B7-H1疫苗增强HER-2疫苗抗肿瘤免疫效应的研究
发布时间：2019-05-18 03:54
【摘要】： 虽然肿瘤疫苗在动物和人体内可诱发特异性的抗肿瘤免疫反应,但其临床治疗效果较差。免疫系统不能引起肿瘤消退是肿瘤疫苗研究中所面临的一个巨大难题。已知肿瘤存在多种逃避免疫系统识别和攻击的机制,研究肿瘤免疫逃逸机制对于提高肿瘤疫苗的治疗效果具有重要意义。 B7-H1是共刺激分子家族新成员之一。通过与T细胞和B细胞表面的PD-1和非PD-1受体结合参与T细胞功能调节和细胞因子分泌。B7-H1蛋白分子在正常组织中几乎不表达,但普遍存在于人肺癌、卵巢癌、结肠癌、肾癌和黑色素瘤等多种肿瘤细胞表面,并且与肿瘤的进展程度相关。在外周组织,肿瘤细胞表面的B7-H1能诱导肿瘤特异性CTL凋亡从而抑制机体对肿瘤的免疫反应;而在淋巴器官内,抗原呈递细胞表面的B7-H1与初始T淋巴细胞相互作用诱导T淋巴细胞的无能。因此,理论上,B7-H1即可以作为一种肿瘤抗原,又是参与肿瘤免疫逃逸的重要分子;阻断B7-H1,既能直接抑制肿瘤的生长,又能抑制肿瘤的免疫逃避,提高初始T细胞的激活能力和CTL的杀伤活性,从而提高其它抗原的免疫反应。 HER2/neu蛋白是一种肿瘤相关抗原,在多种肿瘤细胞中过表达,并且与肿瘤的预后不良相关,是肿瘤免疫治疗的理想靶点。HER-2的单抗Herceptin于1998年被美国FDA批准上市,在治疗HER-2阳性乳腺癌转移患者中取得了明显疗效;但Herceptin有心脏毒性的毒副作用。HER-2的主动免疫治疗——疫苗,能够激活CTL,诱导多克隆抗体反应,从而更好的激发抗体依赖的效应功能,与被动的单抗治疗相比有更大优势。多年来,针对HER-2的疫苗研究层出不穷,类型多样,包括多肽疫苗,基因疫苗,细胞疫苗等。但是由于HER-2是自身抗原,难以突破免疫耐受;肿瘤又存在多种免疫逃避的方式,疫苗实验始终不能诱导有效的免疫反应。 本实验室之前工作已证实B7-H1蛋白疫苗免疫小鼠可诱导出高滴度的抗B7-H1抗体,并能够明显抑制B7-H1+SP20转移瘤的生长.本课题旨在证实B7-H1蛋白疫苗能否切实阻断一般转移瘤的免疫逃避,提高相应肿瘤抗原的免疫反应和抑瘤效果。 在本课题中,我们根据文献报道,选取了HER-2分子中包含抗原表位较多,抗原性较强的胞外区近膜端一段序列,构建HER-2疫苗。将分泌性较强的人Ig的信号肽序列融合HER-2基因片段,插入真核表达载体pRC-CMV中构建了HER-2基因疫苗——pRC-CMV-信号肽-HER2-ECD片段(简称为CH疫苗);又将这段HER-2序列插入原核表达载体pQE-30中,构建了pQE-30-rhHER2重组质粒,转化大肠杆菌,进行了蛋白的表达与纯化,得到HER2-ECD多肽疫苗(简称为pH疫苗)。另外,活化本实验室保存的包含pQE-30-TT-B7-H1IgV重组质粒的菌种,按之前的工艺表达纯化B7-H1IgV蛋白。 分别用CH疫苗和pH疫苗与B7-H1疫苗联合免疫BALB/c小鼠,用ELISA,ELISPOT方法检测抗体滴度和CTL反应,观察疫苗是否具有协同效应,结果显示B7-H1疫苗能增强CH疫苗和pH疫苗诱导的体液和细胞免疫反应。进一步观察疫苗联合应用能否具有更强的抗肿瘤效果,小鼠免疫后,接种HER-2/neu+小鼠乳腺癌细胞株EMT6,结果显示疫苗能够抑制转移瘤的生长,并且疫苗联合应用抑瘤效果最好,说明疫苗具有协同作用,B7-H1疫苗能增强CH疫苗和pH疫苗的抑瘤效果。又观察了疫苗对荷瘤小鼠的治疗作用,结果显示疫苗能在一定程度上抑制荷瘤小鼠肿瘤的生长。虽然荷瘤小鼠的肿瘤个体差异很大,但能看出疫苗联合应用更有效的抑制了肿瘤的生长。 最后,本实验分析了疫苗应用的毒理作用。常规免疫BALB/c小鼠,分别观察各实验组小鼠的进食,体重等一般状况,血液学和血清生化学指标,重要脏器组织的大体病理学,组织病理学情况及相对重量,CD4/CD8细胞百分率和比值。结果显示基因疫苗组的小鼠体重减轻,说明基因疫苗有一定毒性。血液学和血清生化学检查有个别组的个别指标上下浮动;各脏器未发生显著的异常所见,也未发现小鼠明显的组织病理学病变。免疫组的CD4+细胞百分率普遍增高,说明引起了免疫反应。 本实验证实了B7-H1疫苗与HER-2肿瘤疫苗联合应用具有协同作用,B7-H1疫苗能显著增强HER-2肿瘤疫苗诱导的体液和细胞免疫反应,并能进一步提高HER-2肿瘤疫苗的抑瘤效果。为构建既能提高抗肿瘤特异性免疫反应,又可阻断肿瘤免疫逃避的肿瘤疫苗提供新的设计思路和实验依据。
[Abstract]:Although the tumor vaccine can induce specific anti-tumor immune response in animals and human bodies, the clinical treatment effect of the tumor vaccine is poor. The immune system cannot cause tumor regression to be a great challenge for tumor vaccine research. It is known that a variety of mechanisms for avoiding the identification and attack of the immune system exist in the tumor, and the research on the tumor immune escape mechanism is of great significance to the improvement of the therapeutic effect of the tumor vaccine. B7-H1 is a new member of the costimulatory molecule family One of the genes involved in T cell function regulation and cytokine by binding to PD-1 and non-PD-1 receptors on T-cell and B-cell surface The secretion of B7-H1 protein is almost non-expressed in normal tissues, but is common in various tumor cell surfaces such as human lung cancer, ovarian cancer, colon cancer, renal cell carcinoma and melanoma, and the degree of progression of the tumor In the peripheral tissue, the B7-H1 on the surface of the tumor cell can induce the apoptosis of the tumor-specific CTL to inhibit the immune response of the body to the tumor, and the B7-H1 on the surface of the antigen-presenting cell and the initial T-lymphocyte interact with the initial T-lymphocyte to induce the T-lymphocyte in the lymphoid organ. Therefore, in theory, B7-H1 can be used as a tumor antigen and is an important molecule involved in the immune escape of the tumor; the B7-H1 is blocked, so that the growth of the tumor can be directly inhibited, the immune escape of the tumor can be inhibited, the activation capacity of the initial T cell and the killing of the CTL can be improved, the activity, thereby enhancing the immunity of other antigens, The HER2/ neu protein is a tumor-related antigen that is overexpressed