肝素酶HLA-A2.1限制性CTL表位的预测、鉴定及其抗肿瘤的免疫效应研究
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摘要
[背景和目的]
生物治疗是继手术、放疗和化疗之后临床治疗恶性肿瘤的第四种治疗模式。随着对机体抗肿瘤免疫机制的深入研究，人们已经认识到诱发体内细胞毒T淋巴细胞(Cytotic T Lymphocyte，CTL)发挥抗瘤作用的并不是整个肿瘤抗原分子，而是与MHC分子结合的短肽即CTL表位(epitopes)，因此，寻找特异性肿瘤抗原及其相应的CTL表位成为肿瘤免疫治疗的关键。树突状细胞(Dendritic cells,DC)作为体内最强大的抗原提呈细胞，是免疫应答过程的始动者和调节者。负载CTL表位的DC疫苗因其免疫原性好、副作用小、便于应用等优势而成为目前抗肿瘤治疗研究的一个热点。
肝素酶(Heparanase，Hpa)是近年来新发现的一个肿瘤转移相关基因，在正常组织中仅低表达于淋巴细胞和骨髓等免疫组织，但在几乎所有的转移性恶性肿瘤细胞中普遍存在，而且表达的水平与肿瘤的转移、TMN分期等临床预后不良指标明显相关，肝素酶已成为中晚期肿瘤治疗的一个良好靶位。那么肝素酶能否作为一种肿瘤相关抗原用于肿瘤的免疫治疗?我们过去的研究表明，肝素酶全长基因负载的DC体外可以诱导肝素酶特异性CTL，对肝素酶阳性且MHC相匹配的胃癌细胞具有杀伤作用，而对肝素酶阳性但MHC不相匹配的胃癌细胞不具有杀伤效应，提示肝素酶氨基酸全长序列中一定存在能诱导特异性CTL反应的抗原表位。
本研究的目的在于寻找存在于肝素酶全长序列中的能够有效激发机体抗肿瘤效应的CTL表位。鉴于HLA-A2．1是中国人群中最为常见的HLAⅠ类分子的型别，阳性率高达50％，因此鉴定肿瘤转移相关抗原Hpa的HLA-A2．1限制性CTL表位将会对中国人群中肝素酶表达阳性的恶性肿瘤提供免疫治疗的基础。
[方法]
1、采用超基序和量化基序相结合的方法对肝素酶的HLA-A2．1限制性CTL表位进行预测，并用分子模拟技术模拟表位肽与MHC分子结合的空间构象，分析结合参数以进一步提高预测的准确性，然后从结果中选取得分较高的5条肝素酶表位肽进行合成、纯化、分子量鉴定，利用T_2细胞的特点对合成的候选肽与HLA-A2．1分子的进行亲和力分析；采用标准~(51)Cr释放实验检测上述肝素酶表位肽负载DC后诱导的肝素酶特异性CTL对KATO-Ⅲ胃癌细胞的免疫杀伤效应，从而筛选出能够有效激活CTL反应的肝素酶抗原表位。
2、采用密度梯度离心法分离并得到人外周血单个核细胞，利用rhGM-CSF、rhIL-4及rhTNF-α诱导扩增人成熟树突状细胞，并从形态学、细胞表面CD分子进行鉴定；用上述筛选的候选肽负载DC诱导产生特异性CTL，采用标准~(51)Cr释放实验检测肝素酶特异性CTL对KATO-Ⅲ胃癌细胞(Hpa~+，HLA-A2．1~+)、U-2OS骨肉瘤细胞(Hpa~+，HLA-A2．1~+)、SW480结肠癌细胞(Hpa~+，HLA-A2．1~+)、MCF-7乳腺癌细胞(Hpa~-，HLA-A2．1~+)、HepG2肝癌细胞(Hpa~+，HLA-A2．1~-)以及转染了肝素酶全长基因的MCF-7／Hpa乳腺癌细胞(Hpa~+，HLA-A2．1~+)和转染了HLA-A2．1基因的HepG2／HLA-A2．1肝癌细胞(Hpa~+，HLA-A2．1~+)的免疫杀伤效应；采用HLA-A2．1单克隆抗体封闭上述靶细胞的HLA-A2．1位点，或以CD8单克隆抗体封闭效应细胞的CD8位点，再用肝素酶表位特异性CTL杀伤KATO-Ⅲ胃癌细胞，以研究筛选的肝素酶表位是否受HLA-A2．1限制以及CTL效应细胞的来源；采用标准~(51)Cr释放实验研究肝素酶特异性CTL对自体淋巴细胞的免疫杀伤活性，以确定其可能存在的毒副作用；采用ELISPOT技术检测肝素酶特异的效应细胞IFN-γ释放情况。
3、将上述筛选的肝素酶候选表位负载C57BL／6-Tg(HLA-A2．1)1Enge／J小鼠骨髓来源的DC(mDC)后，免疫小鼠三次，取其脾淋巴细胞作为效应细胞，采用~(51)Cr释放实验检测肝素酶特异性CTL对KATO-Ⅲ、U2OS、SW480、MCF-7、MCF-7／Hpa、HepG2、HepG2／HLA-A2．1细胞以及自体淋巴细胞和mDC的杀伤效应；ELISPOT技术检测肝素酶特异的效应细胞IFN-γ释放情况。
[结果]
1、采用超基序和量化基序方法联合预测出5条得分最高肝素酶表位肽：Hpa(525-533)(PAFSYSFFV)、Hpa(353-361)(PLLSDTFAA)、Hpa(277-285)(KMLKSFLKA)、Hpa(400-408)(PLPDYWLSL)、Hpa(405-413)(WLSLLFKKL)；T_2细胞亲和力分析发现预测的5条多肽均能与T_2细胞有效结合；采用标准~(51)Cr释放实验检测上述肝素酶表位肽负载DC后诱导的肝素酶特异性CTL对KATO-Ⅲ胃癌细胞的免疫杀伤效应，结果表明，Hpa(525-533)、Hpa(277-285)、Hpa(405-413)诱导的CTL对KATO-Ⅲ胃癌细胞具有明显的杀伤效应，而Hpa(353-361)和Hpa(400-408)诱导的CTL对KATO-Ⅲ胃癌细胞的杀伤效应与阴性肽相同，提示Hpa(525-533)、Hpa(277-285)、Hpa(405-413)可能是肝素酶特异性抗原表位。
2、采用密度梯度离心法分离HLA-A2．1阳性健康志愿者外周血中的单个核细胞(PBMC)，用重组人细胞因子GM-CSF、IL-4及TNF-α联合诱导扩增，经形态学观察以及采用流式细胞术对细胞表面CD分子进行鉴定，成功制备了PBMC来源的成熟DC；采用标准~(51)Cr释放实验检测Hpa(525-533)、Hpa(277-285)、Hpa(405-413)表位负载DC后诱导的肝素酶特异性CTL对不同来源肿瘤细胞的免疫杀伤效应，结果表明，肝素酶特异性CTL对肝素酶阳性且HLA-A2．1阳性的KATO-Ⅲ、U2OS和SW480细胞具有明显的杀伤效应，而对肝素酶阴性但HLA-A2．1阳性的MCF-7细胞和肝素酶阳性但HLA-A2．1阴性的HepG2细胞均不具有杀伤效应，但对转染了肝素酶全长基因的MCF-7细胞(MCF-7／Hpa)和转染了HLA-A2．1全长基因的HepG2细胞(HepG2／HLA-A2．1)重新产生明显的免疫杀伤效应，提示肝素酶抗原表位诱导的CTL反应是肝素酶特异、且受HLA-A2．1限制；用HLA-A2．1和CD8分子的单抗分别封闭靶细胞表面的HLA-A2．1位点和效应细胞表面的CD分子后再进行杀伤实验，结果表明肝素酶特异性CTL对封闭了HLA-A2．1的KATO-Ⅲ细胞无明显杀伤效应，封闭了CD8的效应细胞对KATO-Ⅲ细胞亦无明显杀伤效应，进一步提示这种杀伤效应是HLA-A2．1限制，CTL效应细胞来源于CD8阳性的T淋巴细胞；采用标准~(51)Cr释放实验验检测上述肝素酶抗原表位诱导的肝素酶特异性CTL对自体淋巴细胞的免疫杀伤效应，结果表明肝素酶特异性CTL对自体淋巴细胞无明显杀伤效应，提示肝素酶多肽疫苗的安全性(表1)；采用ELISPOT技术检测肝素酶特异性效应细胞IFN-γ的分泌能力，结果表明，肝素酶抗原表位能促进效应细胞IFN-γ的分泌。
