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

Elife. 2021 Apr 20:10:e62019. doi: 10.7554/eLife.62019.

Abstract

Proteasome-catalyzed peptide splicing (PCPS) of cancer-driving antigens could generate attractive neoepitopes to be targeted by T cell receptor (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.

Keywords: TCR gene therapy; biochemistry; chemical biology; human; immunology; inflammation; mouse; proteasome processing; spliced epitopes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigen Presentation
  • Antigens, Neoplasm / genetics
  • Antigens, Neoplasm / immunology
  • Antigens, Neoplasm / metabolism*
  • CD8-Positive T-Lymphocytes / immunology
  • CD8-Positive T-Lymphocytes / metabolism*
  • Epitopes*
  • HEK293 Cells
  • HLA-A2 Antigen / immunology
  • HLA-A2 Antigen / metabolism
  • Humans
  • K562 Cells
  • Mice
  • Mice, Transgenic
  • Mutation
  • Neoplasms / genetics
  • Neoplasms / immunology
  • Neoplasms / metabolism*
  • Proof of Concept Study
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Processing, Post-Translational*
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Proto-Oncogene Proteins p21(ras) / immunology
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • RAC2 GTP-Binding Protein
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / immunology
  • Receptors, Antigen, T-Cell / metabolism*
  • rac GTP-Binding Proteins / genetics
  • rac GTP-Binding Proteins / immunology
  • rac GTP-Binding Proteins / metabolism*

Substances

  • Antigens, Neoplasm
  • Epitopes
  • HLA-A*02:01 antigen
  • HLA-A2 Antigen
  • KRAS protein, human
  • Receptors, Antigen, T-Cell
  • Proteasome Endopeptidase Complex
  • Proto-Oncogene Proteins p21(ras)
  • rac GTP-Binding Proteins

Associated data

  • Dryad/10.5061/dryad.jq2bvq88b

Grants and funding

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