A newly identified MAGE-3-derived epitope recognized by HLA-A24-restricted cytotoxic T lymphocytes

Five MAGE-3-derived peptides carrying an HLA-A24-binding motif were synthesized. Binding capacity of these peptides was analyzed by an HLA-class-I stabilization assay. Two of the 5 peptides bound to HLA-A*2402 molecule with high affinity, and 3 peptides with low affinity. Peripheral-blood mononuclear cells (PBMC) depleted of CD4+T cells were stimulated with the peptides to determine whether these peptides would induce cytotoxic T lymphocytes (CTL) from PBMCs obtained from 7 healthy HLA-A*2402+ donors. Peptide M3-p97 (TFPDLESEF; corresponding to amino-acid residues 97-105 of MAGE-3), with high binding capacity to the HLA-A*2402 molecule, elicited the peptide-specific and HLA-A24-restricted CD8+CTL lines in 2 of the 7 donors, while none of the 4 other peptides induced CTL specific for the corresponding peptide in any of the donors. CTL lines induced by stimulation with peptide M3-p97 exhibited cytolytic activities against HLA-A*2402 transfectant cell lines (C1R-A*2402) in the presence of peptide M3-p97, but not in unloaded or irrelevant peptide-pulsed C1R-A*2402 cells. The CTL lines and a cloned CD8+CTL isolated from one of the bulk populations by limiting dilution could lyse MAGE-3+/HLA-A*2402+ squamous-cell-carcinoma(SCC) lines but neither MAGE-3-/HLA-A*2402+ nor MAGE-3+/HLA-A*2402- SCC lines, indicating that M3-p97 can be naturally processed and presented on the tumor-cell surface in association with HLA-A*2402 molecules. Combined with the 4 currently reported CTL epitopes derived from MAGE-3 and presented by HLA-A1, HLA-A2, HLA-A24 or HLA-B44, identification of this CTL epitope presented by the HLA-A*2402 molecule will extend the application of MAGE-3-derived peptides for immunotherapy for cancer patients.     

Analysis of individual specific cytotoxic T lymphocytes for two MAGE-3-derived epitopes presented by HLA-A24

BACKGROUND: The human MAGE-3 gene encodes tumor-specific antigens that are recognized by cytotoxic T lymphocytes (CTLs) and expressed in a high percentage of various malignant tumors. Of the five MAGE-3-derived CTL epitopes identified to date, two nonapeptides (TFPDLESEF and IMPKAGLLI, designated MAGE-3.A24a and MAGE-3.A24b, respectively) can be expressed on the tumor surface by binding to the HLA-A24 molecule, which is the most frequent HLA class I molecule in Asian populations. To compare the immunogenecities of the two peptides, individual specific CTL lines were generated for each peptide (MAGE-3.A24a and MAGE-3.A24b). METHODS: Peripheral blood mononuclear cells (PBMCs) from four HLA-A24+ healthy donors were stimulated in vitro with autologous dendritic cells pulsed with MAGE-3.A24a, MAGE-3.A24b or both and were subsequently cultivated with a cytokine combination including interleukin-2. RESULTS: We succeeded in generating peptide-specific CTL lines in two of the four donors. The two CTL lines showed similar cytolytic levels against three MAGE-3+/HLA-A24+ cancer cell lines and also target cells pulsed with the corresponding peptide. Cytolytic activities were blocked by either anti-CD8 or anti-HLA-A24 monoclonal antibodies. CONCLUSIONS: The results suggest that MAGE-3.A24a and MAGE-3.A24b peptides have equal potential in inducing MAGE-3-specific and HLA-A24-restricted CTLs.     

联合表位肽诱导HLA-A2+人PBMC产生抗原特异性CTL及杀伤活性研究

目的:探讨MAGE-3,MAGE-n抗原表位体外联合诱导的CTL,并研究其特异性杀伤活性。方法:候选抗原表位以固相多肽合成技术合成,并用HPLC进行纯化,质谱法(MS)鉴定,以流式细胞仪筛选HLA-A2 人外周血PBMC,T2细胞负载抗原肽反复刺激活化诱导抗原特异性CTL,LDH检测其杀伤活性。结果:联合表位肽体外刺激人PBMC,能够较强地诱导抗原特异性CTL并产生特异性杀伤。结论:MAGE-3与MAGE-n的HLA-A2限制性CTL表位肽的联合应用能够产生较强的体外抗肿瘤免疫反应。     