3、用上述肝素酶表位肽负载C57BL／6-Tg(HLA-A2．1)1Enge／J小鼠骨髓来源的DC(mDC)，然后免疫小鼠三次，取其脾淋巴细胞作为效应细胞，以不同来源的多种肿瘤细胞作为靶细胞，用~(51)Cr释放法检测杀伤效应，实验结果表明，肝素酶表位特异性的CTL对肝素酶阳性且HLA-2阳性的KATO-Ⅲ、U2OS和SW480细胞具有明显的杀伤效应，而对肝素酶阴性但HLA-A2．1阳性的MCF-7细胞和肝素酶阳性但HLA-A2．1阴性的HepG2细胞均不具有杀伤效应，但对感染了肝素酶全长基因的重组腺病毒(rAd-Hpa)的MCF-7乳腺癌细胞(MCF-7／rAd-Hpa)和转染HLA-A2．1的HepG2／HLA-A2．1细胞亦重新产生明显的杀伤效应，对自体淋巴细胞和mDC亦无明显杀伤效应(表1)。ELISPOT技术检测肝素酶抗原表位能有效促进肝素酶特异性效应细胞分泌IFN-γ。
[结论]
1、采用超基序和量化基序法并通过体外杀伤实验从肝素酶的全长氨基酸序列中筛选出了3条HLA-A2．1限制的CTL表位，即Hpa(525-533)(PAFSYSFFV)、Hpa(277-285)(KMLKSFLKA)、Hpa(405-413)(WLSLLFKKL)，以上肝素酶抗原表位与德国学者的报道不同。
2、从体外及动物实验二方面证实肝素酶抗原表位Hpa(525-533)、Hpa(277-285)、Hpa(405-413)可以诱导产生肝素酶特异性CTL，对肝素酶阳性且HLA-A2．1相匹配的肿瘤细胞具有很强的免疫杀伤活性，对肝素酶阴性或HLA-A2．1阴性的肿瘤细胞不具有杀伤效应，但对转染了肝素酶或HLA-A2．1的肿瘤细胞具有杀伤效应，提示肝素酶抗原表位诱导的CTL反应是肝素酶特异且受HLA-A2．1限制，肝素酶特异性CTL主要来源于CD8阳性T淋巴细胞。
3、从体外及动物实验二方面证实肝素酶特异性CTL对自体淋巴细胞和／或DC无明显的免疫杀伤活性，提示肝素酶多肽疫苗临床应用的安全性。
4、从体外及动物实验二方面证实肝素酶抗原表位Hpa(525-533)、Hpa(277-285)、Hpa(405-413)负载的DC可以促进效应细胞IFN-γ释放。
以上研究表明，人肝素酶特异性抗原表位[Hpa(525-533)、Hpa(277-285)、Hpa(405-413)]不仅能激发机体特异性的抗肿瘤效应，而且还能诱导一个非特异性抗肿瘤的良性循环，这种肝素酶多肽疫苗具有广谱、高效、特异、安全的优点，为肝素酶表位多肽疫苗的临床应用提供了理论依据。
Backgrounds and objectives:
The biotherapy for malignant tumors has become the forth treatment model followed surgery, chemical therapy, and radiotherapy. As the study on anti-tumor immunity and its mechanisms enhanced, researchers have recognized that the substance which induces the anti-tumor activities of CTL in human body is the CTL epitopes (the peptides binding with MHC) rather than the entire neoplasm antigen. Therefore, the key to tumor immunotherapy is to find the specific tumor antigen and its epitopes. Dendritic cell(DC), as the strongest antigen presenting cell, is a motivator and pacificator in the immune response. The DC vaccine loaded with CTL epitopes which has strong immunogenicity and little side effects therefor is becoming one of the hotest topics in the study of the malignant tumor therapy.
Heparanase(Hpa) is an endo-β-D-glucuronidase that can cleave heparan sulfate proteoglycans(HSPG). It has been implicated in tumor angiogenesis and metastasis. Heparanase can be expressed in almost all the malignant tumors and it is associated with poor prognostic parameters, such as metastasis, TMN phase and so on. The broad expression of heparanase in advanced tumors indicates that heparanase can serve as a tumor associated antigen(TAA) in the immunotherapy of advanced tumors. Our previous study have demonstrated that the DC modified with full-length cDNA of heparanase could elicit heparanase-specific CTLs against HLA-matched and heparanase-positive gastric cancer