Screening Of A TCR Gene Against Mutated Survivin Epitope Peptide And Study Of Its Anti-hepatoma Effect

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world, which represents the third leading cause of cancer death world-wide. China is the high incidence of HCC and the number of new onset and death accounted for more than50%of the world. Nearly30million people a year die from liver cancer in China. Resection, chemo-embolization, local ablation and radiotherapy are the most commonly curative treatments in the clinic. However, these treatments have not remarkably shown a survival advantage for HCC patients.In recent years, tumor immunotherapy has been demonstrated as an attractive therapeutic option for malignant tumors, which has the advantages of strong anti-tumor immunogenicity and weak side effects compared with conventional therapy. At present, tumor immunotherapy is used to kill tumor cells by excitation of an effective anti-tumor immune response in tumor patients, which include tumor-associated antigens (TAA) peptide vaccine therapy and adoptive immune cells treatment. The anti-tumor immune response is divided into two types, non-specific immune response and specific immune response. Cytokine-induced killer cell(CIK) is one of the representative adoptive cell therapy methods, which has been in clinical application and played a therapeutic role in some patients. TAA peptide vaccine treatment and T cell receptor (TCR) gene modified cell therapy have been the focus and new directions of current research, which can elicit specific anti-tumor immune response against TAA.TAA have been shown to be associated with the development of tumor, which are usually significantly higher expression in cancerous cells, but not expression or trace expression in normal tissue. This feature enables the TAA may have potential application in cancer therapy. An ideal universal TAA for tumor vaccine should have the following characteristics:1) present on the majority of human cancers but rarely on normal tissues,2) include peptide sequences that bind to major histocompatibility complex (MHC) molecules effectively, and3) be recognized by the T-cell repertoire of tumor patients in an MHC-restricted fashion and can elicit specific T-cell response. In the past three decades, researchers have identified more than170varieties of antigenic peptides from60kinds of human TAA, and confirmed that these antigenic peptides could be presented by MHC molecules and recognized by T cells, which were are considered to be a potential target for cancer therapy. There have been carried out a variety of clinical trials for TAA vaccines. Although the first cancer vaccine, Sipuleucel-T (Provenge) that is specific against prostate cancer, has received the FDA approval in2010, in most of clinical trials to date, TAA peptide-based vaccines rarely eliminated tumors. This makes the study of specific tumor immunotherapy have experienced great difficulty.In recent years, some new reasons about the poor efficacy of tumor vaccine have been understood with the deepening of the study. Because the tumors are malignant transformation from autologous cells, most of the TAA are autoantigens. Due to the central mechanism of immune tolerance, TCR high affinity T cells that are agaist TAA have been immune removed early in the development. Most of the remaining TAA-recognized mature T cells in vivo are TCR low-affinity naive T cells, which are difficult to be activated by TAA peptides. To solve this problem, we need for new ideas in the design of the TAA peptides for tumor-specific immunotherapy.At the same time, we need to realize that ideal TAA peptides are still difficult to activate an effective anti-tumor immune because of the abnormal immune function that already exists in cancer patient. Some studies have shown that adoptive transfusion with tumor antigen-specific CTL clones that were induced in vitro may be a new anti-tumor therapeutic approach. However, proportion of antigen-specific T cells is only10-6～10-4in the T cell repertoire with the MHC same genotype. It is difficoult to rapidly produce cytotoxic T lymphocytes against a TAA in vitro and obtain a sufficient number of cells, which may affect the subsequent adoptive anti-tumor efficacy.A number of TAAs epitope peptides, including a-fetoprotein (AFP), NY-ESO1and MAGE-A, have been the productions of anti-HCC vaccine to conduct clinical trials in the last decade. However, the vast majority of trial efficacies were not prominent. Survivin belongs to the inhibitory apoptotic protein family (IAP), which has been implicated in blocking mitochondrial-dependent apoptosis by targeting caspase9and promoting tumor progression. Survivin is aberrantly expressed in virtually every human cancer but is undetectable in normal differentiated adult tissues. Among