Dominant neoantigen verification in hepatocellular carcinoma by a single-plasmid system coexpressing patient HLA and antigen

BACKGROUND: Previous studies confirmed that most neoantigens predicted by algorithms do not work in clinical practice, and experimental validations remain indispensable for confirming immunogenic neoantigens. In this study, we identified the potential neoantigens with tetramer staining, and established the Co-HA system, a single-plasmid system coexpressing patient human leukocyte antigen (HLA) and antigen, to detect the immunogenicity of neoantigens and verify new dominant hepatocellular carcinoma (HCC) neoantigens. METHODS: First, we enrolled 14 patients with HCC for next-generation sequencing for variation calling and predicting potential neoantigens. Then, the Co-HA system was established. To test the feasibility of the system, we constructed target cells coexpressing HLA-A*11:01 and the reported KRAS G12D neoantigen as well as specific T-cell receptor (TCR)-T cells. The specific cytotoxicity generated by this neoantigen was shown using the Co-HA system. Moreover, potential HCC-dominant neoantigens were screened out by tetramer staining and validated by the Co-HA system using methods including flow cytometry, enzyme-linked immunospot assay and ELISA. Finally, antitumor test in mouse mode and TCR sequencing were performed to further evaluate the dominant neoantigen. RESULTS: First, 2875 somatic mutations in 14 patients with HCC were identified. The main base substitutions were C>T/G>A transitions, and the main mutational signatures were 4, 1 and 16. The high-frequency mutated genes included HMCN1, TTN and TP53. Then, 541 potential neoantigens were predicted. Importantly, 19 of the 23 potential neoantigens in tumor tissues also existed in portal vein tumor thrombi. Moreover, 37 predicted neoantigens restricted by HLA-A*11:01, HLA-A*24:02 or HLA-A*02:01 were performed by tetramer staining to screen out potential HCC-dominant neoantigens. HLA-A*24:02-restricted epitope 5'-FYAFSCYYDL-3' and HLA-A*02:01-restricted epitope 5'-WVWCMSPTI-3' demonstrated strong immunogenicity in HCC, as verified by the Co-HA system. Finally, the antitumor efficacy of 5'-FYAFSCYYDL-3'-specific T cells was verified in the B-NDG-B2m(tm1)Fcrn(tm1(mB2m)) mouse and their specific TCRs were successfully identified. CONCLUSION: We found the dominant neoantigens with high immunogenicity in HCC, which were verified with the Co-HA system.