Prediction And Identification Of Hla-a2/a3 Restricted Ctl Epitopes Derived From Mage-4

Background and ObjectivesAt the present time, malignant tumor have already threaten the health of our people, now the main therapy including surgery, chemotherapy and radiation, these treatment measures have some curative effects, but also have some side effects. Esophageal carcinoma (ECC) is one of the most frequently occurred malignant cancers in northern china, it is high sensitive to radiation but low sensitive to chemotherapy on account of its unique biological characters, surgery is still main measure to improve survival rate. The 5-year survival rate in patients with cancer especially with esophageal carcinoma is less than 10%. So, novel treatment options of ECC are urgently needed.Due to the development of immunology in recent decades, people have already recognized that the immune system play an important role in controlling tumor. Immunotherapy is becoming to play more and more important role as a new measure to deal with tumors.Tumor-specific antigen (TSA) and tumor-associated antigen (TAA) are very important for early detection, diagnosis and treatment. Further research in immune response molecule mechanisms made people recognized that the immunocyte couldn't response to various antigens, it is only to all kinds of antigen epitopes. Epitope is not only the specific chemical groups in the decision of antigen's specificity, but TCR / BCR basic binding unit, which plays a key role in the antigen . People will design the specific antibody or vaccine against this antigen if they find new tumor antigen epitope. Now, it is clear that T cell epitopes have tempting application in the future as a new generation of anti-cancer vaccines. Antigen detection and identification of T cell epitopes has become one of the hottest topics in the field of molecular immunology.MAGEs are the first group of tumor-associated antigens founding in Melanoma, MAGE gene do not express in normal tissues (except testes and embryos) but mainly expressed in malignant tissue, its expression have a very close relationship with the occurrence, development and prognosis of tumor. Researches showed that MAGE gene has different levels of expression in a variety of digestive tumors. Their gene product is tumor rejecting antigens, and can binding to human leukocyte antigen (HLA) molecules in the surface of the tumor cell by the recognition of specific CTLs to play specific anti-tumor effect.MAGE-4 is one member of the MAGE gene family. It has higher levels of expression in esophageal squamous cell carcinoma (74%), lung cancer (59%), head and neck cancer (53%), bladder cancer (45%). Zambon and his group found that at least one MAGE gene express in 67 % esophageal squamous cell carcinoma and 37.5% esophageal adenocarcinoma, MAGE-4 express more in squamous cell carcinoma, which can be considered as a specific target of esophageal squamous cell carcinoma. So searching for MAGE-4 HLA restricted epitope will be provides more treatment options for esophageal cancer and other malignant tumors.Methods 1. Accessing SYFPEITHI Home (http://www.syfpeithi.de/scripts/MHCServer.dll/ home.htm) in Internet, going into the predict interface by knock "Epitope prediction", predicting the target antigen epitopes by choosing "HLA-A*0201"and "HLA-A*03". Selecting the peptide which is more than 20 percentile and inputting the target antigen sequence in FASTA format to analysis the C-terminal restriction site analysis in proteasomal cleavage motifs forecast program (NetChop3.0) tools. Analyzing structural compatibility on the structure of epitope prediction algorithm (MHC-Pred) tools.2. According with the standard Fmoc method of synthesize peptide, HPLC purify the synthesized candidate peptides. Electrospray ionization mass Spectrometry (ESI-MS) identification of the molecular weight.3. RT-PCR detect the expression of MAGE-4 in tumor cells EC-1, EC-109 and EC-9706.4. T2 cells were using to analysis the binding ability of the candidate peptide with HLA-A2/A3.5. Choose the peptide with high binding ability to induced specific CTLs and determine its cytolytic activity by MTT assay.Results1. We have predicted five candidate peptides as epitopes from tumor-associated antigen MAGE-4 using bioinformatics methods.2. Using standard Fmoc solid-phase synthesis, we have synthesized five-epitope peptides, purified by HPLC and its purity was greater than 90%. Mass Spectrometry analysis showed that the actual molecular weight is consistent with the theoretical molecular weight.3. RT-PCR assay showed that all the three tumor cells expressed MAGE-4, the expression of MAGE-4 in EC-109 was significantly weaker than EC-1 and EC-9706. 4. All candidate peptides have no binding ability with HLA-A2 molecule, P22 (ALGLVGAQA) and P286 (KVLEHVVRV) have a weak binding ability with HLA-A3 (FI = 0.81, FI = 0.84).5. The specific CTLs induced by P22 and P286 have the significantly tumor cells cytolytic activity to EC-1, EC-9706, but weak cytolytic activity to EC-109 in which the MAGE-4 expression is lower than EC-1 and EC-9706, P22 could specifically lyse EC-1 (55.13±5.07%) and EC-9706 cell lines (62.66±2.49%), P286 could specifically lyse EC-1 (59.02±1.83 %) and EC-9706 cell lines (62.17±2.82%). (P<0.05).ConclusionsP22 (ALGLVGAQA) and P286 (KVLEHVVRV) may be the new potential HLA-A3 restricted CTL epitopes from MAGE-4.