Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma

Aviyah Peri; Erez Greenstein; Michal Alon; Joy A. Pai; Tamir Dingjan; Shlomit Reich-Zeliger; Eilon Barnea; Chaya Barbolin; Ronen Levy; Claudia Arnedo-Pac; Shelly Kalaora; Bareket Dassa; Ester Feldmesser; Ping Shang; Polina Greenberg; Yishai Levin; Gil Benedek; Mitchell P. Levesque; David J. Adams; Michal Lotem; James S. Wilmott; Richard A. Scolyer; Göran B. Jönsson; Arie Admon; Steven A. Rosenberg; Cyrille J. Cohen; Masha Y. Niv; Nuria Lopez-Bigas; Ansuman T. Satpathy; Nir Friedman; Yardena Samuels

doi:10.1172/JCI129466

Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma

Aviyah Peri, Erez Greenstein, Michal Alon, Joy A. Pai, Tamir Dingjan, Shlomit Reich-Zeliger, Eilon Barnea, Chaya Barbolin, Ronen Levy, Claudia Arnedo-Pac, Shelly Kalaora, Bareket Dassa, Ester Feldmesser, Ping Shang, Polina Greenberg, Yishai Levin, Gil Benedek, Mitchell P. Levesque, David J. Adams, Michal LotemJames S. Wilmott, Richard A. Scolyer, Göran B. Jönsson, Arie Admon, Steven A. Rosenberg, Cyrille J. Cohen, Masha Y. Niv, Nuria Lopez-Bigas, Ansuman T. Satpathy, Nir Friedman, Yardena Samuels

The Mina and Everard Goodman Faculty of Life Sciences

Research output: Contribution to journal › Article › peer-review

20 Scopus citations

Abstract

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-theshelf" precision immunotherapies, alleviating limitations of personalized treatments.

Original language	English
Article number	e129466
Journal	Journal of Clinical Investigation
Volume	131
Issue number	20
DOIs	https://doi.org/10.1172/JCI129466
State	Published - 15 Oct 2021

Bibliographical note

Publisher Copyright:
© 2021 American Society for Clinical Investigation. All rights reserved.

Funding

We acknowledge the NIH Tetramer Facility for their help in producing the tetramers. This publication and the underlying research were partly facilitated by TCGA Research Network, the Hartwig Medical Foundation, and the Center for Personalized Cancer Treatment (CPCT), which generated, analyzed, and made available data for this purpose (32, 33). We are grateful to Astar Shamul and Aviad Pato for teaching us the TCR cloning and electropora-tion procedures and to Zelig Eshhar (The Weizmann Institute of Science, Rehovot, Israel and Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel) and Ugur Sahin (Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Mainz, Germany) for providing the pGEM-4Z-EGFP-A64 plasmid and the MZ2-mel cell line, respectively. This work was supported by the Intramural Research Programs of the NCI and by a National Health and Medical Research Council of Australia (NHMRC) Program Grant (APP1093017, to RAS). YS is supported by the European Union’s Horizon 2020 Research and Innovation Program (no. 770854); ERC-2017-CoG (no. 754282); MRA (no. 622106); The Rising Tide Foundation; the Garvan Institute; the Graf Family Foundation; the Green Family Charitable Foundation; and the Knell Family and Hamburger Family. MYN and YS are supported by grants from the Israel Science Foundation (nos. 494/16, ISF-NSFC 2463/16, and 696/17, respectively). ATS is supported by NIH grant K08CA230188; the Parker Institute for Cancer Immunotherapy; a Technology Impact Award from the Cancer Research Institute; and a Career Award for Medical Scientists from the Burroughs Wellcome Fund. JAP is supported by NIH T32AI007290. RAS is supported by an NHMRC Practitioner Fellowship (APP1141295). Support from colleagues at the Melanoma Institute Australia and the Royal Prince Alfred Hospital and investigators of the Australian Melanoma Genome Project is gratefully acknowledged.

