Proteogenomic platforms establishing personalized and precision neoantigen therapeutics in cancer

Abstract

The evolution of new technologies such as next-generation sequencing has enhanced our ability to search for new immune targets such as mutation-derived antigens (neoantigens), which has accelerated the development of novel immune therapies. Neoantigens are tumour-specific markers that can form when cancer cell-specific mutations encode molecules that differ from ‘self’. Neoantigens may have diverse sources such as viral and mutated proteins. Moreover, posttranslational modifications and altered antigen processing can contribute to the neoantigen landscape. Studies have shown that inhibition of nonsense-mediated decay (NMD) also generates new antigenic determinants in tumour cells. This RNA quality con-trol pathway removes aberrant mRNA containing premature termination codons (PTCs) that arise through mutation or defective splicing. Neoantigens expressed on the surface of tumour cells can be detected and removed by immune cells, thus ne-oantigens can themselves function as immune stimulatory vaccines. Tumour vac-cination aims at stimulating a systemic immune response targeted to, mostly weak, antigens expressed in the disseminated tumour lesions. The main challenge in de-veloping effective vaccination protocols is the identification of potent and broadly expressed tumour rejection antigens and effective adjuvants to stimulate a robust and durable immune response.

The purpose of this project was to create pipelines to identify mutated pro-teins in human and mouse melanoma cell lines from matched genomic, tran-scriptomic, and proteomic data. A proteogenomic pipeline was set up including def-inition of cancer DNA variants, RNA variants (expressed genes under different conditions), and mass spectrometry to identify mutated protein and MHC class I ex-pression. One key question was to what extent can we detect mutated MHC Class I binding peptide based on cancer genome and RNA sequencing. Another key ques-tion was to define to what extent a cancer gene (such as p53) or a drug lead (such as NMD inhibitor or splicing inhibitor) changes the immunopeptidome as well as the mutated immunopeptidome. These pipelines require utilising bioinformatics ap-proach to analyse next generation DNA and RNA sequencing and creating a mu-tant protein reference database specific for the isogenic cell line with or without drug treatment. The thesis outline includes; (i) Developing rules for stop codon readthrough in the presence of NMD inhibitors in order to expand mutant proteins that can be produced; (ii) Analysis of mutant tumour immunopeptidome using an isogenic wild-type and p53 null cell model to define how this tumour suppressor gene status impacts immunopeptidome and how many mutant immunopeptides can be detected when comparing two distinct methodologies for immunopeptidome identification; and finally; (iii) Defining the effects of NMD inhibition or splicing in-hibition on the mutant tumour neoantigen pool in human and mouse melanoma models to form a novel paradigm for developing new drugs to stimulate tumour re-jection. The developed proteogenomic platform resulted in detection of a novel po-tential neoantigenic peptide ASELHTSLY proving the effectiveness of the present-ed approach.