cells in vitro, while there were no killing effects on autologous lymphocytes. These results demonstrated that there must be CTL epitopes which can induce a specific CTL in full-length amino acid sequence of heparanase. The objective of this study is mainly to find these CTL epitopes in heparanase with ability to induce heparanase-specific antitumor immune response. In view of the fact that HLA-A2.1 is the most common phenotype of MHC- I molecules in human, the prediction and identification of HLA A2.1-restricted CTL epitopes in heparanase antigen will provide foundation for immunotherapy for malignant tumor with the positive expression of heparanse.
Methods:
1.The HLA-A2.1-restricted CTL epitopes were predicted with the method of supermotif combined with quantitative motif. The molecular simulation was used to stimulate the space conformation of peptides binding with MHC molecule.The parameters were analyzed to further enhance the accuracy of prediction. Then the five heparanase peptides with the highest score were selected to be synthesized, purified and identified in terms of molecular weight. The analysis on the binding of the synthesized candidate peptides with MHC molecules was carried out based on the feature of T2 cells. The standard ~(51)Cr releasing assay was made to study the specific lysis of CTL induced by the above heparanase epitopes on KATO-Ⅲgastric cancer cells so as to screen out the epitopes which can activate the effective CTL response .
2. The perpheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Mature dendritic cells were co-cultured with cytokine combinations with rhGM-CSF、rhIL-4 and rhTNF-α. It was identified by electron microscope and flow cytometry. Heparanase-specific CTLs were induced by DC pulsed with the above screened candidate peptides. Its tumor-killing activities to KATO-Ⅲgastric cancer cells (Hpa~+, HLA-A2.1~+), U-2OS osteosarcoma cells (Hpa~+, HLA-A2.1~+), colon cancer cells SW480(Hpa~+, HLA-A2.1~+), MCF-7 breast carcer cells(Hpa~-, HLA-A2.1~+), HepG2 hepatocellular carcinoma cells(Hpa~+, HLA-A2.1~-), MCF-7 cells transfected with the full-length cDNA of heparanae gene (MCF-7/Hpa) and HepG2 cells transfected with the plasmid of HLA-A2.1(HepG2/HLA-A2.1) were tested by standard ~(51)Cr releasing assay. In order to find out whether the screened heparanase epitopes were restricteded by HLA-A2.1 and the origin of the effector cells, we furtherly blocked the HLA-A2.1 sites of KATO-Ⅲtarget cells with HLA-A2.1 mono-antibody(mAb) and we blocked CD8 sites of effectors with CD8 mAb, then the specific lysis of effectors on KATO-Ⅲgastric cancer cells was detected. In addition, specific lysis of CTLs induced by the screened epitopes on autologous lymphocytes was involved to study the possible side effects. The IFN-γreleasing of effector cells was tested by ELISPOT.
