Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

An investigation into the neoantigenic landscape of microsatellite instability colorectal cancer using immunopeptidomics (#189)

Jiahui Yu 1 , David Williams 2 , John Mariadason 2 , Shanzou Chung 1 , Sri Ramarathinam 1 , Anthony Purcell 1
  1. Department of Biochemistry and Molecular Biology and Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia
  2. Olivia Newton-John Cancer Research Institute, Austin Hospital, Melbourne, Victoria, Australia

Microsatellite instability (MSI) results from genetic defects of DNA mismatch repair (MMR), leading to DNA replication errors that cannot be repaired. Around 15% of colorectal cancer (CRC) exhibit high-frequency microsatellite instability (MSI-H), the treatment for which still represents an unmet need. Neoantigens are highly specific and can form ideal targets for immunotherapy, with minimal off-target toxicities and the potential to prevent the recurrence of the disease. As the MSI-H phenotype can lead to high mutational burden and is a potential source of neoantigens, we aimed to identify mutant peptides isolated from human leucocyte antigens (HLA) of MSI-H cells using mass spectrometry.

Cell pellets from patient-derived MSI-H CRC cell lines, HCA7 and HCT116, were harvested and the cell surface HLA-A*02:01-peptide complexes were isolated. The peptide cargo was interrogated by LC-MS/MS using Bruker timsTOF PRO mass spectrometer coupled to a nanoElute UHPLC. Data was searched using Peaks X Pro against human proteome appended with a cell line specific mutation database derived from Exome sequencing.

We identified 80302 HLA-A*02:01-bound peptides that included 59 mutant peptides (neopeptides), 4 of which were shared mutant peptides between the two CRC cell lines. We noticed a high proportion of frameshift mutations compared to missense mutations within the mutated peptide sequences. These novel mutation-containing peptides have not been reported previously and form the basis of ongoing immunogenicity studies. The peptides identified and methodology applied have implications for future development of therapeutic interventions in CRC.