Mass spectrometry-based de novo sequencing reveals non-canonical neoantigens with antitumor efficacy in hepatocellular carcinoma
peer-reviewed · JHEP Reports · 2026
| Date | 2026-04-01 |
| Type | peer-reviewed |
| Venue | JHEP Reports |
| Publisher | Elsevier BV |
| Contribution | downstream-application |
| DOI | 10.1016/j.jhepr.2026.101775 |
| Citations (OpenAlex) | 0 |
Abstract
Background & aims Tumor 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. Methods Using 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. Results We 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 Conclusions MS-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.
Methods and tools
- HCC non-canonical neoantigen discovery: Finds non-canonical neoantigens in hepatocellular carcinoma by mass spectrometry-based de novo sequencing and then tests them, addressing the gap that de novo proposes cryptic antigens whose immunogenicity goes unverified.