| 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 51Cr 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 51Cr 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 51Cr 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 51Cr 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 51Cr 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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