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

Gerald Willimsky,Vivian Scheuplein,Peter M Kloetzel,Lena Immisch,Thomas Blankenstein,Hermann-Georg Holzhütter,Christin Beier,George Papafotiou,Andrean Goede

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.

Highlights

Defined anti-tumor CD8+ T cell responses require the proteasome-dependent processing of intracellular proteins and the efficient generation of antigenic peptides presented in the context of HLA class I molecules at the cell surface for TCR recognition
Our view on antigen processing was significantly extended by analysis of cancer patient-derived CD8+ T cells revealing that by proteasome catalyzed peptide splicing (PCPS) proteasomes can fuse excised peptide fragments in a reverse proteolysis reaction, thereby generating new immune reactive spliced epitopes with an amino acid sequence that differs from that of the substrate protein (Hanada et al, 2004; Vigneron et al, 2004)
For the FGF-5 and several splicetopes derived from the tumor differentiation antigen gp100mel in vitro proteasome splicing reactions were found to mimic the in vivo splicing reactions (Ebstein et al, 2016; Warren et al, 2006) suggesting that in vitro PCPS reactions may be a useful tool to discover new spliced epitopes generated from tumor antigens of interest

Summary

Introduction

Defined anti-tumor CD8+ T cell responses require the proteasome-dependent processing of intracellular proteins and the efficient generation of antigenic peptides presented in the context of HLA class I molecules at the cell surface for TCR recognition. To be able to identify splicetopes in in vitro PCPS experiments independent of the availability of patientderived CD8+ T cells, we developed the prediction algorithms ProteaJ (Liepe et al., 2010) and the here described ProtAG. Using these algorithms, we established an inclusion list of potentially immune-relevant spliced peptides theoretically generated from a given antigen, which in combination with the mass spectrometric analysis of the in vitro digest should allow the identification of new splicetopes. Testing the feasibility of such an algorithm aided “reverse immunology” approach, we had isolated CD8+ T cells from phospholipase PlcB-derived splicetopes generated by the proteasome in vitro and in vivo (Platteel et al, 2017)

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Results

Conclusion