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Cancer Exome-Based Identification of Tumor Neo-Antigens Using Mass Spectrometry

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Cancer Immunosurveillance

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1884))

Abstract

Neo-antigens expressed on tumors are targets for development of cancer immunotherapy strategies. Use of prediction algorithms to identify neo-antigens yields a significant number of peptides that must be validated in laborious and time-consuming methods; many prove to be false-positive identifications. The use of HLA peptidomics allows the isolation of the HLA-peptide complexes directly from cells and can be done on fresh tumor, patient-derived xerographs, or cell lines when the tissue sample is limited. This method can be used to identify both HLA class I and HLA class II or any different MHC from different species. Here we describe the steps to create the immune-affinity columns used from the process, the immunoprecipitation procedure, and also the isolation of the peptides that will be analyzed by mass spectrometry.

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References

  1. Klebanoff CA, Rosenberg SA, Restifo NP (2016) Prospects for gene-engineered T cell immunotherapy for solid cancers. Nat Med 22(1):26–36. https://doi.org/10.1038/nm.4015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Tran E, Robbins PF, Rosenberg SA (2017) ‘Final common pathway’ of human cancer immunotherapy: targeting random somatic mutations. Nat Immunol 18(3):255–262. https://doi.org/10.1038/ni.3682

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Schumacher TN, Schreiber RD (2015) Neoantigens in cancer immunotherapy. Science 348(6230):69–74. https://doi.org/10.1126/science.aaa4971

    Article  CAS  PubMed  Google Scholar 

  4. Ott PA, Hu Z, Keskin DB, Shukla SA, Sun J, Bozym DJ, Zhang W, Luoma A, Giobbie-Hurder A, Peter L, Chen C, Olive O, Carter TA, Li S, Lieb DJ, Eisenhaure T, Gjini E, Stevens J, Lane WJ, Javeri I, Nellaiappan K, Salazar AM, Daley H, Seaman M, Buchbinder EI, Yoon CH, Harden M, Lennon N, Gabriel S, Rodig SJ, Barouch DH, Aster JC, Getz G, Wucherpfennig K, Neuberg D, Ritz J, Lander ES, Fritsch EF, Hacohen N, Wu CJ (2017) An immunogenic personal neoantigen vaccine for patients with melanoma. Nature 547(7662):217–221. https://doi.org/10.1038/nature22991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Sahin U, Derhovanessian E, Miller M, Kloke BP, Simon P, Lower M, Bukur V, Tadmor AD, Luxemburger U, Schrors B, Omokoko T, Vormehr M, Albrecht C, Paruzynski A, Kuhn AN, Buck J, Heesch S, Schreeb KH, Muller F, Ortseifer I, Vogler I, Godehardt E, Attig S, Rae R, Breitkreuz A, Tolliver C, Suchan M, Martic G, Hohberger A, Sorn P, Diekmann J, Ciesla J, Waksmann O, Bruck AK, Witt M, Zillgen M, Rothermel A, Kasemann B, Langer D, Bolte S, Diken M, Kreiter S, Nemecek R, Gebhardt C, Grabbe S, Holler C, Utikal J, Huber C, Loquai C, Tureci O (2017) Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature 547(7662):222–226. https://doi.org/10.1038/nature23003

    Article  CAS  PubMed  Google Scholar 

  6. Tran E, Robbins PF, Lu YC, Prickett TD, Gartner JJ, Jia L, Pasetto A, Zheng Z, Ray S, Groh EM, Kriley IR, Rosenberg SA (2016) T-Cell transfer therapy targeting mutant KRAS in cancer. N Engl J Med 375(23):2255–2262. https://doi.org/10.1056/NEJMoa1609279

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lu YC, Robbins PF (2016) Cancer immunotherapy targeting neoantigens. Semin Immunol 28(1):22–27. https://doi.org/10.1016/j.smim.2015.11.002

    Article  CAS  PubMed  Google Scholar 

  8. Kalaora S, Barnea E, Merhavi-Shoham E, Qutob N, Teer JK, Shimony N, Schachter J, Rosenberg SA, Besser MJ, Admon A, Samuels Y (2016) Use of HLA peptidomics and whole exome sequencing to identify human immunogenic neo-antigens. Oncotarget 7(5):5110–5117. https://doi.org/10.18632/oncotarget.6960

    Article  PubMed  PubMed Central  Google Scholar 

  9. Bassani-Sternberg M, Braunlein E, Klar R, Engleitner T, Sinitcyn P, Audehm S, Straub M, Weber J, Slotta-Huspenina J, Specht K, Martignoni ME, Werner A, Hein R, D HB, Peschel C, Rad R, Cox J, Mann M, Krackhardt AM (2016) Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry. Nat Commun 7:13404. https://doi.org/10.1038/ncomms13404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yadav M, Jhunjhunwala S, Phung QT, Lupardus P, Tanguay J, Bumbaca S, Franci C, Cheung TK, Fritsche J, Weinschenk T, Modrusan Z, Mellman I, Lill JR, Delamarre L (2014) Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing. Nature 515(7528):572–576. https://doi.org/10.1038/nature14001