in a variety of tumor cells and is associated with a poor prognosis of the tumor, which is a tumor immunotherapy The ideal target for HER-2 was approved by the US FDA in 1998 and has a clear therapeutic effect in the treatment of HER-2-positive breast cancer metastasis; however, Herceptin has a cardiotoxicity The active immunization therapy _ vaccine of HER-2 can activate the CTL to induce the multi-clone antibody to react, thereby better exciting the effect function of the antibody dependence, and compared with the passive monoclonal antibody treatment, There is a greater advantage. Over the years, there are a number of vaccine studies for HER-2, with a variety of types, including polypeptide vaccines, gene vaccines, Cell vaccine, etc. However, because HER-2 is an autoantigen, it is difficult to break through the immune tolerance. There are many ways of immune escape in the tumor, and the vaccine experiment can not be induced to be effective at all times. It has been confirmed that the B7-H1 protein vaccine immunized mice can induce high-titer anti-B7-H1 antibody and can obviously inhibit the B7-H1 + SP. The purpose of this study is to confirm whether the B7-H1 protein vaccine can effectively block the immune escape of the general metastatic tumor and improve the immunity of the corresponding tumor antigen. In this subject, we have selected HER-2 molecules to contain more antigenic epitopes and stronger antigenicity of the near-membrane end of the extracellular domain, according to the literature. The HER-2 vaccine was constructed by fusion of the signal peptide sequence of the human Ig with stronger secretion. The HER-2 gene vaccine _ pRC-CMV-signal peptide-HER2-ECD fragment (abbreviated as CH vaccine) was constructed in the eukaryotic expression vector pRC-CMV, and the HER-2 sequence was inserted into the prokaryotic expression vector pQE-30 to construct pQE-30. -rhHER2 recombinant plasmid, transforming E. coli, carrying out the expression and purification of the protein, and obtaining the HER2-ECD polypeptide. The vaccine (hereinafter referred to as a pH vaccine). In addition, a strain containing the pQE-30-TT-B7-1 IgV recombinant plasmid, which was stored in the laboratory, was activated and expressed as a result of the previous process B7-H1 IgV was used to immunize BALB/ c mice with CH vaccine and pH vaccine and B7-H1 vaccine, and the antibody titer and CTL response were detected by ELISA and ELISPOT method. The results showed that the B7-H1 vaccine can enhance the CH vaccine and the pH value. The result shows that the vaccine can inhibit the growth of the metastatic tumor and the vaccine can be used in combination with the anti-tumor effect. The best results show that the vaccine has a synergistic effect, and the B7-H1 vaccine can be enhanced. The effect of the vaccine on the tumor-bearing mice was also observed, and the results showed that the vaccine can be used in the treatment of tumor-bearing mice. To some extent, the tumor growth of the tumor-bearing mice was inhibited. Although the individual difference of the tumor in the tumor-bearing mice was large, it can be seen that the vaccine combination The application of the invention can effectively inhibit the growth of the tumor, The toxicological effects of the vaccine application were analyzed in this experiment. The general conditions, such as the feeding and body weight of the mice in the experimental group, the general conditions, the hematology and the serum biochemical indexes, the general pathology, the histopathology and the relative weight of the important organ tissues, were observed in the conventional immunized BALB/ c mice. Volume, percentage and ratio of CD4/ CD8 cells. The results showed that the gene vaccine