Notably, LUAD-7-MT peptide encoded by NANOGNB demonstrated higher immunogenicity in promoting CD8+ T cells proliferation and IFN-γ secretion than the corresponding wildtype peptide. This study provides an in-depth analysis of mutation characteristics of LUAD and establishes a neoantigen screening and validation system for identifying immunogenicity neopeptide across individual patients and diverse foci in the same patient with multifocal LUAD.