In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo

  1. Gerald Willimsky  Is a corresponding author
  2. Christin Beier
  3. Lena Immisch
  4. Georgios Papafotiou
  5. Vivian Scheuplein
  6. Andrean Goede
  7. Hermann-Georg Holzhütter
  8. Thomas Blankenstein
  9. Peter M Kloetzel  Is a corresponding author
  1. Institute of Immunology (Charité - Universitätsmedizin Berlin) and German Cancer Research Center (DKFZ Heidelberg), Germany
  2. Institute of Biochemistry (Charité - Universitätsmedizin Berlin), Germany
  3. Max-Delbrück Center for Molecular Medicine, Germany
  4. Institut für Physiologie (Charité - Universitätsmedizin Berlin), Germany
  5. Max Delbrück Center for Molecular Medicine in Helmholtz Association, Germany
  6. Charité - Universitätsmedizin Berlin, Germany

Abstract

Proteasome catalyzed peptide splicing (PCPS) of cancer-driving antigens could generate attractive neoepitopes to be targeted by TCR-based adoptive T cell therapy. Based on a spliced peptide prediction algorithm TCRs were generated against putative KRASG12V and RAC2P29L derived neo-splicetopes with high HLA-A*02:01 binding affinity. TCRs generated in mice with a diverse human TCR repertoire specifically recognized the respective target peptides with high efficacy. However, we failed to detect any neo-splicetope specific T cell response when testing the in vivo neo-splicetope generation and obtained no experimental evidence that the putative KRASG12V- and RAC2P29L-derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2P29L-derived neo-splicetopes was generated by in vitro PCPS. The experiments pose severe questions on the notion that available algorithms or the in vitro PCPS reaction reliably simulate in vivo splicing and argue against the general applicability of an algorithm-driven 'reverse immunology' pipeline for the identification of cancer-specific neo-splicetopes.

Data availability

Additional source data comprising databases for ProteomDiscoverer, Kras/RAC2 kinetics, cleavage maps and PD2.1 result files have been submitted to Dryad under DOI:10.5061/dryad.jq2bvq88b

The following data sets were generated

Article and author information

Author details

  1. Gerald Willimsky

    Experimental and Translational Cancer Immunology, Institute of Immunology (Charité - Universitätsmedizin Berlin) and German Cancer Research Center (DKFZ Heidelberg), Berlin, Germany
    For correspondence
    gerald.willimsky@charite.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9693-948X
  2. Christin Beier

    Biochemistry, Institute of Biochemistry (Charité - Universitätsmedizin Berlin), Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Lena Immisch

    Experimental and Translational Cancer Immunology, Institute of Immunology (Charité - Universitätsmedizin Berlin) and German Cancer Research Center (DKFZ Heidelberg), Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Georgios Papafotiou

    Experimental and Translational Cancer Immunology, Institute of Immunology (Charité - Universitätsmedizin Berlin) and German Cancer Research Center (DKFZ Heidelberg), Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Vivian Scheuplein

    Molecular Immunology, Max-Delbrück Center for Molecular Medicine, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Andrean Goede

    Physiologie (, Institut für Physiologie (Charité - Universitätsmedizin Berlin), Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9044-9869
  7. Hermann-Georg Holzhütter

    Biochemistry, Institute of Biochemistry (Charité - Universitätsmedizin Berlin), Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Thomas Blankenstein

    Max Delbrück Center for Molecular Medicine in Helmholtz Association, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Peter M Kloetzel

    Institut für Biochemie, Charité - Universitätsmedizin Berlin, Berlin, Germany
    For correspondence
    p-m.kloetzel@charite.de
    Competing interests
    The authors declare that no competing interests exist.

Funding

Deutsche Forschungsgemeinschaft (SFB-TR36)

  • Gerald Willimsky
  • Thomas Blankenstein

Deutsche Krebshilfe (111546)

  • Gerald Willimsky

Berlin Institute of Health (CRG-1)

  • Thomas Blankenstein
  • Peter M Kloetzel

DKTK joint funding (NEO-ATT)

  • Gerald Willimsky

Berliner Krebsgesellschaft

  • Peter M Kloetzel

Helmholtz-Gemeinschaft, Zukunftsthema 'Immunology and Inflammation'

  • Gerald Willimsky
  • Thomas Blankenstein

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal experiments were performed according to institutional and national guidelines and regulations. The experiments were approved by the governmental authority (Landesamt für Gesundheit und Soziales, Berlin, H0086/16).

Reviewing Editor

  1. Vincenzo Cerullo, University of Helsinki, Finland

Publication history

  1. Received: August 11, 2020
  2. Accepted: April 15, 2021
  3. Accepted Manuscript published: April 20, 2021 (version 1)
  4. Accepted Manuscript updated: April 22, 2021 (version 2)
  5. Version of Record published: May 26, 2021 (version 3)

Copyright

© 2021, Willimsky et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Gerald Willimsky
  2. Christin Beier
  3. Lena Immisch
  4. Georgios Papafotiou
  5. Vivian Scheuplein
  6. Andrean Goede
  7. Hermann-Georg Holzhütter
  8. Thomas Blankenstein
  9. Peter M Kloetzel
(2021)
In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo
eLife 10:e62019.
https://doi.org/10.7554/eLife.62019
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