group was small The weight loss of the mouse indicated that the gene vaccine had a certain toxicity. The hematology and serum biochemical tests showed that the individual indexes of the individual groups were floating up and down; no significant abnormal findings were observed in the organs No obvious histopathological changes in mice were found. CD4 + cells in the immune group The results show that the B7-H1 vaccine can significantly enhance the humoral and cellular immune response induced by the HER-2 tumor vaccine, and the B7-H1 vaccine can significantly enhance the humoral and cellular immune response induced by the HER-2 tumor vaccine. The anti-tumor effect of the HER-2 tumor vaccine can be further improved, and the tumor-inhibiting effect of the HER-2 tumor vaccine can be improved,
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392;R730.5
本文编号：2479647
[Abstract]:Although the tumor vaccine can induce specific anti-tumor immune response in animals and human bodies, the clinical treatment effect of the tumor vaccine is poor. The immune system cannot cause tumor regression to be a great challenge for tumor vaccine research. It is known that a variety of mechanisms for avoiding the identification and attack of the immune system exist in the tumor, and the research on the tumor immune escape mechanism is of great significance to the improvement of the therapeutic effect of the tumor vaccine. B7-H1 is a new member of the costimulatory molecule family One of the genes involved in T cell function regulation and cytokine by binding to PD-1 and non-PD-1 receptors on T-cell and B-cell surface The secretion of B7-H1 protein is almost non-expressed in normal tissues, but is common in various tumor cell surfaces such as human lung cancer, ovarian cancer, colon cancer, renal cell carcinoma and melanoma, and the degree of progression of the tumor In the peripheral tissue, the B7-H1 on the surface of the tumor cell can induce the apoptosis of the tumor-specific CTL to inhibit the immune response of the body to the tumor, and the B7-H1 on the surface of the antigen-presenting cell and the initial T-lymphocyte interact with the initial T-lymphocyte to induce the T-lymphocyte in the lymphoid organ. Therefore, in theory, B7-H1 can be used as a tumor antigen and is an important molecule involved in the immune escape of the tumor; the B7-H1 is blocked, so that the growth of the tumor can be directly inhibited, the immune escape of the tumor can be inhibited, the activation capacity of the initial T cell and the killing of the CTL can be improved, the activity, thereby enhancing the immunity of other antigens, The HER2/ neu protein is a tumor-related antigen that is overexpressed in a variety of tumor cells and is associated with a poor prognosis of the tumor, which is a tumor immunotherapy The ideal target for HER-2 was approved by the US FDA in 1998 and has a clear therapeutic effect in the treatment of HER-2-positive breast cancer metastasis; however, Herceptin has a cardiotoxicity The active immunization therapy _ vaccine of HER-2 can activate the CTL to induce the multi-clone antibody to react, thereby better exciting the effect function of the antibody dependence, and compared with the passive monoclonal antibody treatment, There is a greater advantage. Over the years, there are a number of vaccine studies for HER-2, with a variety of types, including polypeptide vaccines, gene vaccines, Cell vaccine, etc. However, because HER-2 is an autoantigen, it is difficult to break through the immune tolerance. There are many ways of immune escape in the tumor, and the vaccine experiment can not be induced to be effective at all times. It has been confirmed that the B7-H1 protein vaccine immunized