| Research BackgroundCancer has always been the primary threats of human life. Recent years, benefit from the development of the understanding and clinical applying, the cancer immunotherapy strategy has made remarkable achievements. Along with the progress of the interdisciplinary fields which combined bioinformatics and immunology, the prediction and construction of CTL epitopes that have been derived from tumor associated antigen now attract more and more attention. Choosing the highly specific tumor associate antigen is the most critical concern for the peptide vaccine based strategy. Currently, most antigen that are used for the development of peptide vaccine are expressed by tumor cell itself. In our study, based on the FAP (Fibroblast Activation Protein a) that is specifically expressed in the tumor stromal fibroblast, we did peptide epitopes prediction and evaluation. FAP, which was recognized as a member of the post-propyl peptidase family, was fairly discover in the CAF (Cancer-associated Fibroblasts) cells in 1986, and was officially named as FAP in 1990. FAP has found to be expressed in the cell membrane and cytoplasm of hte stromal fibroblasts cells in more than 90% of the epithelial tumors, while not expressed in normal fibroblasts. FAP now is well accepted to be a highly specific tumor associate antigen that can be used as a target for the tumor diagnosis and treatment. The involvement of FAP in a wide range of tumor development, including facilitating the formation of primary tumors, helping tumor cells invasion and metastasis, and inhibiting tumor immune function, has been proven during recent years. Development of novel anti-tumor strategy by targeting FAP now become a promising area in tumor research area. Therefore, in the present study, FAP was used as a target antigen for epitope identification and immunogenicity research. Research MethodsFirstly, three online algorisms, SYFPEITHI, BIMAS and NetCTL, were used to predict HLA-A2 restricted epitopes that were derived from FAP. All the predicted epitopes were synthesized with Fmoc synthesis protocol and subsequently purified by RP-HPLC, and identified with mass spectrum. Binding ability and stability experiment were then performed to screen out the epitope peptides with high affinity and stability with HLA-A2. To determine the immune activity, ELISPOT and Intracellular cytokine staining (ICS) assay was conducted to evaluate the capability of stimulating IFN-γ release of CD8+ CTL, and LDH target cell killing assay was used to detect the CTLs cytoxicity. Then the in vivo immune activity of the peptides epitopes were investigated using HLA-A2.1/Kb transgenic mice model. Splenocytes and leg ministry lymph nodes cells were isolated to stimulate. ELISPOT, ICS, LDH assay were performed to analysis of the ability of inducing and activating the CTLs by each candidate epitope peptide. Meanwhile, mice peripheral blood serum and medium were isolated to perform ELISA assay to evaluate IFN-γ releasing. Research ResultsEight epitope candidates were firstly predicted by using SYFPEITHI, BIMAS, and NetCTL. And then were chemically synthesized with Fmoc solid phase protocol. RP-HPLC was then used to purify all the above epitope peptides. Mass spectra identification was implemented to make sure that we have acquired the expected epitopes. Three FAP derived epitopes:P120 (Lys-Leu-Trp-Arg-Tyr-Ser-Tyr-Thr-Ala), P265 (Phe-Ile-Ile-Asp-Thr-Thr-Tyr-Pro-Ala), P639 (Gly-Leu-Phe-Lys-Cys-Gly-He-Ala-Val) were further screened out for the high HLA-A2 affinity (0.66, 1.13,1.25 respectively), and stability (>4h,>6h,>6h respectively). On the basis of which, immunoactivity was further analyzed. The peripheral blood was isolated from three HLA-A2+ donors. PBMCs were harvested and then induced by those three epitopes to get the antigen specific CTLs which were then co-incubated with epitope loaded T2A2 cells to conduct the ELISPOT assay. The result proved that all the three peptide epitopes were capable to induce a large number of IFN-γ positive CTLs in HLA-A2+ donors. The LDH assay results indicated that CTLs were effectively activated by P120, P265, and P639. Their cytotoxicity were significantly higher than the negative control group with the same E:T ratio. Furthermore, ICS assay revealed that all the three peptides could better induce IFN-y releasing by CD8+ CTL than the negative control group. Specifically, as for the P120, P265, P639, the IFN-γ+CD8+ CTL proportions were 2.62%,3.47%,9.70% respectively.In the HLA-A2.1/Kb transgenic mice model, the results of ELISPOT, LDH and ICS assay were highly consistent with the results got from human PBMCs. And ELISA assay was used to confirm the IFN-γ level in the mice peripheral blood serum and the medium. Compared with the Th epitope and PBS control (254.28 pg/mL,187.64 pg/mL), P120, P265, P639 induced higher IFN-γ releasing level (337.97 pg/mL,876.99 pg/mL,534.52 pg/mL) in the serum of mice peripheral blood. As for the IFN-y in medium supernatant, IFN-γ level appeared much higher and achieved 43.73μg/mL, 54.25μg/mL,50.03μg/mL respectively by stimulating with P120, P265, and P639. IFN-γ releasing in Th epitope and PBS control group were only 24.20μg/mL,19.60 μg/mL. Besides, mice bodyweight kept a stable level. Together with the consistent mice liver, kidney,heart and lung index among different groups (P>0.05), we draw the conclusion that those three epitopes showed no significant side effects. ConclusionIn conclusion, three peptide epitopes, P120, P265 and P639, which were derived from FAP, were screened out by performing a series in vitro and in vivo experiments. All the three peptides not only were found to be able to activate the immune response to a certain extent, and also were proven to process certain immune activity in the HLA-A2.1/Kb transgenic mice model. Our present study provide solid support for the development of novel peptide vaccine based anti-tumor strategy by targeting tumor stromal cells. |
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