Proteomic and proteogenomic identification of targetable T-cell antigens in multiple myeloma

Multiple myeloma (MM) accounts for approximately 12% of all hematologic malignancies and remains incurable despite therapeutic advances. Targeting tumor-associated antigens (TAAs) and tumor-specific neoantigens presented on human leukocyte antigen (HLA) molecules by vaccination or T cell receptor (TCR)-redirected T cells offers a promising novel immunotherapeutic strategy. However, knowledge of targetable tumor antigens, especially neoantigens, is limited. We applied a mass spectrometry (MS)-based immunopeptidomics approach on primary MM samples (n = 8) and MM cell lines (n = 3), integrating whole-exome and RNA sequencing to comprehensively characterize the MM immunopeptidome and systematically search for TAAs and neoantigens. Using our approach, we detected cancer-associated phosphopeptide HLA ligands as well as multiple cancer testis antigen (CTA)-derived HLA peptides, some shared between tumor samples. Proteogenomic analysis revealed primarily personalized patient-specific Immunoglobulin (Ig)-derived neoantigens generated by V(D)J recombination and somatic hypermutation (SHM), which were present in all samples. Three patients showed clustering of related MHC class II peptide ligands within the hypermutated region. In addition, we identified non-Ig-derived neoantigens in both primary MM samples and cell lines. Approximately 30% of the cell line-derived peptides elicited CD8⁺ T-cell responses in allogeneic HLA-matched healthy donor PBMCs. In contrast, none of the patient-derived neoantigens were immunogenic in autologous PBMCs or tumor-infiltrating lymphocytes, reflecting severe immune dysfunction. This study provides the first proteogenomic-based identification of neoantigens in MM, extends the observation of presented Ig-derived neoantigens from B cell lymphoma to MM, and highlights both actionable antigens and barriers to effective TCR-based immunotherapy.