Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens

ABSTRACT The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts

tumour cell immunogenicity1,2,3,4,5,6, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion1,2,7. Yet, clinically apparent cancers

still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte

associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells8,9. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1

(checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies10,11,12,13. However, little is known about the identity

of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly

tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or

anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection

comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with

anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal

that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe

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IMMUNOTHERAPY Article 24 June 2020 ANTIGEN PRESENTATION IN CANCER: INSIGHTS INTO TUMOUR IMMUNOGENICITY AND IMMUNE EVASION Article 09 March 2021 REGULATORY T CELL-MEDIATED IMMUNOSUPPRESSION

ORCHESTRATED BY CANCER: TOWARDS AN IMMUNO-GENOMIC PARADIGM FOR PRECISION MEDICINE Article 29 February 2024 ACCESSION CODES PRIMARY ACCESSIONS GENE EXPRESSION OMNIBUS * GSE62771 DATA DEPOSITS

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Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to K. Murphy for the _Batf3__−/−_ mice, T. Hansen for providing MHC class I antibodies and the H-2Kb construct, D.

Fremont for the human β2m construct, and the National Institutes of Health (NIH) Tetramer Core Facility for producing MHC class I tetramers. We also thank R. Ahmed and M. Hashimoto for the

multiplex staining strategy used to define functional and dysfunctional T cells. We thank A. Bensimon, O. Schubert and P. Kouvonen for instrument maintenance and for technical support with

the mass spectrometry measurements and R. Vanganipuram, M. Selby and J. Valle for generating and supplying anti-PD-1 and anti-CTLA-4 in endotoxin-free sterile form. We also thank K. Sheehan,

P. Allen, G. Dunn and R. Chan for constructive criticisms and comments, all members of the Schreiber laboratory for discussions, and the many members of The Genome Institute at Washington

University School of Medicine. We would also like to thank W. Song for his assistance with the bioinformatics approaches, P. Kvistborg for assistance with tetramer combinatorial coding, and

Christopher Nelson for advice with peptide-MHC monomer purification. This work was supported by grants to R.D.S. from the National Cancer Institute (RO1 CA043059, U01 CA141541), the Cancer

Research Institute and the WWW.W Foundation; to R.D.S. and W.E.G. from The Siteman Cancer Center/Barnes-Jewish Hospital (Cancer Frontier Fund); to W.E.G. from Susan G. Komen for the Cure

(Promise grant); to E.R.M. from the National Human Genome Research Institute; to G.J.F. from the National Institute of Health (P50 CA101942, P01 AI054456, P50 CA101942); to A.H.S. from the

National Institute of Health (P50 CA101942); and to T.N.S. from the Dutch Cancer Society (Queen Wilhelmina Research Award). E.C. is supported by a Marie Curie Intra-European Fellowship

within the Seventh Framework Programme of the European Community for Research. M.M.G. was supported by a postdoctoral training grant (T32 CA00954729) from the National Cancer Institute and

is currently supported by a postdoctoral training grant (Irvington Postdoctoral Fellowship) from the Cancer Research Institute. Aspects of studies at Washington University were performed

with assistance by the Immunomonitoring Laboratory of the Center for Human Immunology and Immunotherapy Programs and the Siteman Comprehensive Cancer Center. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, Missouri 63110, USA, Matthew M. Gubin, Jeffrey P. Ward, 

Takuro Noguchi, Yulia Ivanova, Cora D. Arthur, Matthew D. Vesely, Samuel S. K. Lam, Erika L. Pearce, Maxim N. Artyomov & Robert D. Schreiber * Department of Surgery, Washington

University School of Medicine, 660 South Euclid Avenue, St Louis, Missouri 63110, USA, Xiuli Zhang & William E. Gillanders * Department of Immunology, Institute of Cell Biology, and

German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ) Partner Site Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany, Heiko Schuster & Hans-Georg Rammensee *

Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland, Etienne Caron & Ruedi Aebersold * Department of Medicine, Division of Oncology,

Washington University School of Medicine, 660 South Euclid Avenue, St Louis, Missouri 63110, USA, Jeffrey P. Ward * The Genome Institute, Washington University School of Medicine, 4444

Forest Park Avenue, St Louis, Missouri 63108, USA, Jasreet Hundal & Elaine R. Mardis * ISA Therapeutics B.V., 2333 CH Leiden, The Netherlands, Willem-Jan Krebber, Gwenn E. Mulder & 