them, approximately90%of patients with hepatocellular carcinoma were confirmed overexpression of Survivin in some studies. Some Clinical trials based on Survivin-peptide vaccine have performed on advanced melanoma, breast, colon and urothelial cancer patients. Published partial results have shown that in several cases there was induction of antigen-specific T cell responses to the Survivin-peptide vaccine. However, currently there are not yet reported on the Survivin-peptide vaccine for treatment of primary HCC up to date.In this study, based on the latest research progress in related fields abroad and our previous research achievements, we conducted a three-part study that were related to the specific anti-HCC immune experimental work against Survivin antigen. Firstly, in order to solve the problem of TAA peptides with low immunogenicity, we used bioinformatics techniques to analyze the possible Survivin CTL epitopes and screened the point mutation Survivin CTL epitopes that owned the higher affinities to the MHC molecules and TCR molecules. Secondly, in order to solve the problem that TAA peptide is difficult to effectively activate the immune response in cancer patient, we took advantage of the screened mutant Survivin antigen peptide to stimulate T cell activation and established reactive CTL clones. Finally, we analysed the expression profile changes of TCR gene subfamilies of mutant Survivin antigen peptide-induced CTL clone, and indentified the specific reactive TCR gene. After transfected with TCR gene, the gene-modified heathy lymphocytes had the specific anti-HCC function. Through this study, we may provide a tumor-specific immunotherapy new program that help to break tumor immune tolerance.1. Computational analysis of point-mutated Survivin CTL epitopesIn the anti-tumor immune response, CTLs specifically recognize the epitope peptides presented by MHC-I molecules (HLA-I molecules in human) through its TCR molecules on surface and kill tumor cells. So epitope design quality is very important for a tumor vaccine preparation. Some studies have reported that, there were more than50%of HCC patients with positive expression of HLA-A2molecule in high incidence area of China. HLA-A2genotype may be a predisposing factor associated with tumorigenesis. Therefore, we thought of HLA-A2-restricted Survivin epitope peptides as our research objects. In this section, we planned to screen ideal candidate point-mutated Survivin epitope peptides using computational analysis and experimental verification. At first, two HLA-peptide biding prediction computational methods BIMAS (http://www-bimas.cit.nih.gov/molbio/hla_bind/) and SYFPEITHI (http://www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm) were performed to analyze the HLA-A2restricted Survivin CTL epitope nonapeptides. We screened the top20-scored peptides to identify the low scores epitope peptide that the amino acid residues of the second bit is not hydrophobic amino acids, such as V, L, I, M, T, etc. Finalized, Sur79(KHSSGCAFL) was confirmed as a candidate for the wild-type Survivin epitope peptide to carry out the artificial mutation of the amino acid sites. Through the comparison the online rating changes between before and after mutation, we identified two point-mutated peptides Sur79L2and Sur79M2for subsequent experiments. We then synthesized five survivin epitope peptides in vitro and done the MHC-peptide binding stability experiments with T2cells. The results showed that point-mutated peptides Sur79L2and Sur79m2had the higher affinity to MHC molecular that was consistent with the computational analysis results. Thus, we got two point-mutated Survivin peptides Sur79L2and Sur79M2used for follow-up experiments.2. Establishment of the anti-HCC-specific CTL clones induced with point-mutated Survivin peptides CTLs are the effector CD8+T cells with cytotoxicity function, which can directly kill target cells by secreting perforin and granzyme (Granzyme B, GzmB) and induce target cell apoptosis by Fas/FasL pathway. CTLs can repeatedly kill target cells without cells self-injury and have the killing characteristics of efficiency, the antigen specificity and MHC restriction. CTLs and natural killer cells constitute an important line of defense of the anti-tumor immune. In this section, we planned to establish point-mutated peptides Sur79L2and Sur79M2-induced CTLs clones and verify their cytotoxic effect against hepatocellular carcinoma cell lines. Density gradient centrifugation was performed to separate mononuclear cells from an ascites samples derived from a HLA-A2+Survivin+HCC patients. Adherent mononuclear cells were induced into dendritic cells (Dendritic cell, DC) in vitro for10days and the mature DC surface markers were detected by flow cytometry at day10. Ascites-derived tumor-associated