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 KRAS^G12V and RAC2^P29L 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 KRAS^G12V- and RAC2^P29L-derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2^P29L-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.