B7-H1疫苗增強(qiáng)HER-2疫苗抗腫瘤免疫效應(yīng)的研究
發(fā)布時間：2019-05-18 03:54
【摘要】： 雖然腫瘤疫苗在動物和人體內(nèi)可誘發(fā)特異性的抗腫瘤免疫反應(yīng),但其臨床治療效果較差。免疫系統(tǒng)不能引起腫瘤消退是腫瘤疫苗研究中所面臨的一個巨大難題。已知腫瘤存在多種逃避免疫系統(tǒng)識別和攻擊的機(jī)制,研究腫瘤免疫逃逸機(jī)制對于提高腫瘤疫苗的治療效果具有重要意義。 B7-H1是共刺激分子家族新成員之一。通過與T細(xì)胞和B細(xì)胞表面的PD-1和非PD-1受體結(jié)合參與T細(xì)胞功能調(diào)節(jié)和細(xì)胞因子分泌。B7-H1蛋白分子在正常組織中幾乎不表達(dá),但普遍存在于人肺癌、卵巢癌、結(jié)腸癌、腎癌和黑色素瘤等多種腫瘤細(xì)胞表面,并且與腫瘤的進(jìn)展程度相關(guān)。在外周組織,腫瘤細(xì)胞表面的B7-H1能誘導(dǎo)腫瘤特異性CTL凋亡從而抑制機(jī)體對腫瘤的免疫反應(yīng);而在淋巴器官內(nèi),抗原呈遞細(xì)胞表面的B7-H1與初始T淋巴細(xì)胞相互作用誘導(dǎo)T淋巴細(xì)胞的無能。因此,理論上,B7-H1即可以作為一種腫瘤抗原,又是參與腫瘤免疫逃逸的重要分子;阻斷B7-H1,既能直接抑制腫瘤的生長,又能抑制腫瘤的免疫逃避,提高初始T細(xì)胞的激活能力和CTL的殺傷活性,從而提高其它抗原的免疫反應(yīng)。 HER2/neu蛋白是一種腫瘤相關(guān)抗原,在多種腫瘤細(xì)胞中過表達(dá),并且與腫瘤的預(yù)后不良相關(guān),是腫瘤免疫治療的理想靶點。HER-2的單抗Herceptin于1998年被美國FDA批準(zhǔn)上市,在治療HER-2陽性乳腺癌轉(zhuǎn)移患者中取得了明顯療效;但Herceptin有心臟毒性的毒副作用。HER-2的主動免疫治療——疫苗,能夠激活CTL,誘導(dǎo)多克隆抗體反應(yīng),從而更好的激發(fā)抗體依賴的效應(yīng)功能,與被動的單抗治療相比有更大優(yōu)勢。多年來,針對HER-2的疫苗研究層出不窮,類型多樣,包括多肽疫苗,基因疫苗,細(xì)胞疫苗等。但是由于HER-2是自身抗原,難以突破免疫耐受;腫瘤又存在多種免疫逃避的方式,疫苗實驗始終不能誘導(dǎo)有效的免疫反應(yīng)。 本實驗室之前工作已證實B7-H1蛋白疫苗免疫小鼠可誘導(dǎo)出高滴度的抗B7-H1抗體,并能夠明顯抑制B7-H1+SP20轉(zhuǎn)移瘤的生長.本課題旨在證實B7-H1蛋白疫苗能否切實阻斷一般轉(zhuǎn)移瘤的免疫逃避,提高相應(yīng)腫瘤抗原的免疫反應(yīng)和抑瘤效果。 在本課題中,我們根據(jù)文獻(xiàn)報道,選取了HER-2分子中包含抗原表位較多,抗原性較強(qiáng)的胞外區(qū)近膜端一段序列,構(gòu)建HER-2疫苗。將分泌性較強(qiáng)的人Ig的信號肽序列融合HER-2基因片段,插入真核表達(dá)載體pRC-CMV中構(gòu)建了HER-2基因疫苗——pRC-CMV-信號肽-HER2-ECD片段(簡稱為CH疫苗);又將這段HER-2序列插入原核表達(dá)載體pQE-30中,構(gòu)建了pQE-30-rhHER2重組質(zhì)粒,轉(zhuǎn)化大腸桿菌,進(jìn)行了蛋白的表達(dá)與純化,得到HER2-ECD多肽疫苗(簡稱為pH疫苗)。