3. DCs derived from the C57BL/6-Tg(HLA-A2.1)1Enge/J mice's bone marrow(mDC) were loaded with the screened heparanase epitopes. We subcutaneously immunized mice by this DC vaccine for three times . The spleen lymphocyte was drawn as effector cells. The standard ~(51)Cr releasing assay was carried out to study the killing activity of the heparanase-specific CTLs on KATO-Ⅲ, U2OS, SW480, MCF-7, MCF-7/Hpa, HepG2 and HepG2/HLA-A2.1 tumor cell lines, as well as autologous lymphocytes and mDC. The IFN-γreleasing of effector cells was tested by ELISPOT.
Results:
1. The 5 highest score heparanase epitopes predicted by supermotif combined with quantitative motif were Hpa (525-533) (PAFSYSFFV), Hpa(353-361) (PLLSDTFAA), Hpa(277-285) (KMLKSFLKA), Hpa(400-408) (PLPDYWLSL) and Hpa(405-413) (WLSLLFKKL). Analysis on binding ability of the peptides indicated that they could be effectively binded with HLA-A2 on the surface of T2 cells. The standard ~(51)Cr releasing assay was made to study the specific lysis of CTL induced by the above screened epitopes on KATO-Ⅲgastric cancer cells. The results demonstrated that only Hpa(525-533), Hpa(277-285) and Hpa(405-413) could elicit potent killing effects to KATO-Ⅲgastric cancer cells, while the CTLs induced by Hpa(353-361) and Hpa(400-408) remained the same killing activities as negative peptides. These findings indicated that the Hpa(525-533)、Hpa(277-285)、Hpa(405-413) are probably the specific CTL epitopes of heparanase.
2. The PBMCs from healthy donors with positive HLA-A2.1 were isolated by density gradient centrifugation and were co-cultured with cytokine combinations of GM-CSF、IL-4 and TNF-α. The DCs were furtherly verified by observation under optical and electron microscope, molecular markers detection through flow cytometry. CTLs primed by Hpa(525-533)、Hpa(277-285)、Hpa(405-413) exhibited potent specific lysis to KATO-Ⅲ, U2OS and SW480 cells which were heparanase and HLA-A2 positive measured by standard ~(51)Cr releasing assay. Meanwhile, there were no notable killing effects to heparanase negative MCF-7 cells and HLA-A2 negative HepG2 cells. But this specific lysis was re-occurred after MCF-7 cells transfected with full-length cDNA of heparanase or HepG2 cells transfected with the plasmid of HLA-A2. These results indicated that the immune response induced by heparanase epitopes was heparanase specific and restricted by HLA-A2.1. After blocking the HLA-A2.1 sites on the surface of target cells with HLA-A2.1 mAb or blocking the CD8 molecules on the surface of effectors cells with CD8 mAb, the specific killing effects of CTLs disappeared. These results furtherly indicated that effectors were HLA-A2.1 restricted and the source of effectors is raised from CD8~+ T lymphocytes. Athough autologous lymphocytes were both heparanase and HLA-A2 positive, no obvious lysis was found after co-cultured with heparanase-specific CTLs(Tab 1). ELISPOT test showed that the heparanase-specific epitopes were capable of enhancing IFN-γrelease effectively.
3.Animal experiment showed that spleen lymphocytes from the transgenic mice (HLA-A2.1~+) immunized with peptides-pulsed mDC could effectively lyse KATO-Ⅲ, U2OS , SW480, MCF-7/Hpa and HepG2/HLA-A2 tumor cell lines, while there was not obvious lysis to tumor cell lines which were only heparanase or HLA-A2 positive ( MCF-7or HepG2 cells). Further study demonstrated that spleen lymphocyte from the transgenic mice could not lyse autologous lymphocytes or mDC, although they were both positive for heparanase and HLA-A2(Tab 1). The ELISPOT test showed that the heparanase epitopes were capable of enhancing IFN-γrelease effectively. The above results from animal experiment were consistent with the in vitro experiment results.
Conclusions:
1.There are 3 HLA-A2.1-restricted CTL epitopes screened out from the full-length amino acid sequence of heparanase using method of supermotif combined with quantitative motif. They are Hpa(525-533)(PAFSYSFFV), Hpa(277-285) (KMLKSFLKA) and Hpa(405-413)(WLSLLFKKL), which are different from German scientist's report.
2.The experiments in vitro and in vivo prove that the above heparanase epitopes can effectively elicit heparanae-specific CTLs against various heparanase positive and HLA-A2 matched tumor cell lines, while there are no specific lysis on heparanase negative or HLA-A2 unmatched tumor cell lines. These data suggest that heparanase peptide vaccine can induce anti-tumor immunity against various tumor cells expressing heparanase in a HLA-A2 restricted fashion. The source of heparanase specific CTLs is raised from CD8~+ T lymphocyte.
3.The in vitro and in vivo experiments prove that heparanase-specific CTLs have no killing effects on autologous lymphocytes and/or DCs, which suggests the heparanase epitope vaccines are probably safe for the future clinic application.
4.The in vitro and in vivo experiments proved that heparanase epitopes can enhance IFN-γreleasing effectively, which indicates that heparanase epitopes not only elicit a specific immune response, but also elicit a non-specific immune response against tumors.
In conclusion, the above studies show for the first time that the heparanase epitopes Hpa(525-533), Hpa(277-285) and Hpa(405-413) are capable of eliciting a specific anti-tumor immune response by heparanase-specific CTLs, as well as a non-specific anti-tumor immune response by enhancing cytokines releasing. These findings show that heparanase epitope vaccines have various advantages such as broad-spectrum, high-effect, high specificity and safety, which will provide theoretical evidence for their clinical application.
