Investigating tumour antigens in mismatch repair proficient gastrointestinal cancers through patient-derived organoids

Abstract

Immunotherapy of gastrointestinal (GI) cancers with immune-checkpoint inhibitors (ICIs) has shown clinical progress in mismatch repair deficient (MMRd) GI cancers, but not in MMRp GI cancers. It is understood that the difference in mutation load and associated neoantigen presentation is behind this difference in immunotherapy sensitivity. Therefore, this thesis focuses on advancing the understanding of peptide and neoantigen presentation in MMRp GI cancers through a multi-omics approach of transcriptomics, proteomics, and peptidomics, alongside patient-derived organoid (PDO) immune cell co-cultures.

Immunopeptidomics of 5 MMRp colorectal cancer (CRC) PDOs was able to define a sparse neoantigen landscape, and demonstrate that perturbation with IFN or trametinib treatment could not promote the presentation of novel neoantigens. Combining proteomics data with the immunopeptidomics data allowed me to resolve features that influenced peptide abundance change under IFN exposure. The data showed peptides generated by chymotryptic activity or presented on HLA-B were more likely to increase in intensity. Analysis also highlighted proline within the core of a peptide (positions 4-6) could promote its downregulation under IFN exposure. This data could be used to inform peptide selection for cancer vaccines, as it would allow for specific selection of peptides which are presented both in the presence or absence of IFN, and do not get downregulated. Furthermore, optimisation of an endoplasmic reticulum (ER) isolation protocol has illustrated that it is possible to detect peptides and neoantigens from organelle preparations. This should support the future study of neoantigen loss in the ER.

Generation of PBMCs and PDOs from gastro-oesophageal adenocarcinoma (GOA) patients enabled the testing of tumour-specific T-cell expansion from MMRp patient PBMCs. Whilst for the patient tested, no tumour-specific T-cell reactivity was seen, the study showed the expansion protocol generates T-cells which can then be interrogated for reactivity, and sensitivity to different immunotherapy combinations.