vivo against individual 15- to 16-mer ASP.
Deconvolution of T-cell reactivity against individual ASP by ex vivo IFN-γ ELISPOT using the week 16 post-vaccination PBMCs, tested in duplicate or triplicate wells per peptide (error bars, s.e.m.). *Responses were scored positive if >55 spot-forming cells (SFC) were detected and were at least 1.5 s.d. over the DMSO control (>3 s.d. over background for patients 5 and 6). a, Patient 3 deconvolution. Inset: examples of positive IMP/ASP sequences; red, ASP stimulating reactivity; boxed, the mutated amino acids. b, Deconvolution for all other patients. c, IFN-γ ELISPOT response of patient 3 _CIT_-specific CD8+ T-cell line from week 16 (7 × 104 CD8+ T cells per well) against mutated (Mut) versus corresponding wild-type peptide across a range of concentrations (10 pg ml−1 to 10 μg ml−1) pulsed on 1 × 104 T-cell-depleted PBMCs (hereafter referred to as APCs) per well.
Extended Data Figure 5 Mapping of CD4+ and CD8+ T-cell responses to individual ASP and EPT to the IMP for all six patients.
ASP and EPT covering the IMP are shown for the IMP that induced T-cell responses. T cells from week 16 PBMCs were tested. Red bold and shading: mutated amino acids, absent in those IMP arising from neoORFs. Blue underline: for class I epitopes, predicted epitopes (IC50 < 300 nM) based on NetMHCpan36,45; for class II epitopes, predicted epitopes <10th percentile based on the Immune Epitope Database and Analysis Resource (IEDB)-recommended consensus approach combining NN-align, SMM-align, and CombLib if allele predictions are available, otherwise NetMHCIIpan (Supplementary Information 6)45. Red font, peptides that generated an ex vivo CD4+ T-cell response; blue font, peptides that generated a T-cell response after one round of pre-stimulation with peptides, Triangle, ASP- or EPT-specific T cells that also recognized a corresponding mutated minigene; *T-cell responses against minigenes that were blocked by pan anti-HLA-DR or anti-HLA class I blocking antibodies for CD4+ or CD8+ T cells, respectively.
Extended Data Figure 6 IFN-γ response of pre- and post-vaccination PBMCs against individual ASP, and IFN-γ response and CD107αβ degranulation by neoantigen-specific T-cell response against expressed minigenes.
PBMCs from week 16 after vaccination were pre-stimulated with ASP and EPT for measuring CD4+ and CD8+ T-cell responses, respectively. a, Detection of IFN-γ responses by ELISPOT of CD4+ T cells against individual ASP after one round of pre-stimulation with ASP pools in matched pre- and 16-week post-vaccination samples for patients 1–6 (1 × 104 APCs and 1 × 104 T cells per well), tested in duplicate or triplicate wells per peptide (error bars, s.e.m.). b, Autologous B cells were nucleofected with in vitro translated RNA generated from mutated (Mut) or wild-type minigenes (MG). T-cell lines were cultured with mutated or wild-type minigene nucleofected B cells, mutated minigene nucleofected B cells with anti-HLA class I or anti-HLA-DR antibodies, and non-nucleofected B cells with DMSO in IFN-γ ELISPOT (8 × 104 B cells per well were plated with 5 × 103 T cells per well and 1 × 104 CD8+ T cells per well for CD4+ and CD8+ T-cell response detection, respectively), tested in duplicate or triplicate wells per each condition (error bars, s.e.m.). c, T-cell lines were cultured with mutated minigene nucleofected B cells or non-nucleofected B cells followed by CD107αβ degranulation assay. Representative FACS plot of patient 3 CD107αβ degranulation assay for _VPS16_-specific CD8+ T-cell lines (top) and other neoantigen-specific CD4+ and CD8+ T-cell lines (bottom) from week 16 against autologous B cells nucleofected or not with mutated minigenes. FACS plots were pre-gated on CD8+ T cells.