mice can induce high-titer anti-B7-H1 antibody and can obviously inhibit the B7-H1 + SP. The purpose of this study is to confirm whether the B7-H1 protein vaccine can effectively block the immune escape of the general metastatic tumor and improve the immunity of the corresponding tumor antigen. In this subject, we have selected HER-2 molecules to contain more antigenic epitopes and stronger antigenicity of the near-membrane end of the extracellular domain, according to the literature. The HER-2 vaccine was constructed by fusion of the signal peptide sequence of the human Ig with stronger secretion. The HER-2 gene vaccine _ pRC-CMV-signal peptide-HER2-ECD fragment (abbreviated as CH vaccine) was constructed in the eukaryotic expression vector pRC-CMV, and the HER-2 sequence was inserted into the prokaryotic expression vector pQE-30 to construct pQE-30. -rhHER2 recombinant plasmid, transforming E. coli, carrying out the expression and purification of the protein, and obtaining the HER2-ECD polypeptide. The vaccine (hereinafter referred to as a pH vaccine). In addition, a strain containing the pQE-30-TT-B7-1 IgV recombinant plasmid, which was stored in the laboratory, was activated and expressed as a result of the previous process B7-H1 IgV was used to immunize BALB/ c mice with CH vaccine and pH vaccine and B7-H1 vaccine, and the antibody titer and CTL response were detected by ELISA and ELISPOT method. The results showed that the B7-H1 vaccine can enhance the CH vaccine and the pH value. The result shows that the vaccine can inhibit the growth of the metastatic tumor and the vaccine can be used in combination with the anti-tumor effect. The best results show that the vaccine has a synergistic effect, and the B7-H1 vaccine can be enhanced. The effect of the vaccine on the tumor-bearing mice was also observed, and the results showed that the vaccine can be used in the treatment of tumor-bearing mice. To some extent, the tumor growth of the tumor-bearing mice was inhibited. Although the individual difference of the tumor in the tumor-bearing mice was large, it can be seen that the vaccine combination The application of the invention can effectively inhibit the growth of the tumor, The toxicological effects of the vaccine application were analyzed in this experiment. The general conditions, such as the feeding and body weight of the mice in the experimental group, the general conditions, the hematology and the serum biochemical indexes, the general pathology, the histopathology and the relative weight of the important organ tissues, were observed in the conventional immunized BALB/ c mice. Volume, percentage and ratio of CD4/ CD8 cells. The results showed that the gene vaccine group was small The weight loss of the mouse indicated that the gene vaccine had a certain toxicity. The hematology and serum biochemical tests showed that the individual indexes of the individual groups were floating up and down; no significant abnormal findings were observed in the organs No obvious histopathological changes in mice were found. CD4 + cells in the immune group The results show that the B7-H1 vaccine can significantly enhance the humoral and cellular immune response induced by the HER-2 tumor vaccine, and the B7-H1 vaccine can significantly enhance the humoral and cellular immune response induced by the HER-2 tumor vaccine. The anti-tumor effect of the HER-2 tumor vaccine can be further improved, and the tumor-inhibiting effect of the HER-2 tumor vaccine can be improved,
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392;R730.5
【引证文献】
相关硕士学位论文 前2条
1 郭壮;杯状细胞在共聚焦显微内镜诊断胃黏膜肠化生中的价值研究[D];山东大学;2011年
2 陈霖;重组人B7-H1IgV肿瘤疫苗的发酵纯化工艺研究和抑瘤活性的检测[D];第四军医大学;2010年
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