Massively parallel HLA class I immunopeptidome by DNA sequencing reveals landscape of cancer antigen presentation

Human leukocyte antigens (HLA) are the most polymorphic genes in the human genome. HLA-I alleles control antigen presentation for T cell recognition that is pivotal for autoimmunity, infectious diseases, and cancer. Current knowledge of HLA-bound peptides is limited, skewed, and falls short of population-wide HLA binding profiles for high value targets. Here, we present ESCAPE-seq (Enhanced Single Chain Antigen Presentation sequencing), a massively parallel platform for comprehensive screening of class I HLA-peptide combinations for antigen presentation via deep DNA sequencing. ESCAPE-seq demonstrates programmability, high throughput, sensitivity, and nominated viral and cancer epitopes. ESCAPE-seq simultaneously assessed >75,000 peptide-HLA combinations, revealing broadly presented epitopes from oncogenic driver mutations and fusions across diverse HLA-A, B, and C alleles that cover 90% of the human population. We further identified epitopes that are differentially presented between oncogenic hotspot mutations vs its cognate wild type. ESCAPE-seq enable one-shot population-wide antigen presentation discovery, offering insights into HLA specificity and immune recognition of genomic mutations.