另外,活化本實驗室保存的包含pQE-30-TT-B7-H1IgV重組質(zhì)粒的菌種,按之前的工藝表達(dá)純化B7-H1IgV蛋白。 分別用CH疫苗和pH疫苗與B7-H1疫苗聯(lián)合免疫BALB/c小鼠,用ELISA,ELISPOT方法檢測抗體滴度和CTL反應(yīng),觀察疫苗是否具有協(xié)同效應(yīng),結(jié)果顯示B7-H1疫苗能增強(qiáng)CH疫苗和pH疫苗誘導(dǎo)的體液和細(xì)胞免疫反應(yīng)。進(jìn)一步觀察疫苗聯(lián)合應(yīng)用能否具有更強(qiáng)的抗腫瘤效果,小鼠免疫后,接種HER-2/neu+小鼠乳腺癌細(xì)胞株EMT6,結(jié)果顯示疫苗能夠抑制轉(zhuǎn)移瘤的生長,并且疫苗聯(lián)合應(yīng)用抑瘤效果最好,說明疫苗具有協(xié)同作用,B7-H1疫苗能增強(qiáng)CH疫苗和pH疫苗的抑瘤效果。又觀察了疫苗對荷瘤小鼠的治療作用,結(jié)果顯示疫苗能在一定程度上抑制荷瘤小鼠腫瘤的生長。雖然荷瘤小鼠的腫瘤個體差異很大,但能看出疫苗聯(lián)合應(yīng)用更有效的抑制了腫瘤的生長。 最后,本實驗分析了疫苗應(yīng)用的毒理作用。常規(guī)免疫BALB/c小鼠,分別觀察各實驗組小鼠的進(jìn)食,體重等一般狀況,血液學(xué)和血清生化學(xué)指標(biāo),重要臟器組織的大體病理學(xué),組織病理學(xué)情況及相對重量,CD4/CD8細(xì)胞百分率和比值。結(jié)果顯示基因疫苗組的小鼠體重減輕,說明基因疫苗有一定毒性。血液學(xué)和血清生化學(xué)檢查有個別組的個別指標(biāo)上下浮動;各臟器未發(fā)生顯著的異常所見,也未發(fā)現(xiàn)小鼠明顯的組織病理學(xué)病變。免疫組的CD4+細(xì)胞百分率普遍增高,說明引起了免疫反應(yīng)。 本實驗證實了B7-H1疫苗與HER-2腫瘤疫苗聯(lián)合應(yīng)用具有協(xié)同作用,B7-H1疫苗能顯著增強(qiáng)HER-2腫瘤疫苗誘導(dǎo)的體液和細(xì)胞免疫反應(yīng),并能進(jìn)一步提高HER-2腫瘤疫苗的抑瘤效果。為構(gòu)建既能提高抗腫瘤特異性免疫反應(yīng),又可阻斷腫瘤免疫逃避的腫瘤疫苗提供新的設(shè)計思路和實驗依據(jù)。
[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,
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392;R730.5
本文編號：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,
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392;R730.5
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前2條
1 郭壯;杯狀細(xì)胞在共聚焦顯微內(nèi)鏡診斷胃黏膜腸化生中的價值研究[D];山東大學(xué);2011年
2 陳霖;重組人B7-H1IgV腫瘤疫苗的發(fā)酵純化工藝研究和抑瘤活性的檢測[D];第四軍醫(yī)大學(xué);2010年
，本文編號：2479647
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