热休克蛋白抗原肽复合物疫苗诱导抗肿瘤免疫效应的分子机制研究

Heat shock proteins-peptide complex vaccine (HSP.PC) has become one of the focuses of tumor biological treatment recent years. We extracted heat shock protein70-peptide complex (HSP70.PC) from allogeneic mRNA-DC electrotransfected tumor vaccine as new kind of peptide vaccine, and verified its specific antiosteosarcoma effects both in vitro and in vivo. However, the exact molecular mechanism of the antitumor effects was still not very clear. Our preliminary research found that the immunogenic response induced by HSP70.PC was very similar with that induced through immunogenic cell death (ICD) process therefore it could be speculated that HSP.PC was possibly the existing form of tumor antigen peptides released through ICD process, which should be confirmed from the foundational level of amino acids. There were still some other questions to be answered, such as which specific cell signaling pathways were involved in the process of HSP based tumor antigen presentation in the DC component, and whether this multivalent vaccine achieved the antitumor effect through overcoming tumor-induced immune suppression, et al. Therefore, in this research item, we intend to carry out some further studies to further explore the HSP.PC-based active antitumor effect and its molecular mechanism, using mass spectrometry analysis and some classic Immunoassay techniques, such as adaptive immunity, innate immunity, or regulating immunity, et al. This study is expected to provide further direct theoretical and experimental basis for the device and application of allogeneic tumor cell derived HSP.PC tumor vaccines.

热休克蛋白抗原肽复合物疫苗（HSP.PC）是目前肿瘤生物治疗的热点之一，我们从异体mRNA-DC瘤苗中提取HSP70.PC作为多肽疫苗，并初步证实了该疫苗能够诱导出特异性抗骨肉瘤免疫效应。然而，该疫苗发挥抗肿瘤效应的分子水平机制尚不够清晰。前期研究发现以HSP70.PC为代表的热休克蛋白抗原肽复合物诱导的抗肿瘤效应与肿瘤细胞免疫原性死亡（ICD）效应相似，推测HSP.PC极有可能是ICD过程释放出的肿瘤抗原肽存在形式，这需要从肿瘤抗原肽的氨基酸构成水平提供证据；另外，该肿瘤抗原成分在DC细胞内呈递过程具体有哪些信号通路参与，以及多价瘤苗的抗肿瘤效应是否通过打破免疫抑制而得以实现，上述假设和疑问均需要进一步的实验验证。本研究拟采用二维电泳、质谱分析、适应免疫、固有免疫、调节免疫等分析技术，深入研究HSP.PC瘤苗抗肿瘤效应的分子机制，为HSP.PC瘤苗的设计和应用，提供直接的理论和实验依据。

在前期的研究中，我们将异体肿瘤细胞来源的mRNA转染至树突状细胞制备成异体瘤苗，并验证了其特异性抗骨肉瘤免疫学效应。本研究以上述细胞为基础，从中提取出HSP70.PC作为多肽疫苗，通过体外ELISPOT实验，CTL杀伤效应实验和体内免疫预防保护实验、主动免疫治疗实验等系列研究来检测其特异性抗骨肉瘤免疫效应。我们还对HSP70.PC疫苗的免疫调节作用进行探索，异体mRNA-DC瘤苗来源HSP70.PC组，CD8+ T细胞表面CTLA-4、TIM3、PD-1等“负免疫调节”分子比例较对照组显著减低；其MDSC数量与CD8+ T细胞表面CTLA-4、TIM3、PD-1等“负免疫调节”分子呈正相关，表明其抗肿瘤效应可能是通过克服荷瘤动物体内的“免疫抑制”状态而得以实现。为深入研究HSP70.PC瘤苗抗肿瘤效应的分子机制，我们采用免疫共沉淀结合质谱分析技术对该疫苗的氨基酸组成进行分析，通过与肿瘤抗原肽数据库对比分析，选出三个可能在免疫过程中起到关键作用的目标抗原肽，并通过进一步的比较研究，确定了其中最可能的目标抗原肽KIF20A。差异表达KIF20A的HSP70-PC疫苗的免疫调节作用存在显著差异，过表达KIF20A的HSP70-PC疫苗能与对照组相比，能显著增加T细胞的免疫活化能力；而低表达KIF20A的HSP70-PC疫苗其对T细胞的免疫活化能力明显下降；过表达KIF20A的HSP70-PC疫苗接种瘤荷大鼠后，生存期明显提升；而低表达KIF20A的HSP70-PC疫苗接种瘤荷大鼠后生存期明显下降。本研究通过对荷瘤模型在接受 HSP70.PC 疫苗治疗的前后，机体的免疫抑制状态的比较，来验证HSP70.PC 多价瘤苗的抗肿瘤效是通过打破免疫抑制而得以实现，从而为阐明该疫苗抗肿瘤免疫效应的分子机制研究提供实验依据。