lymphocytes (TAL) were incubated with lOmM different synthetic epitope peptides for14days for the first round of stimulus. In the second round of stimulation,10mM of the antigen peptide,300IU/mL of recombinant human IL-2and the mature DCs were added into culture system and culture was continued for12days. ELISPOT methods were detected the number of activated lymphocytes that effectively secrete interferon-y (IFN-y) after peptide stimulation. Cytotoxicity assays were carried out using peptide-induced CD8+T cells isolated by immunomagnetic beads as effector cells and peptides-loaded T2cells as target cells. After peptide-induced CTLs were generated in vitro, flow cytometry assays were performed to detect the CD8+GzmB+cell ratio and the morphological changes of tumor cells were observed with inverted phase contrast microscope. The rates of tumor cells lysis were assayed using CytoTox96(?) kit. The results showed that Sur79L2and Sur79M2-induced CTLs could release a large number of y-IFN after stimulated with point-mutated peptides and lysis peptides-loaded T2cells. When co-cultured with HCC cell lines, point-mutated peptides-induced CTLs could secrete GzmB and effectively lysis hepatoma cell lines on HLA-A2-restricted manner. Thus, we demonstrated that point-mutated Survivin peptides could induce specific ani-HCC CTLs in vitro.3. Identification of specific anti-HCC TCR gene and verification of its functional characterizationTCR molecule is the transmembrane protein on T cell surface and can specifically recognize peptide-MHC complex that provid the first signal for T-cell activation. Therefore, TCR play the most important role of T cell immune function. According to the type of the TCR, T cell can be divided into TCRαβ+T cell and TCRγδ+T cell. The TCRαβ+T cell accounted for more than95%of the total number of T cell and the TCRαβ+CD8+T cell play an important anti-tumor function in the immune response. Some previous studies had shown that tumor patients lacked of functional TCRαβ+T cells, which may lead to tumor immune tolerance. In recent years, some applications of the antigen specific TCR transgenic technology have been attempted for empowering ordinary mature T lymphocytes specific killing ability. TCR gene-modified adoptive anti-tumor immunotherapy has benn become a hot topic. In this section, we planned to identify the TCR genes that have significant clonal proliferation characteristics from point-mutated peptides-induced CTLs and verify the kill-HCC ability using TCR gene transfer technology. Firstly, the changes of TCR gene subfamilies were detected by flow cytometry assay before and after mutated peptides induced and the percentage significantly higher TCRVP gene subfamilies were identified. We then did more refined TCR gene subfamilies-analysis using GenomeLab GeXP and screened the significantly monoclonal TCR gene amplification. The monoclonal TCR gene was cloned into T-vector and multiple bacteria monoclones were sequenced. By comparing the nucleic acid sequence of the plurality of CDR3regions, we confirmed the peptide-specific TCR gene with same CDR3sequences. The peptide-specific TCR gene was cloned into the adenovirus vector and the recombinant adenovirus infected HLA-A2-restricted healthy people lymphocytes. The expression levels of TCR gene after transfection were detected by flow cytometry. After incubation with TCR gene-modified PBMC, we verify the cytotoxic effects on liver cancer cell lines. The results showed that the TCRVP9and TCRVβ2two gene subfamilies appeared proportion increased in Sur79L2-induced CTLs and Sur79M2-induced CTLs had TCRVβ7and TCRVβ16two gene subfamilies proportion increased. Through further analysed using GEXP system, we finally identified a monoclonal TCRa24gene and a monoclonal TCRβ9gene from Sur79L2-induced CTLs instead of Sur79M2-induced CTLs. After transferred TCRα24β9into HLA-A2restricted healthy PBMC, the TCR gene-modified PBMC could effectively kill the hepatoma cell lines in an HLA-A2-restricted manner. Thus, we identified an point mutated peptide-specific TCRα24β9gene and confirmed its anti-hepatoma effects.In summary, we identified two point-mutated Survivin epitope peptides Sur79L2and Sur79M2depend on bioinformatic analysis combined with experimental verification. Next, Sur79L2and Sur79M2-induced CTLs clone were established in vitro using ascites-derived tumor-associated lymphocytes from a HLA-A2+HCC patient. We further screened a monoclonal amplification TCR gene TCRα24β9from Sur79L2-induced CTLs and demonstrated that TCRα24β9-modified healthy PBMC could specifially lysis hepatoma cell lines in an HLA-A2-restricted manner. This study may provid useful information and reference for new adoptive anti-tumor treatment based on point-mutated TAA peptides-induced T cells and new TCR gene anti-tumor drugs.