通过共表达患者 HLA 和抗原的单质粒系统验证肝细胞癌中的优势新抗原。

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-A11:01 and the reported 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 , and . 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-A11:01, HLA-A24:02 or HLA-A02:01 were performed by tetramer staining to screen out potential HCC-dominant neoantigens. HLA-A24:02-restricted epitope 5'-FYAFSCYYDL-3' and HLA-A02:01-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- 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.

背景：先前的研究证实，大多数算法预测的新抗原在临床实践中并不起作用，实验验证对于确认免疫原性新抗原仍然不可或缺。在这项研究中，我们通过四聚体染色鉴定了具有潜在免疫原性的新抗原，并建立了共表达 HLA 和抗原的 Co-HA 系统，以检测新抗原的免疫原性，并验证新的肝癌（HCC）优势新抗原。

方法：首先，我们招募了 14 名 HCC 患者进行下一代测序，以进行变异调用和预测潜在的新抗原。然后，建立了 Co-HA 系统。为了测试该系统的可行性，我们构建了共表达 HLA-A*11:01 和报道的 G12D 新抗原以及特异性 T 细胞受体（TCR）-T 细胞的靶细胞。通过 Co-HA 系统显示了该新抗原产生的特异性细胞毒性。此外，通过四聚体染色筛选出潜在的 HCC 优势新抗原，并通过 Co-HA 系统使用包括流式细胞术、酶联免疫斑点分析和 ELISA 在内的方法进行验证。最后，在小鼠模型中进行抗肿瘤试验和 TCR 测序，以进一步评估优势新抗原。

结果：首先，在 14 名 HCC 患者中鉴定出 2875 个体细胞突变。主要碱基替换为 C>T/G>A 转换，主要突变特征为 4、1 和 16。高频突变基因包括、和。然后，预测了 541 个潜在的新抗原。重要的是，肿瘤组织中 23 个潜在新抗原中的 19 个也存在于门静脉癌栓中。此外，通过四聚体染色筛选出 37 个受 HLA-A11:01、HLA-A24:02 或 HLA-A02:01 限制的预测新抗原，以筛选出潜在的 HCC 优势新抗原。通过 Co-HA 系统验证，HLA-A24:02 限制的表位 5'-FYAFSCYYDL-3'和 HLA-A*02:01 限制的表位 5'-WVWCMSPTI-3'在 HCC 中具有很强的免疫原性。最后，在 B-NDG-小鼠中验证了 5'-FYAFSCYYDL-3'-特异性 T 细胞的抗肿瘤疗效，并成功鉴定了其特异性 TCR。

结论：我们在 HCC 中发现了具有高免疫原性的优势新抗原，并通过 Co-HA 系统进行了验证。