新抗原疫苗是个性化肿瘤治疗最有效的免疫疗法之一。目前新抗原疫苗的免疫原设计通常基于全基因组测序（WGS）和生物信息学预测，侧重于肽与MHC分子之间的结合亲和力的预测，忽略其他肽呈递相关步骤。这可能会导致较高的预测准确性和相对较低的临床有效性之间的差距。在本研究中，我们设计了一个集成的计算机模拟流程Neo-intline，它从肿瘤样本的SNP和indels出发，通过集成的计算模型来模拟肽在体内的呈现过程。对 TESLA 基准数据集和临床验证的新抗原的验证表明，neo-intline 在样本水平和黑色素瘤水平上都可以优于当前最先进的工具。此外，以小鼠黑色素瘤模型为例，我们验证了20种新抗原的有效性，其中包括10种MHC-I和10种MHC-II肽。体外和体内实验表明，Neo-intline预测的两种肽都可以招募相应的CD4 T细胞和CD8 T细胞来诱导T细胞介导的细胞免疫反应。此外，虽然单独使用新抗原疫苗的治疗效果还不够，但与其他特异性疗法联合使用，例如粒细胞巨噬细胞集落刺激因子（GM-CSF）和聚肌苷-聚胞苷酸（poly（poly））的广谱免疫增强佐剂， I:C)) 或免疫检查点抑制剂，例如 PD-1/PD-L1 抗体，可以说明对黑色素瘤的显着抗癌作用。 Neo-intline 可用作新抗原疫苗免疫原性靶点设计和筛选的基准流程。© 2023。四川大学华西医院。

Neoantigen vaccines are one of the most effective immunotherapies for personalized tumour treatment. The current immunogen design of neoantigen vaccines is usually based on whole-genome sequencing (WGS) and bioinformatics prediction that focuses on the prediction of binding affinity between peptide and MHC molecules, ignoring other peptide-presenting related steps. This may result in a gap between high prediction accuracy and relatively low clinical effectiveness. In this study, we designed an integrated in-silico pipeline, Neo-intline, which started from the SNPs and indels of the tumour samples to simulate the presentation process of peptides in-vivo through an integrated calculation model. Validation on the benchmark dataset of TESLA and clinically validated neoantigens illustrated that neo-intline could outperform current state-of-the-art tools on both sample level and melanoma level. Furthermore, by taking the mouse melanoma model as an example, we verified the effectiveness of 20 neoantigens, including 10 MHC-I and 10 MHC-II peptides. The in-vitro and in-vivo experiments showed that both peptides predicted by Neo-intline could recruit corresponding CD4+ T cells and CD8+ T cells to induce a T-cell-mediated cellular immune response. Moreover, although the therapeutic effect of neoantigen vaccines alone is not sufficient, combinations with other specific therapies, such as broad-spectrum immune-enhanced adjuvants of granulocyte-macrophage colony-stimulating factor (GM-CSF) and polyinosinic-polycytidylic acid (poly(I:C)), or immune checkpoint inhibitors, such as PD-1/PD-L1 antibodies, can illustrate significant anticancer effects on melanoma. Neo-intline can be used as a benchmark process for the design and screening of immunogenic targets for neoantigen vaccines.© 2023. West China Hospital, Sichuan University.