人端粒酶逆转录酶CTL表位多抗原肽（MAP）疫苗的设计及其抗肿瘤活性研究
发布时间：2018-08-15 16:01
【摘要】：[背景和目的] 恶性肿瘤是危害人类健康的主要疾病之一，传统的手术、放、化疗的疗效又不尽人意，这种局限决定了生物治疗必然要参与肿瘤的综合治疗，而肿瘤疫苗是肿瘤生物治疗的重要方法。目前常用的肿瘤疫苗传递方式分别以细胞、蛋白质、多肽、病毒载体为基础。树突细胞(Dendritic cells, DCs)是目前已知的抗原提呈功能最强的免疫细胞，将肿瘤相关抗原(Tumor-asscociated antigen, TAA)负载DC是目前常用的肿瘤免疫治疗的方式之一。但目前发现的肿瘤相关抗原大多具有组织特异性，如AFP只针对肝癌等，以这些抗原来进行免疫治疗，只能对相应的肿瘤起作用，限制了以这些抗原为靶标的肿瘤疫苗的广泛应用。寻找肿瘤特异性的共有抗原是肿瘤免疫治疗的关键问题。 人端粒酶逆转录酶，又名人端粒酶催化亚单位，为端粒酶活性的限速成分，是癌细胞永生化的必要途径，在维持肿瘤继续分裂、增殖和生存中发挥重要作用。国内外许多研究表明，人端粒酶逆转录酶在85％以上的恶性肿瘤及其癌前病变中过度表达，其异常表达或激活是肿瘤形成的重要原因之一，新近研究报道人端粒酶逆转录酶的高表达也与肿瘤的不良预后、浸润转移均密切相关。我们过去的研究表明，带有hTERT全长基因的腺病毒载体负载DC，以及带有hTERT全长基因和隐性表位的颗粒疫苗，在体内外均可以诱导产生hTERT特异性的细胞毒T淋巴细胞(CTLs)，对hTERT阳性的胃癌、结肠癌等多种癌细胞具有一定的杀伤效应，而对表达hTERT阴性的淋巴细胞和DC不具有杀伤效应。与hTERT全基因序列腺病毒负载DC疫苗相比，多肽疫苗没有腺病毒载体的参与，不必考虑载体及其整合的安全性，也不必考虑全长蛋白中某些非优势表位或病理表位的毒副作用；而且，抗原肽长度仅8-15个左右的氨基酸序列，体外可以合成，临床应用前景更广阔。但多肽疫苗也常有其不足之处：最大的问题在于一个多肽疫苗只能代表一个表位，分子量小，结构单一，免疫原性较弱，在体内容易降解。为解决这一弊端，1988年Tam实验室提出了多抗原肽(Multiple antigen peptides,MAP)的设计方案，其聚赖氨酸核心构成一种十分紧密有序的树状结构，它在加强了抗原表位肽链结构特异性的同时，还增大其分子质量，不仅很好的模拟了天然表位的空间构象，而且还不需要再耦联载体蛋白就能诱导较强的免疫反应；同时，其稳定的二级结构不易反转，故降解速度减缓，延长抗原刺激时间，获得更好的杀伤活性。MAP疫苗的设计方案不仅适用于B细胞表位，同样也适用于T细胞表位。 基于以上分析，本实验拟选取人端粒酶逆转录酶HLA-A2限制性CTL表位hTERT-540(ILAKFLHWL), hTERT-865(RLVDDFLLV) and hTERT-572Y(YLFFYRKSV)，构建其四分支多抗原肽，研究其在体外体内的抗肿瘤活性，并与携带hTERT全长基因腺病毒载体疫苗、单肽疫苗诱导的CTL活性进行对比，为hTERT的MAP疫苗抗肿瘤效应的临床应用提供理论依据。 [方法] 1、选取三条人端粒酶逆转录酶CTL表位［hTERT-540(ILAKFLHWL), hTERT-865(RLVDDFLLV) and hTERT-572Y (YLFFYRKSV)］设计其MAP结构，采用标准固相肽合成法(SPSS)合成其四分支肽；反相高压液相色谱仪(RP-HPLC) 分析其纯度；液相色谱/质谱联用(LC/MS)检测多肽分子量。阴性肽选用人 HIV病毒HLA-A2限制性表位(ILLEP VHGV)。 2、按文献所述分离培养人外周血单个核细胞(PBMC)来源的树突细胞。采用光学显微镜观察其形态，并采用流式细胞术鉴定其细胞表型。然后将携带hTERT全长序列的腺病毒载体、人端粒酶逆转录酶MAP肽、及其对应单肽分别负载DC后，诱导产生人端粒酶逆转录酶特异性CTL。采用标准51Cr释放实验检测人端粒酶逆转录酶特异性CTL对不同肿瘤靶细胞[SW480结肠癌细胞(hTERT~+, HLA-A2~+)、KATO-Ⅲ胃癌细胞(hTERT~+, HLA-A2~+)、U2OS骨肉瘤细胞(hTERT-, HLA-A2~+),转染了人端粒酶逆转录酶cDNA的U2OS/hTERT骨肉瘤细胞(hTERT~+, HLA-A2~+), MCF-7乳腺癌细胞(hTERT~+, HLA-A2~+), HepG2肝癌细胞(hTERT~+, HLA-A2-),转染了HLA-A2cDNA的HepG2/HLA-A2肝癌细胞(hTERT~+, HLA-A2~+)的免疫杀伤活性；采取同样方法检测上述人端粒酶逆转录酶特异性CTL对低表达人端粒酶逆转录酶的自体淋巴细胞和树突细胞有无杀伤作用以研究其毒副作用；采用ELISPOT试验检测产IFN-γ效应细胞的数量。 3、按文献所述分离培养C57BL/6-Tg(HLA-A2~+)小鼠骨髓来源的树突细胞(mDC)， 采用光学显微镜观察其形态，采用流式细胞术鉴定其细胞表型。然后将携带hTERT全长序列的腺病毒载体、人端粒酶逆转录酶MAP肽、及其对应单肽分别负载mDC后，免疫小鼠，每周一次，共三次。最后一次免疫7天后取免疫小鼠脾淋巴细胞作为效应细胞，51Cr释放实验检测人端粒酶逆转录酶特异性CTL对上述的肿瘤靶细胞以及自体淋巴细胞和树突状细胞有无杀伤效应；采用ELISPOT试验检测产IFN-γ效应细胞的数量。 4、实验数据采用SPSS17.0统计软件进行t检验，当P㩳0.05时认为差异有统计学意义。 ［结果］ 1、通过反相高压液相色谱仪(RP-HPLC)检测合成的多抗原肽及对应单肽的纯度均在95%以上，达到国际多肽实验标准。液相色谱/质谱联用(LC/MS)检测合成的多抗原肽及对应单肽的分子量，结果表明所得肽的分子量理论值与实测值之间无明显差异，为我们所需的目的多肽。 2、体外(In vitro)和离体(Ex vivo)实验均提示，携带全长hTERT序列的腺病毒载体、人端粒酶逆转录酶特异性CTL对于人端粒酶逆转录酶阳性且HLA-A2阳性的SW480、MCF-7、KATO-Ⅲ细胞具有明显的杀伤作用，且携带全长hTERT序列的腺病毒载体诱导的杀伤效应最强；人端粒酶逆转录酶MAP所诱导的杀伤效应明显高于其对应单肽诱导的杀伤效应；但对HLA-A2阳性但人端粒酶逆转录酶阴性的U2OS细胞或人端粒酶逆转录酶阳性但HLA-A2阴性的HepG2细胞均无杀伤效应，对人端粒酶逆转录酶阳性且HLA-A2.1匹配的U2OS/hTERT细胞和HepG2/HLA-A2.1细胞，提示该CTL反应具有人端粒酶逆转录酶特异性及HLA-A2限制性。同时本实验还发现，无论是人端粒酶逆转录酶MAP肽诱导的CTL，还是其对应单肽诱导的效应细胞对低表达人端粒酶逆转录酶的自体淋巴细胞和DC都不具有杀伤效应，提示人端粒酶逆转录酶MAP肽疫苗的安全性。通过ELISPOT试验检测产IFN-γ效应细胞的数量，结果显示携带全长hTERT序列的腺病毒载体可以诱导效应细胞释放最多IFN-γ；人端粒酶逆转录酶MAP肽较之其对应单肽可以诱导更多的效应细胞产生IFN-γ。但在去除CD4＋T淋巴细胞后，携带全长hTERT序列的腺病毒载体、人端粒酶逆转录酶MAP肽及其对应单肽诱导效应细胞分泌IFN-γ的水平均明显下降，，提示CD4+T淋巴细胞在CD8+T淋巴细胞的免疫应答及应答维持中发挥重要作用。 ［结论］ DC负载的HLA-A2限制性人端粒酶逆转录酶CTL表位MAP疫苗相较于其单肽能在体外体内诱导更强的抗肿瘤免疫效应。这种人端粒酶逆转录酶MAP疫苗具有高效、安全的特点，为人端粒酶逆转录酶MAP肽疫苗的临床应用提供了实验依据。
[Abstract]:[background and purpose]
Malignant tumor is one of the main diseases that endanger human health. The efficacy of traditional surgery, radiotherapy and chemotherapy is unsatisfactory. This limitation determines that biotherapy must participate in the comprehensive treatment of tumor. Cancer vaccine is an important method of tumor biotherapy. Currently, the commonly used delivery methods of tumor vaccine are cell, protein, polypeptide. Dendritic cells (DCs) are the most potent antigen-presenting immune cells. Tumor-associated antigen (TAA) loaded with DCs is one of the most commonly used methods for tumor immunotherapy. Immunotherapy with these antigens can only work on the corresponding tumors, which limits the wide application of tumor vaccines targeting these antigens.
Human telomerase reverse transcriptase, also known as human telomerase catalytic subunit, is the rate-limiting component of telomerase activity and is an essential pathway for immortalization of cancer cells. It plays an important role in maintaining the continued division, proliferation and survival of tumors. Overexpression, abnormal expression or activation of human telomerase reverse transcriptase is one of the important causes of tumor formation. Recent studies have reported that high expression of human telomerase reverse transcriptase is also closely related to the poor prognosis, invasion and metastasis of tumors. Epitope-specific granular