生物治疗是继手术、放疗和化疗之后临床治疗恶性肿瘤的第四种治疗模式。随着对机体抗肿瘤免疫机制的深入研究，人们已经认识到诱发体内细胞毒T淋巴细胞(Cytotic T Lymphocyte，CTL)发挥抗瘤作用的并不是整个肿瘤抗原分子，而是与MHC分子结合的短肽即CTL表位(epitopes)，因此，寻找特异性肿瘤抗原及其相应的CTL表位成为肿瘤免疫治疗的关键。树突状细胞(Dendritic cells,DC)作为体内最强大的抗原提呈细胞，是免疫应答过程的始动者和调节者。负载CTL表位的DC疫苗因其免疫原性好、副作用小、便于应用等优势而成为目前抗肿瘤治疗研究的一个热点。
肝素酶(Heparanase，Hpa)是近年来新发现的一个肿瘤转移相关基因，在正常组织中仅低表达于淋巴细胞和骨髓等免疫组织，但在几乎所有的转移性恶性肿瘤细胞中普遍存在，而且表达的水平与肿瘤的转移、TMN分期等临床预后不良指标明显相关，肝素酶已成为中晚期肿瘤治疗的一个良好靶位。那么肝素酶能否作为一种肿瘤相关抗原用于肿瘤的免疫治疗?我们过去的研究表明，肝素酶全长基因负载的DC体外可以诱导肝素酶特异性CTL，对肝素酶阳性且MHC相匹配的胃癌细胞具有杀伤作用，而对肝素酶阳性但MHC不相匹配的胃癌细胞不具有杀伤效应，提示肝素酶氨基酸全长序列中一定存在能诱导特异性CTL反应的抗原表位。
本研究的目的在于寻找存在于肝素酶全长序列中的能够有效激发机体抗肿瘤效应的CTL表位。鉴于HLA-A2．1是中国人群中最为常见的HLAⅠ类分子的型别，阳性率高达50％，因此鉴定肿瘤转移相关抗原Hpa的HLA-A2．1限制性CTL表位将会对中国人群中肝素酶表达阳性的恶性肿瘤提供免疫治疗的基础。
[方法]
1、采用超基序和量化基序相结合的方法对肝素酶的HLA-A2．1限制性CTL表位进行预测，并用分子模拟技术模拟表位肽与MHC分子结合的空间构象，分析结合参数以进一步提高预测的准确性，然后从结果中选取得分较高的5条肝素酶表位肽进行合成、纯化、分子量鉴定，利用T_2细胞的特点对合成的候选肽与HLA-A2．1分子的进行亲和力分析；采用标准~(51)Cr释放实验检测上述肝素酶表位肽负载DC后诱导的肝素酶特异性CTL对KATO-Ⅲ胃癌细胞的免疫杀伤效应，从而筛选出能够有效激活CTL反应的肝素酶抗原表位。
2、采用密度梯度离心法分离并得到人外周血单个核细胞，利用rhGM-CSF、rhIL-4及rhTNF-α诱导扩增人成熟树突状细胞，并从形态学、细胞表面CD分子进行鉴定；用上述筛选的候选肽负载DC诱导产生特异性CTL，采用标准~(51)Cr释放实验检测肝素酶特异性CTL对KATO-Ⅲ胃癌细胞(Hpa~+，HLA-A2．1~+)、U-2OS骨肉瘤细胞(Hpa~+，HLA-A2．1~+)、SW480结肠癌细胞(Hpa~+，HLA-A2．1~+)、MCF-7乳腺癌细胞(Hpa~-，HLA-A2．1~+)、HepG2肝癌细胞(Hpa~+，HLA-A2．1~-)以及转染了肝素酶全长基因的MCF-7／Hpa乳腺癌细胞(Hpa~+，HLA-A2．1~+)和转染了HLA-A2．1基因的HepG2／HLA-A2．1肝癌细胞(Hpa~+，HLA-A2．1~+)的免疫杀伤效应；采用HLA-A2．1单克隆抗体封闭上述靶细胞的HLA-A2．1位点，或以CD8单克隆抗体封闭效应细胞的CD8位点，再用肝素酶表位特异性CTL杀伤KATO-Ⅲ胃癌细胞，以研究筛选的肝素酶表位是否受HLA-A2．1限制以及CTL效应细胞的来源；采用标准~(51)Cr释放实验研究肝素酶特异性CTL对自体淋巴细胞的免疫杀伤活性，以确定其可能存在的毒副作用；采用ELISPOT技术检测肝素酶特异的效应细胞IFN-γ释放情况。
3、将上述筛选的肝素酶候选表位负载C57BL／6-Tg(HLA-A2．1)1Enge／J小鼠骨髓来源的DC(mDC)后，免疫小鼠三次，取其脾淋巴细胞作为效应细胞，采用~(51)Cr释放实验检测肝素酶特异性CTL对KATO-Ⅲ、U2OS、SW480、MCF-7、MCF-7／Hpa、HepG2、HepG2／HLA-A2．1细胞以及自体淋巴细胞和mDC的杀伤效应；ELISPOT技术检测肝素酶特异的效应细胞IFN-γ释放情况。
[结果]
1、采用超基序和量化基序方法联合预测出5条得分最高肝素酶表位肽：Hpa(525-533)(PAFSYSFFV)、Hpa(353-361)(PLLSDTFAA)、Hpa(277-285)(KMLKSFLKA)、Hpa(400-408)(PLPDYWLSL)、Hpa(405-413)(WLSLLFKKL)；T_2细胞亲和力分析发现预测的5条多肽均能与T_2细胞有效结合；采用标准~(51)Cr释放实验检测上述肝素酶表位肽负载DC后诱导的肝素酶特异性CTL对KATO-Ⅲ胃癌细胞的免疫杀伤效应，结果表明，Hpa(525-533)、Hpa(277-285)、Hpa(405-413)诱导的CTL对KATO-Ⅲ胃癌细胞具有明显的杀伤效应，而Hpa(353-361)和Hpa(400-408)诱导的CTL对KATO-Ⅲ胃癌细胞的杀伤效应与阴性肽相同，提示Hpa(525-533)、Hpa(277-285)、Hpa(405-413)可能是肝素酶特异性抗原表位。
2、采用密度梯度离心法分离HLA-A2．1阳性健康志愿者外周血中的单个核细胞(PBMC)，用重组人细胞因子GM-CSF、IL-4及TNF-α联合诱导扩增，经形态学观察以及采用流式细胞术对细胞表面CD分子进行鉴定，成功制备了PBMC来源的成熟DC；采用标准~(51)Cr释放实验检测Hpa(525-533)、Hpa(277-285)、Hpa(405-413)表位负载DC后诱导的肝素酶特异性CTL对不同来源肿瘤细胞的免疫杀伤效应，结果表明，肝素酶特异性CTL对肝素酶阳性且HLA-A2．1阳性的KATO-Ⅲ、U2OS和SW480细胞具有明显的杀伤效应，而对肝素酶阴性但HLA-A2．1阳性的MCF-7细胞和肝素酶阳性但HLA-A2．1阴性的HepG2细胞均不具有杀伤效应，但对转染了肝素酶全长基因的MCF-7细胞(MCF-7／Hpa)和转染了HLA-A2．1全长基因的HepG2细胞(HepG2／HLA-A2．1)重新产生明显的免疫杀伤效应，提示肝素酶抗原表位诱导的CTL反应是肝素酶特异、且受HLA-A2．1限制；用HLA-A2．1和CD8分子的单抗分别封闭靶细胞表面的HLA-A2．1位点和效应细胞表面的CD分子后再进行杀伤实验，结果表明肝素酶特异性CTL对封闭了HLA-A2．1的KATO-Ⅲ细胞无明显杀伤效应，封闭了CD8的效应细胞对KATO-Ⅲ细胞亦无明显杀伤效应，进一步提示这种杀伤效应是HLA-A2．1限制，CTL效应细胞来源于CD8阳性的T淋巴细胞；采用标准~(51)Cr释放实验验检测上述肝素酶抗原表位诱导的肝素酶特异性CTL对自体淋巴细胞的免疫杀伤效应，结果表明肝素酶特异性CTL对自体淋巴细胞无明显杀伤效应，提示肝素酶多肽疫苗的安全性(表1)；采用ELISPOT技术检测肝素酶特异性效应细胞IFN-γ的分泌能力，结果表明，肝素酶抗原表位能促进效应细胞IFN-γ的分泌。
