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

Abstract

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. 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.

Neo-Splicetopes in Tumor Therapy: A Lost Case?

“…Furthermore, the proteasomal transpeptidation reaction is most likely less frequent than the normal peptide hydrolysis reaction that results in the generation of non-spliced peptide fragments including linear HLA-1 epitopes. This latter point is nicely reflected by the analysis of mutRAC2P29L where in vitro generation of the linear spliced mutRCA2 derived neoepitope is at least 200fold more efficient than the generation of the putative mutRAC2 neosplicetope (43). From these studies we have to acknowledge that an in silico-in vitro pipeline thought to be a straight forward, easy to use approach to identify spliced peptides for therapeutic use cannot be the method of choice (57) and that in silico predictions of spliced epitopes alone or in combination with algorithms calculating HLA-1 binding affinity (60) and in vitro PCPS reactions seem by no means of sufficient reliability to set out for laborious and time consuming TCR generation.…”

“…Furthermore, following the outlined theoretical arguments that identification of suitable tumor specific neosplicetopes for ATT may allow to make neoantigens visible for the immune systems which cannot be recognized otherwise alternative approaches allowing a more direct splicetope seemed worthy to consider. With this in mind the spliced peptide predictions algorithms ProteaJ and ProtAG ( 42 , 43 ) were developed. In combination with mass spectrometric analyses of in vitro PCPS assays these permit the identification spliced peptides that are generated by the 20S proteasome from synthetic polypeptide substrates.…”

Filamentous Bacteriophage—A Powerful Carrier for Glioma Therapy

“…Therefore, the targeted therapy is particularly important for tumor therapy (45)(46)(47). Peptides specifically binding to tumor tissues, as carriers to direct drugs to tumor tissues, significantly improved the accuracy of drug targeting (48)(49)(50)(51). Although monoclonal antibodies as vectors were successfully applied to anti-tumor, the high molecular weight of antibodies might reduce efficiency (52)(53)(54)(55), while the phage peptide library has the benefits of screening for small molecular weight peptides, which can compensate for antibody deficiencies that are widely used in the diagnosis and treatment of glioma.…”

Are There Indeed Spliced Peptides in the Immunopeptidome?

“…Recently, Willimsky et al . ( 49 ) also concluded that the frequency of spliced epitopes is largely overestimated.…”