vaccines can induce hTERT-specific cytotoxic T lymphocytes (CTLs) in vitro and in vivo, and have a certain killing effect on hTERT-positive gastric cancer, colon cancer and other cancer cells, but have no killing effect on hTERT-negative lymphocytes and DC. Polypeptide vaccines have no adenovirus vectors, no need to consider the safety of vectors and their integration, and no need to consider the toxic and side effects of some non-dominant epitopes or pathological epitopes in full-length proteins. Moreover, antigen peptides with only 8-15 amino acid sequences can be synthesized in vitro and have a broader clinical application prospect. The biggest problem is that a polypeptide vaccine can only represent one epitope, with small molecular weight, single structure, weak immunogenicity and easy degradation in vivo. To solve this problem, Tam Laboratory put forward a design scheme of multiple antigen peptides (MAP) in 1988. Its polylysine core constitutes one kind of 10. Tightly ordered dendritic structures not only enhance the structural specificity of antigen epitope peptides, but also increase their molecular weight. They not only simulate the spatial conformation of natural epitopes, but also induce strong immune responses without recombinant carrier proteins. At the same time, their stable secondary structure is not easy to reverse, so the degradation rate is fast. The design of MAP vaccine is not only suitable for B cell epitope, but also for T cell epitope.
Based on the above analysis, we intend to construct four-branched polyantigenic peptides of human telomerase reverse transcriptase HLA-A2 restriction CTL epitopes hTERT-540 (ILAKFLHWL), hTERT-865 (RLVDDFLLV) and hTERT-572Y (YLFFYRKSV) to study their antitumor activities in vitro and in vivo, and to induce them with adenovirus vector vaccine carrying full-length hTERT gene and single peptide vaccine. The comparison of CTL activity provides a theoretical basis for the clinical application of hTERT MAP vaccine.
[method]
1. Three human telomerase reverse transcriptase CTL epitopes (hTERT-540 (ILAKFLHWL), hTERT-865 (RLVDDFLLV) and hTERT-572Y (YLFFYRKSV) were selected to design their MAP structures, and their tetrabranched peptides were synthesized by standard solid-phase peptide synthesis (SPSS); and their tetrabranched peptides were synthesized by RP-HPLC.