Cornelis J. M. Melief * Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands, Mireille Toebes & Ton N. Schumacher * Bristol-Myers Squibb, 700 Bay

Road, Redwood City, 94063, California, USA Alan J. Korman * Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, 77030, Texas, USA James P. Allison *

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, 02115, Massachusetts, USA Gordon J. Freeman * Department of Microbiology and Immunobiology,

Harvard Medical School, Boston, 02115, Massachusetts, USA Arlene H. Sharpe * Faculty of Science, University of Zurich, Zurich, 8093 Zurich, Switzerland, Ruedi Aebersold * Department of

Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333ZA Leiden, The Netherlands, Cornelis J. M. Melief * Department of Genetics, Washington University School of

Medicine, 660 South Euclid Avenue, St Louis, Missouri 63110, USA, Elaine R. Mardis Authors * Matthew M. Gubin View author publications You can also search for this author inPubMed Google

Scholar * Xiuli Zhang View author publications You can also search for this author inPubMed Google Scholar * Heiko Schuster View author publications You can also search for this author

inPubMed Google Scholar * Etienne Caron View author publications You can also search for this author inPubMed Google Scholar * Jeffrey P. Ward View author publications You can also search

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View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.M.G. and R.D.S. were involved in all aspects of this study including planning and

performing experiments, analysing and interpreting data, and writing the manuscript. X.Z. performed peptide binding experiments, helped design and perform the vaccine experiments. H.S.,

E.C., R.A. and H.-G.R. planned and performed the mass spectrometry analyses, interpreted the data and were involved in writing the manuscript. T.N., J.P.W., C.D.A., M.D.V., S.S.K.L. and

E.L.P., participated in assessing the phenotypes of the tumour-specific T-cell lines, interpreting the data and in writing the manuscript. M.T. helped generate MHC class I multimers. A.J.K.,

J.P.A., G.J.F. and A.H.S. provided mAbs, helped plan the checkpoint blockade therapy experiments, and contributed to writing the manuscript. T.N.S. helped generate MHC class I multimers,

analysed data and was involved in writing the manuscript. W.-J.K., G.E.M. and C.J.M.M. produced and purified the synthetic long peptides, participated in the planning of the vaccine

experiments, analysed data and were involved in writing the manuscript. J.H. and E.R.M. were responsible for genomic analyses and epitope prediction and participated in writing the

manuscript. W.E.G. contributed to the design and analysis of peptide binding and vaccine experiments and in writing the manuscript. Y.I. and M.N.A were responsible for optimizing the epitope

prediction method, performing the RNA-sequencing analyses, analysing data and writing the manuscript. R.D.S. oversaw all the work performed. CORRESPONDING AUTHOR Correspondence to Robert D.

Schreiber. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1 INNATE AND ADAPTIVE IMMUNE

COMPONENTS ARE REQUIRED FOR REJECTION OF D42M1-T3 AFTER CHECKPOINT BLOCKADE THERAPY. A, Cohorts of _Rag2_−/−, _Batf3_−/− or wild-type mice were treated with control mAb, anti-CD4, anti-CD8α

or anti-IFN-γ mAbs and then were injected with 1 × 106 d42m1-T3 tumour cells subcutaneously and subsequently treated with anti-CTLA-4 on days 3, 6 and 9 post-transplant. B, C, d42m1-T3 (B)

or F244 (C) tumour cells were injected subcutaneously into wild-type mice (_n_ = 5) that were subsequently treated with anti-PD-1 on days 3, 6, and 9. Fifty days after tumours were rejected,

mice were rechallenged with d42m1-T3 or F244 tumour cells. Data are presented as average tumour diameter ± s.e.m. of 5 mice per group and are representative of at least two independent

experiments. EXTENDED DATA FIGURE 2 H-2DB MUTANT EPITOPES OF D42M1-T3 TUMOURS. A, Missense mutations in d42m1-T3 were subjected to _in silico_ analysis for the potential to form

H-2Db-binding epitopes using three epitope prediction algorithms. The median predicted epitope-binding affinity for each peptide was calculated and expressed as ‘median affinity value’ where

affinity value = 1/IC50 × 100. Predicted epitopes are arrayed along the _x_-axis in alphabetical order based on their protein of origin. B, Unfiltered median affinity values for the four

predicted H-2Db epitopes. C, Median affinity values of remaining two H-2Db epitopes after filtering. D, Tetramer staining of CD8+ TIL from tumour-bearing mice treated with anti-PD-1 using

