Five dominant amino acid substitution signatures shape tumour immunity.

Although numerous mutational processes operate in cancer, their functional impacts are unclear. We hypothesised that certain mutation sources preferentially generate amino acid substitutions that evade immune recognition, producing immune-cold tumours regardless of tissue or mutation load. By analysing 9300 cancer exomes and performing mutagenesis experiments, we mapped links between mutagens, DNA-repair defects, and amino acid substitution signatures (AAS). Surprisingly, the spectrum collapsed into five recurrent AAS with distinct functional profiles. AAS4-generated by alkylating agents and mismatch-repair (MMR) deficiency and enriched in kidney and liver cancers-is less likely to accumulate hydrophobic residues, yielding poorly immunogenic neopeptides. These tumours display immune-desert microenvironments and respond poorly to immunotherapy. However, certain human leukocyte antigen (HLA) class I variants, such as HLA-B*07:02, correlate with immune-hot tumours in this subgroup. HLA-B*07:02, common in Europeans, presents proline-enriched neopeptides derived from AAS4 mutations. Supporting this, B*07:02-positive cancer cells harbouring AAS4-type mutations stimulated T-cell proliferation in vitro. These results show that neoantigen quality, not merely quantity, dictates anti-tumour immunity, explain inconsistent immunotherapy responses in MMR-deficient cancers, and advocate incorporating amino acid substitution patterns into predictive biomarkers and therapy design.