The purity of the peptide was analyzed by liquid chromatography / mass spectrometry (LC/MS).
HIV virus HLA-A2 restricted epitope (ILLEP VHGV).
2. Human peripheral blood mononuclear cells (PBMC) derived dendritic cells were isolated and cultured according to the literature. The morphology of PBMC was observed by optical microscope and its phenotype was identified by flow cytometry. The adenovirus vector carrying full-length sequence of hTERT, human telomerase reverse transcriptase MAP peptide and its corresponding monopeptide were loaded with DC respectively to induce the production of dendritic cells. Human telomerase reverse transcriptase-specific CTL. Human telomerase reverse transcriptase-specific CTL was transfected into human telomerase reverse transcriptase cDNA U2OS/hTER by standard 51Cr release assay in different tumor target cells [SW480 colon cancer cells (hTERT~+, HLA-A2~+), KATO-III gastric cancer cells (hTERT~+, HLA-A2~+), U2OS osteosarcoma cells (hTERT-, HLA-A2~+). Immunocytotoxicity of HPG2/HLA-A2 hepatoma cells (hTERT~+, HLA-A2~+), MCF-7 breast cancer cells (hTERT~+, HLA-A2~+), HepG2 hepatoma cells (hTERT~+, HLA-A2 -) transfected with HLA-A2 cDNA was detected by the same method. The number of IFN-gamma effector cells was detected by ELISPOT assay.
3, according to the literature, the bone marrow derived dendritic cells (mDC) of C57BL/6-Tg (HLA-A2~+) mice were isolated and cultured.
Then the adenovirus vector carrying the full-length sequence of hTERT, human telomerase reverse transcriptase MAP peptide and its corresponding single peptide were loaded with mDC respectively, and the mice were immunized once a week for three times. The spleen lymphocytes of the immunized mice were obtained after the last immunization for seven days. The cytotoxicity of human telomerase reverse transcriptase-specific CTL to tumor target cells, autologous lymphocytes and dendritic cells was detected by 51Cr release assay, and the number of IFN-gamma effector cells was detected by ELISPOT assay.
4, the experimental data were analyzed by SPSS17.0 statistical software for t test. When P? 0.05, the difference was statistically significant.
[results]
1. The purity of the polyantigenic peptides and the corresponding single peptides were all above 95% by RP-HPLC. The molecular weight of the polyantigenic peptides and the corresponding single peptides were determined by liquid chromatography/mass spectrometry (LC/MS). The results showed that there was no obvious difference between the theoretical value and the measured value of the molecular weight of the peptides. Differences are the peptides we need.
2. In vitro and Ex vivo experiments showed that adenovirus vector carrying full-length hTERT sequence and human telomerase reverse transcriptase-specific CTL had significant killing effect on SW480, MCF-7 and KATO-III cells with positive human telomerase reverse transcriptase and HLA-A2, and the adenovirus vector carrying full-length hTERT sequence induced killing effect. The killing effect induced by human telomerase reverse transcriptase MAP was significantly higher than that induced by its corresponding single peptide, but it had no killing effect on HLA-A2 positive U2OS cells or HLA-A2 negative HepG2 cells. LA-A2.1-matched U2OS/hTERT cells and HepG2/HLA-A2.1 cells suggested that the CTL reaction was human telomerase reverse transcriptase-specific and HLA-A2-restrictive. It was also found that either the CTL induced by human telomerase reverse transcriptase MAP peptide or the effector cells induced by its corresponding single peptide could induce autologous lymphoma of low-expression human telomerase reverse transcriptase. The number of IFN-gamma effector cells was detected by ELISPOT assay. The results showed that adenovirus vector carrying full-length hTERT sequence could induce effector cells to release the most IFN-gamma. Human telomerase reverse transcriptase MAP peptide was more safe than its corresponding single peptide. More effector cells could be induced to produce IFN-gamma. However, after removal of CD4+T lymphocytes, adenovirus vector carrying full-length hTERT sequence, the levels of IFN-gamma secreted by human telomerase reverse transcriptase MAP peptide and its corresponding monopeptide-induced effector cells were significantly decreased, suggesting that CD4+T lymphocytes maintained immune response and response in CD8+T lymphocytes. China plays an important role.
