Mass spectrometry-based de novo sequencing reveals non-canonical neoantigens with antitumor efficacy in hepatocellular carcinoma.

BACKGROUND &

aimsTumor neoantigens, especially cryptic antigens from non-canonical translation, are vital for cancer immunotherapy. Mass spectrometry (MS)-based de novo sequencing identifies candidates, but unverified immunogenicity and antitumor efficacy limit clinical applicability. This study aimed to identify novel non-canonical neoantigens in hepatocellular carcinoma (HCC) using MS-based de novo sequencing and rigorously validate their immunogenicity and antitumor efficacy.

methodsUsing a C57BL/6 subcutaneous HCC mouse model, immunopeptides were comprehensively profiled via MHC-I immunoprecipitation combined with MS-based de novo sequencing. Identified high-immunogenicity peptides predicted by deep learning were validated using ex vivo ELISpot assays. Endogenous peptide expression was confirmed using parallel reaction monitoring-targeted quantification. The antitumor efficacy of therapeutic peptide vaccines comprising the seven most immunogenic peptides combined with the adjuvant poly(I:C) was evaluated in vivo in the subcutaneous and orthotopic HCC models.

resultsWe identified 5,576 immunopeptides, with sequence motifs consistent with prior reports. Remarkably, 95% of deep learning-predicted high-immunogenicity peptides were successfully validated by ELISpot (p <0.05). Parallel reaction monitoring confirmed endogenous expression of these peptides. Most significantly, the peptide vaccines (7 peptides + poly(I:C)) demonstrated potent antitumor efficacy in vivo compared to controls (p <0.05).

conclusionsMS-based de novo sequencing combined with computational prioritization enables identification of non-canonical, immunogenic neoantigens in HCC. Selected peptides demonstrated endogenous presentation and measurable antitumor activity in preclinical models. IMPACT AND IMPLICATIONS: This study provides robust experimental validation that mass spectrometry-based de novo sequencing effectively identifies novel, highly immunogenic non-canonical neoantigens in hepatocellular carcinoma, overcoming a key limitation of prior predictive methods and opening avenues for exploring this understudied neoantigen class in other cancers. The findings are critical for cancer immunologists and oncologists developing next-generation immunotherapies, demonstrating a viable discovery-to-validation pipeline for novel therapeutic targets. The validated neoantigens and successful peptide vaccine strategy offer a direct pathway towards developing personalized hepatocellular carcinoma immunotherapies, enabling clinicians to adopt similar integrated approaches for patient-specific neoantigen discovery; however, clinical translation beyond this preclinical murine model requires confirmation in human settings due to potential differences in HLA presentation and the tumor microenvironment.