Introductionĭefined 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. 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.
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Furthermore, only the putative RAC2 P29L-derived neo-splicetopes was generated by in vitro PCPS. 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.
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TCRs generated in mice with a diverse human TCR repertoire specifically recognized the respective target peptides with high efficacy. 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. 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.