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bioRxiv · 10.64898/2025.12.07.692877

Decoding Neoantigen-encoding Tumor-Specific Transcripts Unveils a Shared Target Reservoir for Immunotherapy in Hepatocellular Carcinoma

Lin, P.·Wen, Y.·Zhao, J.·Zhang, F.·Su, Y.·Yu, H.·Li, Q.·Liu, C.·Li, Y.·Hu, Z.·Fang, Z.·Liang, L.·Huang, S.
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Abstract

Background and AimsPrimary liver cancer, predominantly hepatocellular carcinoma (HCC), has limited therapeutic options. While mutation-derived neoantigen vaccine holds promise, its success is hindered by low antigen availability. This study explores transcriptome-derived neoantigens (neoantigen-encoding tumor-specific transcripts, neoTSTs) in HCC, characterizing their features, generation mechanisms, and therapeutic potential. Approach and ResultsWe analyzed RNA-seq data from 1,013 liver cancer patients and constructed a multi-layered reference dataset. Using a customized pipeline, we identified an average of 60 neoTSTs per patient, significantly surpassing mutation-derived neoantigens (neoMuts). NeoTSTs exhibited higher population frequencies, with 73.1% providing multiple epitopes, and were validated through mass spectrometry and HLA transgenic mouse models. Mechanistically, neoTSTs were generated via retained introns, transposable element activation, HNF4A-regulated alternative promoters, and de novo transmembrane domain (TMD) generation. Single-cell analysis revealed neoTSTs cover >75% of tumor cells and identified antigen-presenting cancer-associated fibroblasts (apCAFs) that enriched in immunotherapy responders and amplified CD4 T-cell responses via MHC-II presentation. In murine HCC models, neoTST vaccination outperformed neoMuts, inducing dual MHC-I/II activation and significant tumor growth inhibition. ConclusionsNeoTSTs represent a superior neoantigen source in HCC, compensating for the limitations of mutation-derived targets. Their abundance, sharedness, and dual MHC pathway activation highlight their potential for personalized immunotherapy, particularly in low-TMB tumors. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/692877v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1bedfedorg.highwire.dtl.DTLVardef@5f6c95org.highwire.dtl.DTLVardef@d1deb5org.highwire.dtl.DTLVardef@743e85_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Lin, P., Wen, Y., Zhao, J., Zhang, F., Su, Y., Yu, H., Li, Q., Liu, C., Li, Y., Hu, Z., Fang, Z., Liang, L., Huang, S.. 2025-12-10. Decoding Neoantigen-encoding Tumor-Specific Transcripts Unveils a Shared Target Reservoir for Immunotherapy in Hepatocellular Carcinoma. https://doi.org/10.64898/2025.12.07.692877

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