借CTP实现外源性FoxM1抗原交叉递呈以诱导肝癌特异性CTL应答

The cross-presentation of exogenous antigens in dendritic cells(DCs) is essential to the induction of tumor-specific cytotoxic lymphocyte(CTL) response. The available antigen-loading systems fail to activate the potential of cross- presentation of DCs, which definitely restricts the anti-tumor effect of DC vaccines. It has been well-recognized that the "internalization" of exogenous antigens is the premise of the activation of the cross-presentation potentiality. Inspiringly, cytoplasmic transduction peptide(CTP) has exhibited a clear preference for cytoplasmic localization and dramatic membrane transduction potential, which will efficiently implement the "internalization" of exogenous antigens by localizing exogenous antigens in the cytoplasm of cells. FOXM1, as a tumor-associated antigen, is required for the survival of hepatocellular carcinoma(HCC) cells and is the ideal target antigen for DC-based HCC vaccine. Therefore, we first propose the assumption of "inducing HCC-specific CTL response via the accomplishment of cross-presentation of exogenous FOXM1 antigen through CTP". Through prokaryotic expression and purification of the CTP-FOXM1 fusion protein, we intend to observe its transduction activity, subcellular localization and cross-presentation in DC, and the in vitro and in vivo FOXM1-specific CTL response activated by the DC vaccine. This DC-based strategy of "activating the FOXM1-specific CTL response via the accomplishment of cross-presentation of exogenous FOXM1 antigen through CTP" will show great advantages in good biological safety, efficient activation of the cross-presentation potential of DC, and ideal target antigen.

外源性抗原在DC细胞内发生交叉递呈是诱导肿瘤特异性CTL应答的关键，现有的抗原负载系统不能有效激活DC细胞的交叉递呈潜能，极大地限制了DC疫苗抗肿瘤效力。外源性抗原的"内源化"是激活交叉递呈潜能的前提。CTP是一种具有显著胞浆定位和高效转导潜能的转导肽，可使外源性抗原发生胞浆定位而实现"内源化"，FOXM1是肝癌细胞存活必须的肿瘤相关抗原，是肝癌DC疫苗理想的靶抗原。由此，我们首次提出"借CTP实现外源性FOXM1抗原交叉递呈以诱导肝癌特异性CTL应答"的设想。拟通过原核表达纯化CTP-FOXM1融合蛋白将其转导入DC细胞，观察其在DC细胞内的转导活性、亚细胞定位、交叉递呈水平和DC疫苗在体内外激活FOXM1特异性CTL应答的情况。这种以DC细胞为基础借CTP实现外源性FOXM1抗原交叉递呈，激活肝癌特异性CTL应答的策略具有生物安全性好，可激活DC细胞交叉递呈潜能和靶抗原理想等优势。

叉头框M1 （forkhead box M1, FOXM1）是一种与细胞增殖相关的转录因子，对各种恶性肿瘤的发生以及发展起促进作用。我们和其他课题组的研究都发现明FOXM1 在肝细胞肝癌（hepatocellular carcinoma, HCC）中高表达，提示FOXM1可能为肝细胞肝癌的一个潜在治疗靶点。本项目中，我们以胞浆转导肽（cytoplasmic transduction peptide, CTP）为抗原递呈系统呈递FOXM1到树突状细胞（dendritic cells, DCs），结果发现CTP可将重组表达的FOXM1抗原呈递到DC细胞浆，并且刺激DCs的成熟与活化，负载CTP-FOXM1抗原的树突状细胞能够诱发特异的肝癌细胞杀伤效应。更重要的是，体内动物实验发现：负载CTP-FOXM1抗原的树突状细胞能够有效抑制肝癌的生长和转移。这些结果表明CTP-FoxM1是树突状细胞免疫治疗肝癌的一种新型抗原。. 另外，我们还发现趋化因子CXCL13 在原发性肝癌患者血清中表达增加，其表达水平与肿瘤大小、转移、TNM 分期密切相关，表明CXCL13可作为原发性肝癌诊断治疗及预后评估的血清学标志物。. 上述研究结果在《Oncotarget》和《Cytokine》等期刊发表SCI论文3篇，CSCD论文3篇，培养硕士研究生4人。