3、用上述肝素酶表位肽负载C57BL／6-Tg(HLA-A2．1)1Enge／J小鼠骨髓来源的DC(mDC)，然后免疫小鼠三次，取其脾淋巴细胞作为效应细胞，以不同来源的多种肿瘤细胞作为靶细胞，用~(51)Cr释放法检测杀伤效应，实验结果表明，肝素酶表位特异性的CTL对肝素酶阳性且HLA-2阳性的KATO-Ⅲ、U2OS和SW480细胞具有明显的杀伤效应，而对肝素酶阴性但HLA-A2．1阳性的MCF-7细胞和肝素酶阳性但HLA-A2．1阴性的HepG2细胞均不具有杀伤效应，但对感染了肝素酶全长基因的重组腺病毒(rAd-Hpa)的MCF-7乳腺癌细胞(MCF-7／rAd-Hpa)和转染HLA-A2．1的HepG2／HLA-A2．1细胞亦重新产生明显的杀伤效应，对自体淋巴细胞和mDC亦无明显杀伤效应(表1)。ELISPOT技术检测肝素酶抗原表位能有效促进肝素酶特异性效应细胞分泌IFN-γ。
[结论]
1、采用超基序和量化基序法并通过体外杀伤实验从肝素酶的全长氨基酸序列中筛选出了3条HLA-A2．1限制的CTL表位，即Hpa(525-533)(PAFSYSFFV)、Hpa(277-285)(KMLKSFLKA)、Hpa(405-413)(WLSLLFKKL)，以上肝素酶抗原表位与德国学者的报道不同。
2、从体外及动物实验二方面证实肝素酶抗原表位Hpa(525-533)、Hpa(277-285)、Hpa(405-413)可以诱导产生肝素酶特异性CTL，对肝素酶阳性且HLA-A2．1相匹配的肿瘤细胞具有很强的免疫杀伤活性，对肝素酶阴性或HLA-A2．1阴性的肿瘤细胞不具有杀伤效应，但对转染了肝素酶或HLA-A2．1的肿瘤细胞具有杀伤效应，提示肝素酶抗原表位诱导的CTL反应是肝素酶特异且受HLA-A2．1限制，肝素酶特异性CTL主要来源于CD8阳性T淋巴细胞。
3、从体外及动物实验二方面证实肝素酶特异性CTL对自体淋巴细胞和／或DC无明显的免疫杀伤活性，提示肝素酶多肽疫苗临床应用的安全性。
4、从体外及动物实验二方面证实肝素酶抗原表位Hpa(525-533)、Hpa(277-285)、Hpa(405-413)负载的DC可以促进效应细胞IFN-γ释放。
以上研究表明，人肝素酶特异性抗原表位[Hpa(525-533)、Hpa(277-285)、Hpa(405-413)]不仅能激发机体特异性的抗肿瘤效应，而且还能诱导一个非特异性抗肿瘤的良性循环，这种肝素酶多肽疫苗具有广谱、高效、特异、安全的优点，为肝素酶表位多肽疫苗的临床应用提供了理论依据。
Backgrounds and objectives:
The biotherapy for malignant tumors has become the forth treatment model followed surgery, chemical therapy, and radiotherapy. As the study on anti-tumor immunity and its mechanisms enhanced, researchers have recognized that the substance which induces the anti-tumor activities of CTL in human body is the CTL epitopes (the peptides binding with MHC) rather than the entire neoplasm antigen. Therefore, the key to tumor immunotherapy is to find the specific tumor antigen and its epitopes. Dendritic cell(DC), as the strongest antigen presenting cell, is a motivator and pacificator in the immune response. The DC vaccine loaded with CTL epitopes which has strong immunogenicity and little side effects therefor is becoming one of the hotest topics in the study of the malignant tumor therapy.
Heparanase(Hpa) is an endo-β-D-glucuronidase that can cleave heparan sulfate proteoglycans(HSPG). It has been implicated in tumor angiogenesis and metastasis. Heparanase can be expressed in almost all the malignant tumors and it is associated with poor prognostic parameters, such as metastasis, TMN phase and so on. The broad expression of heparanase in advanced tumors indicates that heparanase can serve as a tumor associated antigen(TAA) in the immunotherapy of advanced tumors. Our previous study have demonstrated that the DC modified with full-length cDNA of heparanase could elicit heparanase-specific CTLs against HLA-matched and heparanase-positive gastric cancer cells in vitro, while there were no killing effects on autologous lymphocytes. These results demonstrated that there must be CTL epitopes which can induce a specific CTL in full-length amino acid sequence of heparanase. The objective of this study is mainly to find these CTL epitopes in heparanase with ability to induce heparanase-specific antitumor immune response. In view of the fact that HLA-A2.1 is the most common phenotype of MHC- I molecules in human, the prediction and identification of HLA A2.1-restricted CTL epitopes in heparanase antigen will provide foundation for immunotherapy for malignant tumor with the positive expression of heparanse.
