| Background and Objectives:Metastasis and invasion often occurred when malignant tumor at advanced stage. There is no curative therapy method for patients with advanced stage cancer, because of high recurrence rate and low exairesis rate. How to extend survival time and subsistence quality of patients with malignant tumor are hot topics of tumor research. Dendritic cells-based immunotherapy for tumors is an effective and promising method. Dendritic cells loading with tumor associated antigen (TAA) induce effectors is the major way of immunotherapy for tumors. However, most TAA we found so far are specifically expressed in certain types of tumors, and can not be generally applied to all types of tumor immunotherapy. It's very important to find an ideal universal TAA as target for tumor immunotherapy.Heparanase (Hpa) is the only endo-?-D-glucuronidase that can cleave heparin sulfate proteoglycans (HSPG) in exracellular matrix (ECM) and basal membrane (BM) and release different kinds of cytokines which promote tumor angiogenesis and tumor metastasis. Hpa is expressed in almost all advanced stage malignant tumor cells, but not in normal mature tissues, except for lymphocytes and dendritic cells expressing Hpa at very low level. Expression of Hpa is associated with poor prognosis of patients with cancers. It is reported that inhibition of Hpa can significantly reduce tumor metastasis.Our previous study indicated that Hpa full-length cDNA pulsed dendritic cells can induce effectors lysis KATO-III gastric carcinoma cells in heparanase specific and HLA-A2.1 restrict manner. The effectors induced by Hpa cDNA could not lysis autologous lymphocyte and dendritic cells. These results suggest that Hpa could served as a tumor associated antigen for cancer immunotherapy, on the other hand, the results also demonstrated that Hpa contain CTL epitopes. We further identified three CTL epitopes from human Hpa [Hpa(525-533)(PAFSYSFFV),Hpa(277-285)(KMLKSF LKA),Hpa (405-413) (WLSLL FKKL)] and two CTL epitopes from murine Hpa [mHpa(398-405)(LSL LFKKL),mHpa(519-526) (FSYGFFVI)] by bioinformatics and reverse immunological techniques. The result showed epitopes from Hpa could induce potent anti-tumor immune response in vitro and in vivo.Epitopes peptides vaccines have the advantages for therapeutic application: the in vitro synthesized peptide vaccines are feasible techniques even at clinical grade, without the virus vectors delivering and can ensure high level of biosafety. There are several shortcomings limited the clinical application of epitopes vaccines such as small molecular weight, single structure and weak immunogenicity. Normally, one epitope could only activate one CTL clone. Hence if we use a hybrid-epitope vaccine to induce immune response, it will stimulate several CTL clones and induce a stronger anti-tumor lysis effect.Based on our previous study, the present study was designed to identify more epitopes from human Hpa and to investigate the anti-tumor immune response induced by hybrid epitopes vaccines from Hpa. The activation of CTL induced by hybrid epitope vaccines was tested by a a standard 4-hour chromium release assay in vitro and ex vivo, compared to single epitope and full length Hpa cDNA.Methods:1. We use quantitative motif combined with supermotif strategy to predict HLA-A2.1 restricted epitopes. Then epitopes peptides were synthesized, the purity coefficient and molecular weight of peptides we synthesized were identified by high pressure liquid chromatography (HPLC) and mass spectroscopy (MS). The binding of epitopes with HLA-A2.1 was performed based on the feature of T2 cells (TAP-deficent). The specific lysis of effectors induced by hybrid epitopes vaccines on KATO-III gastric carcinoma cells was detected by 4-hour chromium release assay.2. Mature dendritic cells from healthy donor perpheral blood mononuclear cells (PBMCs) were induced and proliferated by recombinant rhGM-CSF,rhIL-4 and rhTNF-?. Then identified by microscopy and flow cytometry. CTLs were induced by DC loading with hybrid epitopes peptides. The specific lysis of CTLs on KATO-III gastric carcinoma cells, U2OS osteosarcoma cells and SW480 colon cancer cells, HepG2 hepatocellular cells, MCF-7 breast cancer cells, HepG2/HLA-A2.1 hepatocellular cells, MCF-7/Hpa breast cancer cells, autologous lymphocytes, and dendritic cells were detected by 4-hour chromium release assay. The IFN-?released by effectors was detected by ELISPOT assay.3. C57BL/6-Tg(HLA-A2.1)1Enge/J mice were immunized by hybrid epitopes pulsed DC derived from homogeneity mice bone marrow once a week for three weeks. Then the spleen lymphocytes were performed as effectors. 