Extended Data Figure 7 Surface expression of HLA class I and class II on patient melanoma cell lines and originally resected tumour.
a, Flow cytometric staining of autologous melanoma cell lines generated from primary tumour samples with anti-class I and anti-class II or isotype antibodies. b, Dual chromogenic immunohistochemical staining of excised FFPE tumours (see Methods for details). Representative images of positive staining for HLA class I (patients 4 and 2) and HLA class II (patient 6) and negative staining for class II (patient 2). Red, melanoma transcription factor SOX10; brown, HLA class I or class II. c, Summary of immunohistochemical results of five patients with available FFPE tissue. Semi-quantitative scoring was performed for the intensity of positive staining of melanoma cell membranes for class I or II (0, negative; 1, weak; 2, moderate; 3, strong) and for the percentage of positive staining malignant cells (0–100%). A cumulative H score was obtained by multiplying intensity score by the percentage of malignant cells with positive staining.
Extended Data Figure 8 Repertoire of neoantigen-specific T-cell responses persists and broadens following PD-1 blockade, and summary of all CD4+ and CD8+ T-cell responses against neoantigens across the six patients.
a, Persistent or new responses to mutated peptides after PD-1 blockade (pembrolizumab) are shown. PBMCs from patients 2 and 6 (before vaccination, week 16 after vaccination, or 11.5 months (patient 2) or 8.5 months (patient 6) after initiation of pembrolizumab therapy) were pre-stimulated for 21 days with ASP or EPT pools, and reactivity against individual ASP and EPT was then tested by IFN-γ ELISPOT. Ten thousand APCs per well were plated with 1 × 104 T-cell lines per well and 1 × 104 CD8+ T-cell lines per well for CD4+ and CD8+ T-cell response detection, respectively, tested in duplicate or triplicate wells per peptide (error bars, s.e.m.). Each line represents the IFN-γ response against a single ASP or EPT tested per immunizing peptide. Number for each gene: total number of ASP or EPT tested per immunizing peptide. b, The rings, from outer to inner, show the following: (1) predicted affinity of peptide for HLA; heat map scaled from darkest (lowest half-maximal inhibitory concentration (IC50) (nM), or highest affinity) to lightest (highest IC50 (nM), or lowest affinity); (2) level of transcript expression of the tumour, scaled from darkest (highest number of transcripts per million reads) mapped to lightest (lowest number of transcripts per million reads); (3) reactivity to peptide-pulsed autologous APCs; (4) reactivity to B cells nucleofected with minigenes; (5) reactivity to cultured autologous melanoma cells. c, d, Ex vivo class II tetramer staining of patient 1 CD4+ T cells. Visualization of transcriptomes of single CD4+ T cells before vaccination and tetramer-positive CD4+ T cells after vaccination by _t_-distributed stochastic neighbour embedding (_t_-SNE).
Extended Data Table 1 Characteristics of patients
Extended Data Table 2 Class II tetramer information
Supplementary information
Supplementary Table 1
This table contains QC metrics of whole-exome sequencing and RNA-sequencing for Patients 1-10. Sheet a contains whole exome sequencing. Sheet b contains RNA sequencing. (XLSX 14 kb)
Supplementary Table 2
This table contains somatic mutations identified from Patients 1-10. (XLSX 1128 kb)
Supplementary Table 3
This table contains a summary of the number of identified somatic mutations, predicted HLA binders and synthesized immunizing peptides for Patients 1-10. (XLSX 10 kb)
Supplementary Table 4
This table contains patient information. Sheet a contains HLA allotypes of all subjects. Sheet b contains treatment-related adverse events. (XLSX 29 kb)
Supplementary Table 5
This table contains a summary of expression and class I prediction related to the immunizing peptides for Patients 1-6. (XLSX 44 kb)
Supplementary Table 6
This table a summary of class II prediction related to the immunizing peptides for Patients 1-6. (XLSX 36 kb)
Supplementary Table 7
This table contains differential analysis of single cell gene expression of CD4+ T cells pre-vaccination and tetramer-positive CD4+ T cells post-vaccination for Patients 1 and 4. Sheet a contains Patient 1, sheet b contains Patient 4, sheet c contains Patient 1 and 4 and sheet d Patient 1 and 4 intersection. (XLSX 163 kb)
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Ott, P., Hu, Z., Keskin, D. et al. An immunogenic personal neoantigen vaccine for patients with melanoma.Nature 547, 217–221 (2017). https://doi.org/10.1038/nature22991
- Received: 16 February 2017
- Accepted: 16 May 2017
- Published: 05 July 2017
- Issue Date: 13 July 2017
- DOI: https://doi.org/10.1038/nature22991