Funders	Funder number
ERC-2017-CoG	754282
Graf Family Foundation
Green Family Charitable Foundation
Knell Family and Hamburger Family
National Institutes of Health
National Cancer Institute	K08CA230188
Burroughs Wellcome Fund	APP1141295, T32AI007290
Cancer research institute
Melanoma Research Alliance	622106
Horizon 2020 Framework Programme	770854
Parker Institute for Cancer Immunotherapy
National Health and Medical Research Council	APP1093017
Garvan Institute of Medical Research
Israel Science Foundation	696/17, 494/16, ISF-NSFC 2463/16
Rising Tide Foundation

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Peri, A., Greenstein, E., Alon, M., Pai, J. A., Dingjan, T., Reich-Zeliger, S., Barnea, E., Barbolin, C., Levy, R., Arnedo-Pac, C., Kalaora, S., Dassa, B., Feldmesser, E., Shang, P., Greenberg, P., Levin, Y., Benedek, G., Levesque, M. P., Adams, D. J., ... Samuels, Y. (2021). Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma. Journal of Clinical Investigation, 131(20), Article e129466. https://doi.org/10.1172/JCI129466

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abstract = "Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote {"}off-theshelf{"} precision immunotherapies, alleviating limitations of personalized treatments.",

author = "Aviyah Peri and Erez Greenstein and Michal Alon and Pai, {Joy A.} and Tamir Dingjan and Shlomit Reich-Zeliger and Eilon Barnea and Chaya Barbolin and Ronen Levy and Claudia Arnedo-Pac and Shelly Kalaora and Bareket Dassa and Ester Feldmesser and Ping Shang and Polina Greenberg and Yishai Levin and Gil Benedek and Levesque, {Mitchell P.} and Adams, {David J.} and Michal Lotem and Wilmott, {James S.} and Scolyer, {Richard A.} and J{\"o}nsson, {G{\"o}ran B.} and Arie Admon and Rosenberg, {Steven A.} and Cohen, {Cyrille J.} and Niv, {Masha Y.} and Nuria Lopez-Bigas and Satpathy, {Ansuman T.} and Nir Friedman and Yardena Samuels",

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Peri, A, Greenstein, E, Alon, M, Pai, JA, Dingjan, T, Reich-Zeliger, S, Barnea, E, Barbolin, C, Levy, R, Arnedo-Pac, C, Kalaora, S, Dassa, B, Feldmesser, E, Shang, P, Greenberg, P, Levin, Y, Benedek, G, Levesque, MP, Adams, DJ, Lotem, M, Wilmott, JS, Scolyer, RA, Jönsson, GB, Admon, A, Rosenberg, SA, Cohen, CJ, Niv, MY, Lopez-Bigas, N, Satpathy, AT, Friedman, N & Samuels, Y 2021, 'Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma', Journal of Clinical Investigation, vol. 131, no. 20, e129466. https://doi.org/10.1172/JCI129466

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T1 - Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma

AU - Peri, Aviyah

AU - Greenstein, Erez

AU - Alon, Michal

AU - Pai, Joy A.

AU - Dingjan, Tamir

AU - Reich-Zeliger, Shlomit

AU - Barnea, Eilon

AU - Barbolin, Chaya

AU - Levy, Ronen

AU - Arnedo-Pac, Claudia

AU - Kalaora, Shelly

AU - Dassa, Bareket

AU - Feldmesser, Ester

AU - Shang, Ping

AU - Greenberg, Polina

AU - Levin, Yishai

AU - Benedek, Gil

AU - Levesque, Mitchell P.

AU - Adams, David J.

AU - Lotem, Michal

AU - Wilmott, James S.

AU - Scolyer, Richard A.

AU - Jönsson, Göran B.

AU - Admon, Arie

AU - Rosenberg, Steven A.

AU - Cohen, Cyrille J.

AU - Niv, Masha Y.

AU - Lopez-Bigas, Nuria

AU - Satpathy, Ansuman T.

AU - Friedman, Nir

AU - Samuels, Yardena

PY - 2021/10/15

Y1 - 2021/10/15

N2 - Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-theshelf" precision immunotherapies, alleviating limitations of personalized treatments.

AB - Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-theshelf" precision immunotherapies, alleviating limitations of personalized treatments.

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Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma

Abstract

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