H-2Db tetramers loaded with top 4 H-2Db synthetic peptides. E, IFN-γ and TNF-α intracellular cytokine staining of CD8+ TIL from tumour-bearing mice treated with anti-PD-1 immunotherapy

following co-culture with naive irradiated splenocytes pulsed with the top four H-2Db synthetic peptides added at 1 μM final concentration. Data are presented as per cent of CD8+ TIL

positive for IFN-γ, TNF-α or both cytokines. Data are representative of two independent experiments. EXTENDED DATA FIGURE 3 MLAMA4 AND MALG8 STIMULATE CD8+ T CELL LINES GENERATED AGAINST

D42M1-T3 FOLLOWING ANTI-PD-1 IMMUNOTHERAPY. A, CD8+ T cell lines generated from splenocytes of individual d42m1-T3-tumour-bearing mice that rejected their tumours after anti-PD-1 therapy

were incubated with irradiated d42m1-T3 tumour cells (or F244 tumour cells) treated with blocking mAb specific for H-2Kb, and/or H-2Db and IFN-γ production was quantitated. Data are

presented as means ± s.e.m. and are representative of two independent experiments. Samples were compared using an unpaired, two-tailed Student’s _t_ test (***_P_ < 0.001). B, IFN-γ

release by the CTL 74 T cell line following co-culture with naive irradiated splenocytes pulsed with the top 62 H-2Kb synthetic peptides added at 1 μM final concentration. EXTENDED DATA

FIGURE 4 MLAMA4 AND MALG8 BIND H-2KB AND STIMULATE CD8+ T CELL LINES GENERATED AGAINST D42M1-T3 FOLLOWING ANTI-PD-1 IMMUNOTHERAPY. A, IFN-γ release by CTL 62, CTL 73 or CTL 74 T cell lines

following stimulation with naive irradiated splenocytes pulsed with wild-type or mutant forms of Lama4 or Alg8 peptides. B, RMA-S cells were incubated with 8 amino acid peptides of mLama4 or

mAlg8 and surface expression of H-2Kb or H-2Db was assessed by flow cytometry. Mean fluorescent intensity of H-2Kb and H-2Db was expressed as peptide binding score. Data presented are

representative of at least two independent experiments. EXTENDED DATA FIGURE 5 IDENTIFICATION OF A PEPTIDE BOUND TO H-2KB ON D42M1-T3 TUMOUR CELLS CORRESPONDING TO MUTANT LAMA4. A,

Identification of the peptide, VGFNFRTL, corresponding to mLama4 by discovery mass spectrometry. B, Validation of the mLama4 peptide using an isotope-labelled synthetic peptide (VGFNFRTL

(13C6, 15N1)). EXTENDED DATA FIGURE 6 GENERATION OF SRM ASSAY LIBRARY FOR THE DETECTION OF MUTANT H-2KB PEPTIDES ON D42M1-T3. A, SRM transitions were optimized for 51 of the 62 top predicted

H-2Kb peptides. The 51 peptides chosen were selected based on having physiochemical properties that would allow their detection by mass spectrometry if present. Only Lama4 and Alg8 are

shown here for simplicity. The 51 peptides were synthesized and LC-tandem mass spectrometry acquisition was performed on each peptide to determine the best collision energy and to obtain the

full fragment ion spectrum (left panel); three to seven of the highest intensity peaks were selected to be built into SRM transitions. Optimal SRM transitions displayed as extracted ion

chromatograms are shown (right panel). Q1–Q3 transitions are indicated in parenthesis. The mutated amino acid in the peptide sequence is marked in red. B, F244 tumour cells, which lack the

mLama4 and mAlg8 d42m1-T3 epitope, lack detectable mLama4 or mAlg8 in complex with H-2Kb as assessed by SRM. EXTENDED DATA FIGURE 7 CD8+ T CELLS SPECIFIC FOR MUTANT FORMS OF LAMA4 AND ALG8

INFILTRATE D42M1-T3, BUT NOT F244, TUMOURS. A, Detection of tumour-infiltrating mLama4- or mAlg8-specific T cells infiltrating d42m1-T3 or F244 tumours of mice treated with anti-PD-1.

