重组MUC1-MBP抗肿瘤疫苗临床前毒性及免疫原性研究

发布时间：2018-12-21 20:13

【摘要】：Mucin 1(简称MUC1),是黏蛋白家族的跨膜糖蛋白,由多肽核芯和侧枝糖链构成,主要分布于正常腺管上皮细胞及其来源的癌细胞以及部分血液肿瘤细胞表面,作为癌基因在癌细胞的发生发展中发挥重要作用。由于MUC1在正常组织和癌组织中存在明显的差别,使其成为肿瘤疫苗研究的有效靶点。目前,以MUC1为靶点研制的肿瘤疫苗主要包括糖疫苗、蛋白或多肽疫苗和DNA疫苗。其中重组蛋白疫苗因安全、成本低廉而倍受关注,并且反复动物实验和部分人体实验证实其安全有效。本研究组将编码人类MUC1基因按正确阅读框插入高效表达麦芽糖结合蛋白(maltose binding protein,MBP)的载体中,构建了表达MUC1-MBP的重组质粒,获得稳定表达重组MUC1-MBP融合蛋白的工程菌,建立了中试发酵和蛋白生产工艺。我们前期研究结果显示,重组MUC1-MBP抗肿瘤疫苗可以诱导MUC1特异性Th1型免疫应答,促进MUC1特异性细胞毒性T细胞的杀伤活性,并且可以抑制MUC1+B16细胞在小鼠体内的生长。为了促使重组MUC1-MBP抗肿瘤疫苗进入I期临床试验,本课题对重组MUC1-MBP抗肿瘤疫苗的毒性及免疫原性进行研究,包括小鼠单剂量急性毒性、大鼠反复剂量慢性毒性和免疫原性以及探索性食蟹猴毒性和免疫原性研究。结果显示,除了大鼠中偶发BCG诱导的关节炎和局部结节,在动物体内重组MUC1-MBP抗肿瘤疫苗没有导致明显的器官毒性。另外,重组MUC1-MBP抗肿瘤疫苗在大鼠中显著的诱导IFN-γ、TNF的分泌,揭示Th1细胞被激活。重组MUC1-MBP抗肿瘤疫苗在大鼠和食蟹猴中均诱导了MUC1特异性Ig G抗体。本研究提示重组MUC1-MBP抗肿瘤疫苗对大鼠和小鼠无明显毒性,并且大鼠和食蟹猴体免疫后产生较强的免疫活性。本研究为推动重组MUC1-MBP进入临床试验奠定实验基础,为临床研究方案的设计提供依据。
[Abstract]:Mucin 1 (MUC1), a transmembrane glycoprotein of mucin family, is composed of polypeptide core and side sugar chain. It mainly distributes on the surface of normal adenoepithelial cells, cancer cells derived from them and some blood tumor cells. As a oncogene, it plays an important role in the carcinogenesis and development of cancer cells. Because of the obvious difference between normal tissue and cancer tissue, MUC1 has become an effective target for tumor vaccine research. At present, tumor vaccines targeting MUC1 include sugar vaccine, protein or polypeptide vaccine and DNA vaccine. Among them, recombinant protein vaccine has attracted much attention because of its safety and low cost, and has been proved to be safe and effective by repeated animal experiments and some human experiments. In this study, the human MUC1 gene was inserted into the vector expressing maltose binding protein (maltose binding protein,MBP) according to the correct reading frame, and the recombinant plasmid expressing MUC1-MBP was constructed, and the engineering bacteria stably expressing the recombinant MUC1-MBP fusion protein were obtained. A pilot-scale fermentation and protein production process was established. Our previous studies showed that the recombinant MUC1-MBP antitumor vaccine could induce MUC1 specific Th1 type immune response, promote the cytotoxic T cell killing activity of MUC1 specific cells, and inhibit the growth of MUC1 B16 cells in mice. In order to make recombinant MUC1-MBP antitumor vaccine enter phase I clinical trial, the toxicity and immunogenicity of recombinant MUC1-MBP antitumor vaccine were studied, including single dose acute toxicity in mice. The chronic toxicity and immunogenicity of repeated doses of rats and the toxicity and immunogenicity of exploratory crab-eating monkeys were studied. The results showed that, except for the occasional BCG induced arthritis and local nodules in rats, the recombinant MUC1-MBP antitumor vaccine did not cause significant organ toxicity in animals. In addition, recombinant MUC1-MBP antitumor vaccine significantly induced the secretion of IFN- 纬 and TNF in rats, which revealed that Th1 cells were activated. The recombinant MUC1-MBP antitumor vaccine induced MUC1 specific Ig G antibody in both rat and monkey. This study suggested that the recombinant MUC1-MBP antitumor vaccine had no obvious toxicity to rats and mice, and the immune activity of rats and crab-eating monkeys was stronger. This study provides experimental basis for promoting recombinant MUC1-MBP into clinical trials and provides basis for the design of clinical research scheme.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.3

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