The development of immunotherapies has revolutionized approaches of cancer treatments. These therapies include antibodies against immune checkpoints, and Chimeric-Antigen Receptor T cells (CAR-T cells) therapies. The efficiency of these therapies rely on the recognition by T cells of tumor-specific antigens that differentiates tumour cells from healthy cells. The presentation of antigens by the major histo-compatibility class I (MHC-I) complex requires the processing of endogenous proteins through the proteasome to form peptides presented through the MHC on the cell surface. The antigens presented by MHC-I complex form the immunopeptidome. Genetic mutations in tumour cells expand the repertoire of the immunopeptidome presented to T cells. These mutations generate neo-antigens, increasing the chances of recognition of tumour cells as non-self cells to be destroyed by immune cells. In recent years, studies of the immunoproteasome have allowed the discovery of a new class of neo antigens derived from the modification of the peptidome presented by MHC-I, in which the fusion of small peptides by the immunoproteasome can generate new peptides presented by the MHC complex. These denominated spliced peptides can open new strategies for the development of cancer immunotherapy, and improve the current strategies used against cancer. In this project, we aim to explore the immunopeptidome presented by MHC-I and MHC-II in Non-Small Cell Lung Cancer, elucidating the immunogenicity of identified spliced neoantigens and their recurrency across patients. To achieve this objective, we will use LC-MS to detect the peptides bound to the MHC class I and II present in biopsies obtained from a cohort of patients, donated from multiple hospitals in Victoria. These peptides are compared with the RNAseq derived proteome obtained from the same samples, including mutations and indels present in the transcriptome. These peptides sequenced will be eventually synthetized and their immunogenicity will be measured by challenging PBMCs derived from LUAD patients, measuring the CD8 and CD4 immune responses.