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Cancer Immunology, Immunotherapy

, Volume 65, Issue 11, pp 1377–1393 | Cite as

Mass spectrometric analysis of the HLA class I peptidome of melanoma cell lines as a promising tool for the identification of putative tumor-associated HLA epitopes

  • Andreas Gloger
  • Danilo Ritz
  • Tim Fugmann
  • Dario Neri
Original Article

Abstract

Melanoma is one of the most immunogenic tumors, and extensive lists of potential tumor rejection antigens have been collected during the last decades. By isolating human leukocyte antigen (HLA) class I complexes from five melanoma cell lines (FM-82, FM-93/2, Mel-624, MeWo and SK-Mel-5) and sequencing HLA-eluted peptides by mass spectrometry, we identified over 10,000 unique peptides with high confidence. The majority of the peptides were 8–11 amino acids in length and were predicted to bind to the respective HLA alleles. Over 250 epitopes, corresponding to previously described tumor-associated antigens, were identified, suggesting that HLA peptidome analysis may facilitate the characterization of putative tumor rejection antigens. MeWo and SK-Mel-5 cell lines were further interrogated for neo-epitopes, revealing one peptide from MeWo cells carrying an amino acid mutation. We also observed a remarkable overlap between A*03:01 peptides eluted from Mel-624 cells and A*03:01 peptides recovered from soluble HLA complexes purified from two melanoma patients, shedding light on the similarity of the HLA peptidome in cell lines and in patient-derived material. The reliable characterization of the HLA class I peptidome in melanoma promises to facilitate the identification of tumor rejection antigens and the development of immunotherapeutic strategies.

Keywords

HLA Melanoma Immunopeptidome Tumor-associated antigen Mass spectrometry Immunocapture 

Abbreviations

ACN

Acetonitrile

ATCC

American Type Culture Collection

COSMIC

Catalogue of Somatic Mutations in Cancer

ESTDAB

European Searchable Tumour Line Database

ETH Zurich

Eidgenössische Technische Hochschule Zürich

FDR

False discovery rate

FPLC

Fast protein liquid chromatography

HCD

Higher-energy collisional dissociation

HLA

Human leukocyte antigen

IC50

Half maximal inhibitory concentration

LC-MS

Liquid chromatography–mass spectrometry

MS

Mass spectrometry

MS/MS

Fragment mass spectra

PMSF

Phenylmethylsulfonyl fluoride

sHLA

Soluble human leukocyte antigen

UHPLC

Ultra high performance liquid chromatography

WT

Wild type

Notes

Acknowledgments

We thank Camilla Bacci (Philogen SpA) for providing W6/32 antibody.

Funding

This work was supported financially by ETH Zürich, the Swiss National Science Foundation, the European Union’s Seventh Framework Programme (FP7/2007-2013) under Grant Agreement Nos. 305309 (PRIAT) and 305608 (EURenOmics), the Bonveda Foundation and the European Research Council (ERC advanced Grant “ZAUBERKUGEL”).

Compliance with ethical standards

Conflict of interest

Dario Neri is co-founder of Philogen, shareholder and member of the board. Tim Fugmann and Danilo Ritz are employees of Philochem AG. The authors declare no additional conflict of interest.

Research involving human participants and/or animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

262_2016_1897_MOESM1_ESM.pdf (12.8 mb)
Supplementary material 1 (PDF 13113 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Institute of Pharmaceutical SciencesETH ZurichZurichSwitzerland
  2. 2.Philochem AGOtelfingenSwitzerland

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