Deeply analyzing MHC class I-restricted peptide presentation mechanistics across alleles, pathways, and disease coupled with TCR discovery/characterization - Project Abstract/Summary • Our framing hypothesis is that the combined constraints of peptide processing, HLA allelic specificity, and peptide editing generate a significant “funnel effect”, where the peptidome constitutes only a tiny percentage of all possible peptides obtainable from a cell’s proteome and the ligandome constitutes only a small percentage of all peptides from a cell’s peptidome. Our research focus is understanding how the interplay of differential sequence recognition at each step ultimately informs functional presentation of a highly restricted subset of all possible proteome-derived peptides – and how to identify the most “translatable” pHLA targets from ligandomes. • We will rigorously test this hypothesis using our allele-specific mass-spec platform for peptide discovery, ARTEMIS, to identify relevant, HLA-restricted peptides from Mesothelin and high-risk HPV E6/E7 oncoproteins. Identified peptides will be validated and characterized biochemically. Cell-surface displayed peptide arrays from E6/E7 and Mesothelin will be used to determine the magnitude of the “funnel effect” of peptide processing on peptidome composition. Patient T cell responses to identified Mesothelin peptides will be mapped using state- of-the-art methodologies. Unusual peptides and alternate binding conformations will be characterized crystallo- graphically. We will assess viral immunoevasion mechanisms, HLA-G presentation and receptor interactions, and the presentation of glycosylated peptides. Deliverables include mapping the full Mesothelin and E6/E7 presentomes across multiple alleles and presentation modalities, ranked for future clinical exploitation as immu- notherapy targets. • Significance: From a basic science perspective, assaying cellular peptidomes and ligandomes is fundamental for understanding how the immune system responds to infections and cancer, immunoevasion mechanisms, the recognition rules of MHC proteins, the mechanistics of antigen processing/editing, and how self is defined. From a translational perspective, validated HLA-restricted peptides represent targets for diagnosis and therapy, through therapeutic vaccination, engineered T cell-based adoptive immunotherapies, and antibody-based recog- nition of specific, disease-associated peptide/HLA complexes Our proposed studies advance both objectives by elucidating the underlying molecular principles governing peptide processing and presentation through inte- grated studies of an extended set of classical and non-classical human HLA class I proteins and, in the process, identify specific pHLAs useful for future clinical applications.