Methods:
1.The HLA-A2.1-restricted CTL epitopes were predicted with the method of supermotif combined with quantitative motif. The molecular simulation was used to stimulate the space conformation of peptides binding with MHC molecule.The parameters were analyzed to further enhance the accuracy of prediction. Then the five heparanase peptides with the highest score were selected to be synthesized, purified and identified in terms of molecular weight. The analysis on the binding of the synthesized candidate peptides with MHC molecules was carried out based on the feature of T2 cells. The standard ~(51)Cr releasing assay was made to study the specific lysis of CTL induced by the above heparanase epitopes on KATO-Ⅲgastric cancer cells so as to screen out the epitopes which can activate the effective CTL response .
2. The perpheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Mature dendritic cells were co-cultured with cytokine combinations with rhGM-CSF、rhIL-4 and rhTNF-α. It was identified by electron microscope and flow cytometry. Heparanase-specific CTLs were induced by DC pulsed with the above screened candidate peptides. Its tumor-killing activities to KATO-Ⅲgastric cancer cells (Hpa~+, HLA-A2.1~+), U-2OS osteosarcoma cells (Hpa~+, HLA-A2.1~+), colon cancer cells SW480(Hpa~+, HLA-A2.1~+), MCF-7 breast carcer cells(Hpa~-, HLA-A2.1~+), HepG2 hepatocellular carcinoma cells(Hpa~+, HLA-A2.1~-), MCF-7 cells transfected with the full-length cDNA of heparanae gene (MCF-7/Hpa) and HepG2 cells transfected with the plasmid of HLA-A2.1(HepG2/HLA-A2.1) were tested by standard ~(51)Cr releasing assay. In order to find out whether the screened heparanase epitopes were restricteded by HLA-A2.1 and the origin of the effector cells, we furtherly blocked the HLA-A2.1 sites of KATO-Ⅲtarget cells with HLA-A2.1 mono-antibody(mAb) and we blocked CD8 sites of effectors with CD8 mAb, then the specific lysis of effectors on KATO-Ⅲgastric cancer cells was detected. In addition, specific lysis of CTLs induced by the screened epitopes on autologous lymphocytes was involved to study the possible side effects. The IFN-γreleasing of effector cells was tested by ELISPOT.
3. DCs derived from the C57BL/6-Tg(HLA-A2.1)1Enge/J mice's bone marrow(mDC) were loaded with the screened heparanase epitopes. We subcutaneously immunized mice by this DC vaccine for three times . The spleen lymphocyte was drawn as effector cells. The standard ~(51)Cr releasing assay was carried out to study the killing activity of the heparanase-specific CTLs on KATO-Ⅲ, U2OS, SW480, MCF-7, MCF-7/Hpa, HepG2 and HepG2/HLA-A2.1 tumor cell lines, as well as autologous lymphocytes and mDC. The IFN-γreleasing of effector cells was tested by ELISPOT.
