Targeting the mutanome of HCC by viral inflammation and tumor-directed vaccinations: a model for individualized tumor therapy

溶瘤病毒疗法和免疫疗法为肝细胞癌（HCC）的治疗提供了巨大的潜力。在先前的实验中，我们已经开发了一种创新的和高效的免疫策略，其在肿瘤炎症期间通过溶瘤病毒促进有效的肿瘤靶向树突状细胞疫苗接种（=溶瘤辅助DC疫苗接种，或ODC）。在肺癌细胞的进一步初步工作中，我们建立了一种诊断/治疗程序，包括通过全外显子组测序鉴定肿瘤突变组，然后通过计算机模拟预测潜在免疫原性新表位，并在肿瘤内感染溶瘤腺病毒后通过T细胞分析进行验证。在拟议的项目中，我们希望开发一种用于治疗HCC的个性化治疗策略，包括优化的ODC策略，以引发针对个体肿瘤突变体的免疫原性新表位的强烈抗肿瘤免疫应答。我们希望在临床相关的转基因小鼠模型中解决优化的ODC对免疫原性新表位的治疗功效。首先，我们希望通过全外显子组测序分析选定的HCC细胞系中的非同义单核苷酸变异谱。在确定突变组后，我们使用SYFPEGII算法定义有希望的MHC I类结合新表位。在同基因小鼠模型中皮下肿瘤的溶瘤病毒感染后，通过CD 8 T细胞筛选来鉴定针对免疫原性新表位的CD 8 T细胞应答的成功触发。在这些模型中，我们希望使用这些免疫原性新表位来定义靶向完整突变组的DC疫苗的优化制备规则。将通过ELISpot分析和体内细胞毒性测量来确定抗原特异性免疫应答。将通过监测肿瘤生长、存活和转移形成来评估治疗功效。最后，我们希望在我们的新型转基因肝内肝癌电穿孔模型中研究相应的基于ODC的疫苗的治疗潜力。这些研究将为肝癌个体化免疫治疗的临床试验提供必要的信息。

Oncolytic virotherapy and immunotherapies promise enormous potential for the treatment of hepatocellular carcinoma (HCC). In previous experiments we have developed an innovative and highly effective immunotherapeutic strategy which facilitates an effective tumor-targeted dendritic cell vaccination during inflammation of the tumor by an oncolytic virus (= oncolysis-assisted DC-vaccination, or ODC). In further preliminary work in a lung cancer cell we established a diagnostic/therapeutic procedure involving identification of the tumor mutanome by whole exome sequencing, followed by in-silico prediction of potentially immunogenic neoepitopes and verification by T cell analyses after intratumoral infection with oncolytic adenoviruses. In the proposed project we want to develop a personalized therapy strategy for the treatment of HCC involving an optimized ODC strategy to elicit strong antitumoral immune response against immunogenic neoepitopes of individual tumor mutanomes. We want to address the therapeutic efficacy of optimized ODC against immunogenic neoepitopes in clinically relevant, transgenic mouse models. First, we want to analyse the spectrum of non-synonymous single nucleotide variants in selected HCC cell lines by whole exome sequencing. After determination of the mutanome, we define promising MHC class I-binding neoepitopes using the SYFPEITHI algorithm. Successful triggering of CD8 T cell responses against immunogenic neoepitopes will be identified by CD8 T cell screens after oncolytic virus infection of subcutaneous tumors in a syngeneic mouse model. In these models, we want to use these immunogenic neoepitopes to define the rules for optimized preparation of DC-vaccines targeting a complete mutanome. Antigen-specific immune responses will be determined by ELISpot analyses and in-vivo cytotoxicity measurements. Therapeutic efficacy will be assessed by monitoring tumor growth, survival, and metastases formation. Finally, we want to investigate the therapeutic potential of corresponding ODC-based vaccine in our novel electroporation model for transgenic intrahepatic HCC. These investigations will provide essential informations for a future clinical trial of personalized immunotherapy of HCC.