Tumours were harvested on day 12 post-transplant. Cells were gated on live CD45+ and CD8α+ tumour-infiltrating lymphocytes. Detection of mLama4- or mAlg8-specific T cells was achieved by

staining with peptide-MHC H-2Kb PE-labelled tetramers. Data are representative of at least five independent experiments. B, Detection of mLama4-specific tumour-infiltrating T cells from

tumour-bearing mice treated with anti-PD-1, anti-CTLA-4, both anti-PD-1 plus anti-CTLA-4 or control mAb. Detection of mLama4-specific T cells was achieved by staining with mLama4-MHC H-2Kb

PE-labelled tetramers. Data presented are plotted as the mean mLama4 tetramer-positive as a percent of CD8α+ tumour-infiltrating cells and are representative of at least three independent

experiments. EXTENDED DATA FIGURE 8 MALG8 AND MLAMA4 SLP VACCINE CONTROL D42M1-T3 TUMOUR OUTGROWTH WHEN ADMINISTERED THERAPEUTICALLY OR PROPHYLACTICALLY. A, Tumour growth of d42m1-T3 tumours

from mice therapeutically vaccinated with mLama4 and mAlg8 SLP plus poly(I:C), HPV control SLP plus poly(I:C) or poly(I:C) alone. Data shown are mean ± s.e.m. Mutant Lama4 and mAlg8 SLP

vaccine group was compared to HPV control SLP vaccine group using an unpaired, two-tailed Student’s _t_ test (*_P_ < 0.05 and **_P_ < 0.01). B, Kaplan–Meier survival curves of

d42m1-T3-tumour-bearing mice (7 per group) prophylactically vaccinated with SLP vaccines plus poly(I:C). mLama4 plus mAlg8 compared to HPV control: _P_ = 0.0003 (log-rank (Mantel–Cox) test).

Representative of two independent experiments. C, Cumulative number of mice (7–10 per group) from at least two independent experiments rejecting d42m1-T3 or F244 tumours as a consequence of

SLP or minimal epitope peptide prophylactic vaccination. EXTENDED DATA FIGURE 9 DETECTION OF TIM-3, LAG-3, IFN-Γ AND TNF-Α EXPRESSION IN TUMOUR-INFILTRATING CD8+ T CELLS. A, Representative

histogram of TIM-3 or LAG-3 expression on mLama4-specific CD8+ tumour-infiltrating T cells from tumour-bearing mice treated with anti-PD-1, anti-CTLA-4, both anti-PD-1 and anti-CTLA-4 or

control mAbs. B, TIM-3 and LAG-3 are reduced in mAlg8-specific CD8+ TIL from tumour-bearing mice treated with anti-PD-1, anti-CTLA-4, or both anti-PD-1 and anti-CTLA-4 compared to mice

treated with control mAb. _n_ = 5 mice per group pooled. Data are presented as mean ± s.e.m. of at least three independent experiments. Samples were compared using an unpaired, two-tailed

Student’s _t_ test (*_P_ < 0.05, **_P_ < 0.01). C, Representative dot plots of IFN-γ and TNF-α stained CD8+ tumour-infiltrating T cells from tumour-bearing mice following treatment

with anti-PD-1, anti-CTLA-4, both anti-PD-1 and anti-CTLA-4 or control mAbs. Data presented are representative of at least three independent experiments. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY DATA 1 This file contains MS traces for each of the mutant H-2Kb d42m1-T3 epitopes tested by targeted MS. (PDF 1891 kb) SUPPLEMENTARY INFORMATION This file contains

Supplementary Tables 1-3. Supplementary Table 1 contains a complete list of d42m1-T3 H-2Kb-bound peptides identified by discovery MS. Supplementary Table 2 shows all differentially expressed

genes in mLama4-tetramer-positive CD8+ TILs from mice treated with checkpoint blockade therapy compared to mLama4-tetramer-positive CD8+ TILs from control mice. Supplementary Table 3 shows

differentially regulated pathways (GSEA pathway analysis) in mLama4-tetramer-positive CD8+ TILs from mice treated with checkpoint blockade therapy compared to mLama4-tetramer-positive CD8+

TILs from control mice. (PDF 3029 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 RIGHTS AND PERMISSIONS

Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Gubin, M., Zhang, X., Schuster, H. _et al._ Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.

_Nature_ 515, 577–581 (2014). https://doi.org/10.1038/nature13988 Download citation * Received: 14 May 2014 * Accepted: 22 October 2014 * Published: 26 November 2014 * Issue Date: 27

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