Supplementary mass spectonomy data to analyse generation of spliced mutant Kras/RAC2 epitopes

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