Functional Immunopeptidomics by Characterizing Immunopeptides Against HLAs, TCRs, and Antibodies

Target-directed immunotherapies, including TCR-T cell therapies, targeted antibody therapy, and therapeutic vaccines, rely on the identification of tumor-specific antigens (TSAs). However, classical immunopeptidomics is severely constrained by an inability to distinguish passively presented self-ligands of HLAs from functionally recognized by T cells. To resolve these challenges, we developed immunopeptidomics for receptor-associated antigens, a platform capable of sequentially isolating immunopeptidome and their functions with immune cell receptors from a single clinical tissue specimen. Applied to a cohort of 30 renal cell carcinoma (RCC) specimens (18 primary tumors and 12 paired normal adjacent tissues), this approach generated expansive presentation maps encompassing 38,848 unique HLA-I and 26,961 unique HLA-II peptides. Functional receptor captures (IgG- and TCR-bound) achieved up to 5.3-fold higher tumor-specific peptide (TSP) purity ratios compared to direct baseline HLA isolation. Furthermore, cross-fractional co-identification validated recurrent, high-priority unmutated targets (NNMT, TMBIM6, RPL3, VIM, and ENPP3) alongside tumor-specific phosphopeptide hubs and macrophage-restricted N-glycopeptides (CD68). Integrating companion global proteomics uncovered a fundamental regulatory divergence between MHC classes: HLA-I presentation throughput scales linearly (R = 0.883) via a STAT1-orchestrated transcriptional program driving the antigen presentation machinery (APM), whereas HLA-II presentation is uncoupled from parental protein abundance (R = 0.302) due to asynchronous downstream endolysosomal processing and proteolytic over-cleavage kinetics. Together, this platform establishes a micro-input framework that unifies physical presentation with native functional characterization and microenvironmental regulation to accelerate target discovery in low-TMB human cancers.