Results:
1. The 5 highest score heparanase epitopes predicted by supermotif combined with quantitative motif were Hpa (525-533) (PAFSYSFFV), Hpa(353-361) (PLLSDTFAA), Hpa(277-285) (KMLKSFLKA), Hpa(400-408) (PLPDYWLSL) and Hpa(405-413) (WLSLLFKKL). Analysis on binding ability of the peptides indicated that they could be effectively binded with HLA-A2 on the surface of T2 cells. The standard ~(51)Cr releasing assay was made to study the specific lysis of CTL induced by the above screened epitopes on KATO-Ⅲgastric cancer cells. The results demonstrated that only Hpa(525-533), Hpa(277-285) and Hpa(405-413) could elicit potent killing effects to KATO-Ⅲgastric cancer cells, while the CTLs induced by Hpa(353-361) and Hpa(400-408) remained the same killing activities as negative peptides. These findings indicated that the Hpa(525-533)、Hpa(277-285)、Hpa(405-413) are probably the specific CTL epitopes of heparanase.
2. The PBMCs from healthy donors with positive HLA-A2.1 were isolated by density gradient centrifugation and were co-cultured with cytokine combinations of GM-CSF、IL-4 and TNF-α. The DCs were furtherly verified by observation under optical and electron microscope, molecular markers detection through flow cytometry. CTLs primed by Hpa(525-533)、Hpa(277-285)、Hpa(405-413) exhibited potent specific lysis to KATO-Ⅲ, U2OS and SW480 cells which were heparanase and HLA-A2 positive measured by standard ~(51)Cr releasing assay. Meanwhile, there were no notable killing effects to heparanase negative MCF-7 cells and HLA-A2 negative HepG2 cells. But this specific lysis was re-occurred after MCF-7 cells transfected with full-length cDNA of heparanase or HepG2 cells transfected with the plasmid of HLA-A2. These results indicated that the immune response induced by heparanase epitopes was heparanase specific and restricted by HLA-A2.1. After blocking the HLA-A2.1 sites on the surface of target cells with HLA-A2.1 mAb or blocking the CD8 molecules on the surface of effectors cells with CD8 mAb, the specific killing effects of CTLs disappeared. These results furtherly indicated that effectors were HLA-A2.1 restricted and the source of effectors is raised from CD8~+ T lymphocytes. Athough autologous lymphocytes were both heparanase and HLA-A2 positive, no obvious lysis was found after co-cultured with heparanase-specific CTLs(Tab 1). ELISPOT test showed that the heparanase-specific epitopes were capable of enhancing IFN-γrelease effectively.
3.Animal experiment showed that spleen lymphocytes from the transgenic mice (HLA-A2.1~+) immunized with peptides-pulsed mDC could effectively lyse KATO-Ⅲ, U2OS , SW480, MCF-7/Hpa and HepG2/HLA-A2 tumor cell lines, while there was not obvious lysis to tumor cell lines which were only heparanase or HLA-A2 positive ( MCF-7or HepG2 cells). Further study demonstrated that spleen lymphocyte from the transgenic mice could not lyse autologous lymphocytes or mDC, although they were both positive for heparanase and HLA-A2(Tab 1). The ELISPOT test showed that the heparanase epitopes were capable of enhancing IFN-γrelease effectively. The above results from animal experiment were consistent with the in vitro experiment results.
Conclusions:
1.There are 3 HLA-A2.1-restricted CTL epitopes screened out from the full-length amino acid sequence of heparanase using method of supermotif combined with quantitative motif. They are Hpa(525-533)(PAFSYSFFV), Hpa(277-285) (KMLKSFLKA) and Hpa(405-413)(WLSLLFKKL), which are different from German scientist's report.
2.The experiments in vitro and in vivo prove that the above heparanase epitopes can effectively elicit heparanae-specific CTLs against various heparanase positive and HLA-A2 matched tumor cell lines, while there are no specific lysis on heparanase negative or HLA-A2 unmatched tumor cell lines. These data suggest that heparanase peptide vaccine can induce anti-tumor immunity against various tumor cells expressing heparanase in a HLA-A2 restricted fashion. The source of heparanase specific CTLs is raised from CD8~+ T lymphocyte.
3.The in vitro and in vivo experiments prove that heparanase-specific CTLs have no killing effects on autologous lymphocytes and/or DCs, which suggests the heparanase epitope vaccines are probably safe for the future clinic application.
4.The in vitro and in vivo experiments proved that heparanase epitopes can enhance IFN-γreleasing effectively, which indicates that heparanase epitopes not only elicit a specific immune response, but also elicit a non-specific immune response against tumors.
In conclusion, the above studies show for the first time that the heparanase epitopes Hpa(525-533), Hpa(277-285) and Hpa(405-413) are capable of eliciting a specific anti-tumor immune response by heparanase-specific CTLs, as well as a non-specific anti-tumor immune response by enhancing cytokines releasing. These findings show that heparanase epitope vaccines have various advantages such as broad-spectrum, high-effect, high specificity and safety, which will provide theoretical evidence for their clinical application.
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