Massively parallel immunopeptidome by DNA sequencing provides insights into cancer antigen presentation

Abstract Human leukocyte antigens (HLAs) are encoded by the most polymorphic genes in the human genome. HLA class I alleles control antigen presentation for T cell recognition, which 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. We simultaneously assessed over 75,000 peptide–HLA combinations, revealing broadly presented epitopes from oncogenic driver mutations and fusions across diverse HLA-A , HLA-B and HLA-C alleles that cover 90% of the human population. We further identified epitopes that are differentially presented, comparing oncogenic hotspot mutations versus wild type. ESCAPE-seq enables one-shot population-wide antigen presentation discovery, offering insights into HLA specificity and immune recognition of genomic mutations.