[Conclusion]
DC-loaded HLA-A2 restriction human telomerase reverse transcriptase CTL epitope MAP vaccine can induce stronger anti-tumor immune effect in vitro than its single peptide. This human telomerase reverse transcriptase MAP vaccine has the characteristics of high efficiency and safety, which provides experimental basis for clinical application of human telomerase reverse transcriptase MAP peptide vaccine.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2184700
[Abstract]:[background and purpose]
Malignant tumor is one of the main diseases that endanger human health. The efficacy of traditional surgery, radiotherapy and chemotherapy is unsatisfactory. This limitation determines that biotherapy must participate in the comprehensive treatment of tumor. Cancer vaccine is an important method of tumor biotherapy. Currently, the commonly used delivery methods of tumor vaccine are cell, protein, polypeptide. Dendritic cells (DCs) are the most potent antigen-presenting immune cells. Tumor-associated antigen (TAA) loaded with DCs is one of the most commonly used methods for tumor immunotherapy. Immunotherapy with these antigens can only work on the corresponding tumors, which limits the wide application of tumor vaccines targeting these antigens.
Human telomerase reverse transcriptase, also known as human telomerase catalytic subunit, is the rate-limiting component of telomerase activity and is an essential pathway for immortalization of cancer cells. It plays an important role in maintaining the continued division, proliferation and survival of tumors. Overexpression, abnormal expression or activation of human telomerase reverse transcriptase is one of the important causes of tumor formation. Recent studies have reported that high expression of human telomerase reverse transcriptase is also closely related to the poor prognosis, invasion and metastasis of tumors. Epitope-specific granular vaccines can induce hTERT-specific cytotoxic T lymphocytes (CTLs) in vitro and in vivo, and have a certain killing effect on hTERT-positive gastric cancer, colon cancer and other cancer cells, but have no killing effect on hTERT-negative lymphocytes and DC. Polypeptide vaccines have no adenovirus vectors, no need to consider the safety of vectors and their integration, and no need to consider the toxic and side effects of some non-dominant epitopes or pathological epitopes in full-length proteins. Moreover, antigen peptides with only 8-15 amino acid sequences can be synthesized in vitro and have a broader clinical application prospect. The biggest problem is that a polypeptide vaccine can only represent one epitope, with small molecular weight, single structure, weak immunogenicity and easy degradation in vivo. To solve this problem, Tam Laboratory put forward a design scheme of multiple antigen peptides (MAP) in 1988. Its polylysine core constitutes one kind of 10. Tightly ordered dendritic structures not only enhance the structural specificity of antigen epitope peptides, but also increase their molecular weight. They not only simulate the spatial conformation of natural epitopes, but also induce strong immune responses without recombinant carrier proteins. At the same time, their stable secondary structure is not easy to reverse, so the degradation rate is fast. The design of MAP vaccine is not only suitable for B cell epitope, but also for T cell epitope.
Based on the above analysis, we intend to construct four-branched polyantigenic peptides of human telomerase reverse transcriptase HLA-A2 restriction CTL epitopes hTERT-540 (ILAKFLHWL), hTERT-865 (RLVDDFLLV) and hTERT-572Y (YLFFYRKSV) to study their antitumor activities in vitro and in vivo, and to induce them with adenovirus vector vaccine carrying full-length hTERT gene and single peptide vaccine. The comparison of CTL activity provides a theoretical basis for the clinical application of hTERT MAP vaccine.
[method]
1. Three human telomerase reverse transcriptase CTL epitopes (hTERT-540 (ILAKFLHWL), hTERT-865 (RLVDDFLLV) and hTERT-572Y (YLFFYRKSV) were selected to design their MAP structures, and their tetrabranched peptides were synthesized by standard solid-phase peptide synthesis (SPSS); and their tetrabranched peptides were synthesized by RP-HPLC.
The purity of the peptide was analyzed by liquid chromatography / mass spectrometry (LC/MS).
HIV virus HLA-A2 restricted epitope (ILLEP VHGV).