    Article  CAS  PubMed  Google Scholar 

  11. Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J, Arthur CD, Krebber WJ, Mulder GE, Toebes M, Vesely MD, Lam SS, Korman AJ, Allison JP, Freeman GJ, Sharpe AH, Pearce EL, Schumacher TN, Aebersold R, Rammensee HG, Melief CJ, Mardis ER, Gillanders WE, Artyomov MN, Schreiber RD (2014) Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature 515(7528):577–581. https://doi.org/10.1038/nature13988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tran E, Turcotte S, Gros A, Robbins PF, Lu YC, Dudley ME, Wunderlich JR, Somerville RP, Hogan K, Hinrichs CS, Parkhurst MR, Yang JC, Rosenberg SA (2014) Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 344(6184):641–645. https://doi.org/10.1126/science.1251102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tran E, Ahmadzadeh M, Lu YC, Gros A, Turcotte S, Robbins PF, Gartner JJ, Zheng Z, Li YF, Ray S, Wunderlich JR, Somerville RP, Rosenberg SA (2015) Immunogenicity of somatic mutations in human gastrointestinal cancers. Science 350(6266):1387–1390. https://doi.org/10.1126/science.aad1253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Robbins PF, Lu YC, El-Gamil M, Li YF, Gross C, Gartner J, Lin JC, Teer JK, Cliften P, Tycksen E, Samuels Y, Rosenberg SA (2013) Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med 19(6):747–752. https://doi.org/10.1038/nm.3161

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Cohen CJ, Gartner JJ, Horovitz-Fried M, Shamalov K, Trebska-McGowan K, Bliskovsky VV, Parkhurst MR, Ankri C, Prickett TD, Crystal JS, Li YF, El-Gamil M, Rosenberg SA, Robbins PF (2015) Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes. J Clin Invest 125(10):3981–3991. https://doi.org/10.1172/JCI82416

    Article  PubMed  PubMed Central  Google Scholar 

  16. Stronen E, Toebes M, Kelderman S, van Buuren MM, Yang W, van Rooij N, Donia M, Boschen ML, Lund-Johansen F, Olweus J, Schumacher TN (2016) Targeting of cancer neoantigens with donor-derived T cell receptor repertoires. Science 352(6291):1337–1341. https://doi.org/10.1126/science.aaf2288

    Article  CAS  PubMed  Google Scholar 

  17. Ishihama Y, Rappsilber J, Andersen JS, Mann M (2002) Microcolumns with self-assembled particle frits for proteomics. J Chromatogr A 979(1–2):233–239

    Article  CAS  PubMed  Google Scholar 

  18. Mrakovcic M, Meindl C, Roblegg E, Frohlich E (2014) Reaction of monocytes to polystyrene and silica nanoparticles in short-term and long-term exposures. Toxicol Res (Camb) 3(2):86–97. https://doi.org/10.1039/C3TX50112D

    Article  CAS  Google Scholar 

  19. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372. https://doi.org/10.1038/nbt.1511

    Article  CAS  PubMed  Google Scholar 

  20. Hoof I, Peters B, Sidney J, Pedersen LE, Sette A, Lund O, Buus S, Nielsen M (2009) NetMHCpan, a method for MHC class I binding prediction beyond humans. Immunogenetics 61(1):1–13. https://doi.org/10.1007/s00251-008-0341-z

    Article  CAS  PubMed  Google Scholar 

  21. Nielsen M, Andreatta M (2016) NetMHCpan-3.0; improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length datasets. Genome Med 8(1):33. https://doi.org/10.1186/s13073-016-0288-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

YS is supported by the Israel Science Foundation grant number 696/17. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 754282), the ERC (StG-335377), the MRA (#402024), the Knell Family and the Hamburger Family , by the Henry Chanoch Krenter Institute for Biomedical Imaging and Genomics and the estates of Alice Schwarz-Gardos and John Hunter.

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Authors and Affiliations

  1. Molecular Cell Biology Department, Weizmann Institute of Science, Rehovot, Israel

    Shelly Kalaora & Yardena Samuels

Authors
  1. Shelly Kalaora
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  2. Yardena Samuels
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Correspondence to Yardena Samuels .

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Editors and Affiliations

  1. Departamento de Biología Funcional, Inmunología, Universidad de Oviedo, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Oviedo, Spain

    Alejandro López-Soto

  2. Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Oviedo, Asturias, Spain

    Alicia R. Folgueras

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Kalaora, S., Samuels, Y. (2019). Cancer Exome-Based Identification of Tumor Neo-Antigens Using Mass Spectrometry. In: López-Soto, A., Folgueras, A. (eds) Cancer Immunosurveillance. Methods in Molecular Biology, vol 1884. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8885-3_14

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  • DOI: https://doi.org/10.1007/978-1-4939-8885-3_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8884-6

  • Online ISBN: 978-1-4939-8885-3

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