负载gp96-Ki67肽复合物的DC疫苗对肾癌的抗肿瘤效应研究

Renal cell carcinoma is a highly malignant tumor of urology surgical system, developed rapidly. The prognosis is poor and not sensitive to conventional radiotherapy and chemotherapy. DC vaccine for RCC has been extensively studied in rencent years. The two remarkable factors influence the curative effects of vaccine. Firstly，determine the optimal CTL-epitope peptide; secondly，choose the right immune adjunvant to improve immuogenicity..Our Previous research found Ki-67280-288 (LQGETQLLV) had the strongest binding affinity with HLA-A*0201. Further study revealed that Ki-67280-288 increased the frequency of IFN-γ-producing T cells. Gp96 functions as molecular chaperone and can associate with antigenic peptides elicit cancer-specific protective T cell immune response that is recallable, which is a prerequisite for gp96 as a therapeutic vaccine against cancers. So this project will assemble gp96-ki67 compelx in vitro, loaded on dendritic cell. We will confirm the DC vaccine can elicit ki67-spcecific immune response through cytotoxicity test in vivo and in vitro. Simultaneously clarify the anti-tumor mechanism of gp96-ki67 compelx vaccine. The study will provide theoretical and experimental basis on the clinical application of vaccine to treat renal cell carcinoma.

肾癌恶性程度高，发展迅速，并对放疗和化疗不敏感，近年来利用DC疫苗治疗肾癌的研究备受关注。而影响DC疫苗疗效的两个关键因素：一是确定合适的肿瘤抗原，特别是抗原CTL表位肽。二是运用免疫佐剂提高多肽疫苗的免疫原性。.本项目基于前期研究证实位于280-288位置的LQGETQLLV多肽是Ki-67抗原的HLA-A*0201限制性CTL表位，免疫原性良好。同时鉴于热休克蛋白gp96作为高效的免疫佐剂能特异性结合抗原肽，介导并增强抗原递呈，本研究拟将Ki67280-288表位肽与纯化的gp96在体外组装成gp96-Ki67抗原肽复合物并负载DC，通过体内外细胞毒性试验证实该疫苗是否能诱发针对Ki67的特异性免疫效应，同时阐明gp96-Ki67复合物抗肿瘤机制，为负载gp96-Ki67复合物DC疫苗治疗肾癌的临床应用提供理论和实验依据。

申请者前期利用抗原表位预测软件预测人Ki67抗原CTL表位，并结合功能试验证实位于280-288位置的LQGETQLLV多肽是Ki67抗原HLA-A*0201限制性CTL表位肽，免疫原性良好。该蛋白作为肿瘤多肽疫苗治疗中的新靶点，拓展了肿瘤疫苗治疗的临床应用。本研究将Ki67280-288表位肽与纯化的gp96在体外组装成gp96-Ki67280-288抗原肽复合物；将此复合物负载于树突状细胞制备成树突状细胞疫苗，研究该疫苗的体内外杀伤效应。体外杀伤实验证实该疫苗诱导产生的抗原特异性CTL对HLA-A2+/Ki67+的肿瘤细胞有明显的抑制作用；体外用ELISA法检测IFN-γ的浓度结果显示负载gp96-Ki67280-288抗原肽复合物组CTL细胞释放的IFN-γ浓度高于对照组。裸鼠肾癌移植瘤实验证实负载gp96-Ki67280-288抗原肽复合物的树突状细胞疫苗能抑制肿瘤细胞生长，诱导肿瘤细胞凋亡。本课题的研究初步揭示该树突状细胞疫苗能诱导针对Ki67特异性的免疫反应，为以Ki67为靶点的肿瘤免疫治疗提供理论依据。