2. Human peripheral blood mononuclear cells (PBMC) derived dendritic cells were isolated and cultured according to the literature. The morphology of PBMC was observed by optical microscope and its phenotype was identified by flow cytometry. The adenovirus vector carrying full-length sequence of hTERT, human telomerase reverse transcriptase MAP peptide and its corresponding monopeptide were loaded with DC respectively to induce the production of dendritic cells. Human telomerase reverse transcriptase-specific CTL. Human telomerase reverse transcriptase-specific CTL was transfected into human telomerase reverse transcriptase cDNA U2OS/hTER by standard 51Cr release assay in different tumor target cells [SW480 colon cancer cells (hTERT~+, HLA-A2~+), KATO-III gastric cancer cells (hTERT~+, HLA-A2~+), U2OS osteosarcoma cells (hTERT-, HLA-A2~+). Immunocytotoxicity of HPG2/HLA-A2 hepatoma cells (hTERT~+, HLA-A2~+), MCF-7 breast cancer cells (hTERT~+, HLA-A2~+), HepG2 hepatoma cells (hTERT~+, HLA-A2 -) transfected with HLA-A2 cDNA was detected by the same method. The number of IFN-gamma effector cells was detected by ELISPOT assay.
3, according to the literature, the bone marrow derived dendritic cells (mDC) of C57BL/6-Tg (HLA-A2~+) mice were isolated and cultured.
Then the adenovirus vector carrying the full-length sequence of hTERT, human telomerase reverse transcriptase MAP peptide and its corresponding single peptide were loaded with mDC respectively, and the mice were immunized once a week for three times. The spleen lymphocytes of the immunized mice were obtained after the last immunization for seven days. The cytotoxicity of human telomerase reverse transcriptase-specific CTL to tumor target cells, autologous lymphocytes and dendritic cells was detected by 51Cr release assay, and the number of IFN-gamma effector cells was detected by ELISPOT assay.
4, the experimental data were analyzed by SPSS17.0 statistical software for t test. When P? 0.05, the difference was statistically significant.
[results]
1. The purity of the polyantigenic peptides and the corresponding single peptides were all above 95% by RP-HPLC. The molecular weight of the polyantigenic peptides and the corresponding single peptides were determined by liquid chromatography/mass spectrometry (LC/MS). The results showed that there was no obvious difference between the theoretical value and the measured value of the molecular weight of the peptides. Differences are the peptides we need.
2. In vitro and Ex vivo experiments showed that adenovirus vector carrying full-length hTERT sequence and human telomerase reverse transcriptase-specific CTL had significant killing effect on SW480, MCF-7 and KATO-III cells with positive human telomerase reverse transcriptase and HLA-A2, and the adenovirus vector carrying full-length hTERT sequence induced killing effect. The killing effect induced by human telomerase reverse transcriptase MAP was significantly higher than that induced by its corresponding single peptide, but it had no killing effect on HLA-A2 positive U2OS cells or HLA-A2 negative HepG2 cells. LA-A2.1-matched U2OS/hTERT cells and HepG2/HLA-A2.1 cells suggested that the CTL reaction was human telomerase reverse transcriptase-specific and HLA-A2-restrictive. It was also found that either the CTL induced by human telomerase reverse transcriptase MAP peptide or the effector cells induced by its corresponding single peptide could induce autologous lymphoma of low-expression human telomerase reverse transcriptase. The number of IFN-gamma effector cells was detected by ELISPOT assay. The results showed that adenovirus vector carrying full-length hTERT sequence could induce effector cells to release the most IFN-gamma. Human telomerase reverse transcriptase MAP peptide was more safe than its corresponding single peptide. More effector cells could be induced to produce IFN-gamma. However, after removal of CD4+T lymphocytes, adenovirus vector carrying full-length hTERT sequence, the levels of IFN-gamma secreted by human telomerase reverse transcriptase MAP peptide and its corresponding monopeptide-induced effector cells were significantly decreased, suggesting that CD4+T lymphocytes maintained immune response and response in CD8+T lymphocytes. China plays an important role.
[Conclusion]
DC-loaded HLA-A2 restriction human telomerase reverse transcriptase CTL epitope MAP vaccine can induce stronger anti-tumor immune effect in vitro than its single peptide. This human telomerase reverse transcriptase MAP vaccine has the characteristics of high efficiency and safety, which provides experimental basis for clinical application of human telomerase reverse transcriptase MAP peptide vaccine.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 王kH;诱导免疫应答的肿瘤抗原[J];生命科学;2002年01期
本文编号：2184700
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