4-hour chromium release assay was employed once again to detect the specific lysis of effectors on tumor cells. The IFN-?secreted by effectors was tested by ELISPOT assay.Results:1. Twenty-five epitope candidates were predicted by quantitative motif combined with supermotif strategy: Hpa(8-16)(ALPPPLMLL), Hpa(363-371)(FMWLDKLGL), Hpa(184-192)(LIFGLNALL), Hpa(1-9)(LLRSKPAL), Hpa(315-323)(VLDIFISSV), Hpa (174-182)(YTFANCSGL), Hpa(494-502)(QLNGLTLKM), Hpa(456-464) (NLHNVTKYL), Hpa(310-318)(FLNPDVLDI), Hpa(51-59)(LVSPSFLSV), Hpa(16-24) (LLLGPLGPL), Hpa(241-249)(QLGEDFIQL), Hpa(487-495)(GLLSKSVQL), Hpa(412-420) (KLVGT KVLM), Hpa(82-90) (TLARGLSPA) , Hpa(365-373) (WLDKLGLSA) , Hpa(64-72) (NLATDPRFL) , Hpa(380-388)(VMRQVFFGA) , Hpa(449-457)(DLTLYAINL) , Hpa (303-311) (RTATREDFL),Hpa(72-80) (LILLGSPKL),Hpa(66-74) (ATDPRFLIL),Hpa (393-401)(LVDENFDPL),Hpa(322-330) (SVQKVFQVV),Hpa(13-21) (LMLLLLGPL). The result of HLA-A2.1 binding assay showed epitopes from Hpa could effectively bind with HLA-A2.1 molecular except for Hpa184, Hpa456, and Hpa51. The result of 4-hour chromium release assay indicated that Hpa16, Hpa8, Hpa310, Hpa315 and Hpa363 could induce potent immune response; the effectors induced by these epitopes could lysis KATO-III gastric carcinoma cells which expressing Hpa and HLA-A2.1.2. Effectors were induced by hybrid epitopes vaccines, full length Hpa cDNA, and single epitope pulsed mature DC from PBMC, respectively. Our results indicated that effectors could lysis KATO-III gastric carcinoma cells, U2 OS osteosarcoma cells and SW480 colon cancer cells, which expressing both Hpa and HLA-A2.1. Effectors could not lysis HepG2 hepatocellular cells, MCF-7 breast cancer cells, which not expressing HLA-A2.1 or Hpa, but effectors could lysis HepG2 hepatocellular cells transfected with HLA-A2.1 cDNA or MCF-7 breast cancer cells transfected with Hpa cDNA. These results indicated that the effectors kill tumor cells is in Hpa-specific and HLA-A2.1-restricted manner. Furthermore,the specific lysis rate of effectors induced by hybrid epitopes is higher than single epitopes, but lower than the Hpa cDNA induced effectors. The results indicated hybrid epitopes could elicit much more potent immune response than the single epitope. The result of ELISPOT assay showed that effectors induced by Hpa epitopes were capable of enhancing IFN-?release effectively.3. Spleen lymphocytes from hybrid epitopes vaccines, full length Hpa cDNA, and single epitope pulsed mature DC immunized C57BL/6-Tg(HLA-A2.1)1Enge/J mice could lysis KATO-III gastric carcinoma cells, U2 OS osteosarcoma cells and SW480 colon cancer cells, which expressing both Hpa and HLA-A2.1. The lymphocytes could not lysis HepG2 hepatocellular cells or MCF-7 breast cancer cells, which not expressing HLA-A2.1 or Hpa, but could lysis HepG2 hepatocellular cells transfected with HLA-A2.1 cDNA or MCF-7 breast cancer cells transfected with Hpa cDNA. These results are similar to in vitro experiment, and further indicated that the effectors kill tumor cells was in Hpa-specific and HLA-A2.1-restricted manner. Furthermore,the specific lysis rate of effectors induced by hybrid epitopes is higher than single epitopes, but lower than the Hpa cDNA induced effectors. The results indicated hybrid epitopes could elicit much more potent immune response than the single epitope. ELISPOT test demonstrated that effectors induced by Hpa epitopes were capable of enhancing IFN-?release effectively.Conclusions:1. Eight CTL epitopes were screened out from human heparanase by supermotif combined with quantitative motif strategy. Five of them were identified in previous study, all of these epitopes could elicit potent anti-tumor immune response in vitro.2. Hybrid epitopes vaccines Hpa525+277+405+16 and Hpa8+310+315+363 could elicit much more potent immune response against tumor cells than single epitopes from heparanase in vitro and ex vivo.3. Effectors induced by hybrid epitopes vaccines from Hpa could lysis tumor cells from different tissues, which expressing both Hpa and HLA-A2.1. The CTLs induced by hybrid epitopes vaccines from Hpa killing tumor cells in Hpa-specific and HLA-A2.1 restricted manner.4. The results of side effects study indicated that hybrid epitopes vaccines from Hpa induced effectors could not lysis autologous lymphocytes and dendritic cells in vitro and ex vivo. The results confirmed the safety of hybrid epitopes vaccines from Hpa.5. ELISPOT test demonstrated that effectors induced by hybrid epitopes vaccines from Hpa were capable of enhancing IFN-?release effectively in vitro and ex vivo, enhance the non-specific anti-tumor immunityIn conclusions, compared with single epitopes vaccines, the hybrid epitopes vaccines from heparanase could elicit much more potent Hpa-specific immune response against tumor from different tissues; the hybrid epitopes vaccines from heparanase also enhance non-specific anti-tumor immunity by increasing secretion of IFN-?. Our results provide solid evidence for clinical application of hybrid epitopes vaccines from heparanase for immunotherapy for patients with cancers. |
PDF Full Text Request