Becker, J. P. | Search bioRxiv

bioRxiv2023

optiPRM: A targeted immunopeptidomics LC-MS workflow with ultra-high sensitivity for the detection of mutation-derived tumor neoepitopes from limited input material

Personalized cancer immunotherapies such as vaccines and T cell receptor (TCR)-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen (HLA) class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. For their detection by liquid chromatography mass spectrometry (LC-MS), we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM. Optimization of collision energy using optiPRM allows for improved detection of low abundant peptides that are very hard to detect using standard parameters. Applying this to immunopeptidomics, we detected a neoepitope in a patient-derived xenograft (PDX) from as little as 2.5x106 cells input. Application of the workflow on small patient tumor samples allowed for the detection of five mutation-derived neoepitopes in three patients. One neoepitope was confirmed to be recognized by patient T cells. In conclusion, we here present optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, which allowed for the identification of actionable neoepitopes from sample sizes usually available in the clinic.

immunology↗

bioRxiv2020

Pharmacological inhibition of nonsense-mediated RNA decay augments HLA class I-mediated presentation of neoepitopes in MSI CRC

Microsatellite-unstable (MSI) colorectal cancer is characterized by the accumulation of somatic insertion/deletion (InDel) mutations potentially generating tumor-specific, frameshifted protein sequences. Such mutations typically generate premature translation termination codons targeting the affected mRNAs to degradation by nonsense-mediated RNA decay (NMD), limiting the synthesis and HLA class I-mediated presentation of tumor-specific InDel neoepitopes. We reasoned that the NMD inhibitor 5-azacytidine (5AZA) could serve to increase the expression of NMD-sensitive neoepitopes and analyzed the immunopeptidome of MSI HCT-116 cells using a proteogenomic approach. After immunoprecipitation of HLA:peptide complexes, we identified more than 10,000 HLA class I-presented peptides by LC-MS/MS including five InDel neoepitopes. The InDel neoepitopes were verified on the genomic, transcriptomic, and peptidomic level. Treatment with 5AZA increased the expression of the corresponding frameshift- and premature termination codon-bearing mRNAs and enhanced the presentation of peptides originating from known NMD targets and one of the identified InDel neoepitopes. By analyzing an array of MSI colorectal cancer cell lines and patient samples, we found the underlying frameshift mutation to be highly recurrent and immunization with the corresponding neoepitope induced strong CD8+ T cell responses in an HLA-A*02:01 transgenic mouse model. Our data directly show that peptides originating from frameshifted open reading frames due to InDel mutations in mismatch repair-deficient cells are presented on the cell surface via HLA class I. Moreover, we demonstrate the utility of NMD inhibitor-enhanced HLA class I-mediated presentation of InDel neoepitopes as well as their immunogenicity, uncovering the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies. One Sentence SummaryImmunopeptidomics identified increased HLA class I-mediated presentation of immunogenic, frameshift-derived neoepitopes following NMD inhibition.

cancer biology↗