Project Summary Antibody-based immunotherapies have become an established paradigm for treating cancers. Only a small portion of patients have benefited from these agents, though, and many are only effective for treating a small set of tumors. This is due, in part, to a lack of validated cell surface targets. Only a dozen of surface proteins has been approved for antibody intervention, and most are not truly exclusive to malignant cells. There has been growing interesting in searching the cellular immunopeptidome for tumor-exclusive targets. The immunopeptidome is a pool of peptides generated from proteolysis of intracellular proteins and displayed on the cell surface as a complex with the human leukocyte antigens (HLA). It is thought that this peptide repertoire reflects the status of the intracellular proteome and can be modulated by driver oncogenes. Epitopes harboring driver mutations, or neo-antigens, have been detected as peptide-HLA complexes (pHLAs) from tumor samples. However, the mechanism of how oncogene signaling shape the immunopeptidome remains uncharacterized. The goals of this proposal are to develop technologies to map changes in the immunopeptidome unique to specific ongene, and tools to mount an immune response against them. These findings will ultimately aid in developing antibody-based biologics with better safety profile and more applicable to diverse types of cancers. Here, we hypothesized that proliferative oncogenes globally alter the immunopeptidome via transcriptional remodeling and alternative splicing, and induce unique pHLAs beyond mutated fragments of parent oncoproteins. To test this hypothesis, we will first develop a new mass spectrometry technique to map diseased immunopeptidome in an allele-specific manner. Next, we will combine this technique with a reductionist cell model approach to study the immunopeptidome of isogenic cell lines overexpressing specific oncogene. The immunopeptidomics data will be complemented by protein databases and transcript analyses to reveal key contributors of oncogene-specific pHLAs. Collectively, the proposed work will provide insight to how a single genetic alteration can impact mechanisms of antigen presentation and produce neo-antigens on the cell surface. To build on these discoveries, we will also engineer antibodies to recognize pHLAs of interest with high affinity and peptide specificity. These reagents will be further elaborated into chimeric antigen receptors to arm cytotoxic T cells and mount antitumor response against cells displaying oncogene-specific pHLAs. These reagents will be tested in cultured cells, and humanized mice models. In the long term, the basic biological insights and antibody tools generated from the proposed studies will bolster ongoing efforts to understanding the cancerous immunopeptidome and lay the foundation for future therapeutics targeting neo-antigens.