Development of HLA-A2402/K(b) transgenic mice

HLA-transgenic mice have been developed to facilitate studies of HLA-restricted cytotoxic responses, e.g., for the identification of immunodominant HLA-restricted CTL epitopes and the optimization of peptide or DNA vaccine constructs for human use. We have developed HLA-A2402/K(b)-transgenic mice expressing chimeric human (alpha1 and alpha2 domains of HLA-A2402) and mouse (alpha3, transmembrane and cytoplasmic domains of H-2K(b)) class I molecules. Immunization of these HLA-A2402/K(b)-transgenic mice with various known HLA-A24-restricted immunodominant cancer CTL epitope peptides derived from gp100, MAGE-1, MAGE-3, Her2/neu, CEA and TERT induced HLA-A24-restricted, peptide-specific CTLs. Using these transgenic mice, we identified a novel HLA-A24-restricted CTL epitope, PSA(152-160), encoded by human prostate-specific antigen. Staining with HLA tetramers showed that the cytotoxic activity induced by immunizing with PSA(152-160) in HLA-A2402/K(b) transgenic mice was HLA-A2402-restricted and CD8-dependent. Therefore, PSA(152-160) might be a candidate peptide for vaccination of HLA-A24(+) patients with prostate cancer. Our results suggest that HLA-A2402/K(b) transgenic mice might be useful in the search for HLA-A24-restricted CTL epitopes functioning as human cancer antigens and for the development of peptide-based cancer immunotherapy.     

Prediction and analysis of HLA-A2/A24-restricted cytotoxic T-lymphocyte epitopes of the tumor antigen MAGE-n using the artificial neural networks method on NetCTL1.2 Server

Cancer immunotherapy has become one of the most important therapeutic approaches to cancer in the past two decades. Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). Antigen-specific CTLs induced by MAGE-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumor. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma. It is highly homologous to the MAGE-A gene subfamily, particularly to MAGE-3 (93%). MAGE-n-derived peptide QLVFGIEVV is a novel HLA-A2.1-restricted CTL epitope that induces MAGE-n-specific CTLs in vitro. Identification of these CTL epitopes may lead to clinical applications of these peptides as cancer vaccines for patients with MAGE-n(+)/HLA-A2(+) tumors. In the present study, HLA-A/A24-restricted CTL epitopes of antigen MAGE-n were predicted using the NetCTL1.2 Server on the web, COMB >0.85. The results showed that the NetCTL1.2 Server prediction method improved prediction efficacy and accuracy. Additionally, 8 HLA-A2- and 9 HLA-A24-restricted CTL epitope candidates (nonamers) derived from the tumor antigen MAGE-n were predicted. These nonamers, following identification via experimentation, may contribute to the development of potential antigen peptide tumor vaccines.     

Identification of HLA-A24-restricted CTL epitope from cancer-testis antigen, NY-ESO-1, and induction of a specific antitumor immune response.

PURPOSE: For the development of peptide-based, cancer-specific immunotherapy, the identification of CTL epitopes from additional tumor antigens is very important. NY-ESO-1, a cancer-testis antigen, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A24-expressing individuals cover >60% in the population of Japan, we aim at identifying NY-ESO-1-encoded peptide presented by HLA-A24. EXPERIMENTAL DESIGN: In our study, a HLA-A24-restricted CTL epitope was identified by using the following four-step procedure: (a) computer-based epitope prediction from the amino acid sequence of NY-ESO-1 antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A24 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward various carcinoma cells expressing NY-ESO-1 antigen and HLA-A24. RESULTS: Of the tested peptides, effectors induced by a peptide of NY-ESO-1 at residue position 158-166 lysed three kinds of carcinoma cells expressing both NY-ESO-1 and HLA-A24. Our results indicate that peptide NY-ESO-1 (158-166) (LLMWITQCF) is a new HLA-A24-restricted CTL epitope capable of inducing NY-ESO-1-specific CTLs in vitro mediating HLA class I-restricted manner. CONCLUSIONS: We identified a novel HLA-A24-restricted NY-ESO-1-derived epitope peptide (LLMWITQCF) that could induce specific CTLs from the peripheral blood mononuclear cells of HLA-A24(+) healthy donors. This peptide would be useful in further evaluating the clinical utility of peptide-based, cancer-specific immunotherapy against various histological tumors.     

HER-2, gp100, and MAGE-1 are expressed in human glioblastoma and recognized by cytotoxic T cells

It has recently been demonstrated that malignant glioma cells express certain known tumor-associated antigens, such as HER-2, gp100, and MAGE-1. To further determine the possible utilization of these antigens for glioma immunotherapy and as surrogate markers for specific tumor antigen cytotoxicity, we characterized the presence of mRNA and protein expression in 43 primary glioblastoma multiforme (GBM) cell lines and 7 established human GBM cell lines. HER-2, gp100, and MAGE-1 mRNA expression was detected in 81.4%, 46.5%, and 39.5% of the GBM primary cell lines, respectively. Using immunoreactive staining analysis by flow cytometry, HER-2, gp100, and MAGE-1 protein expression was detected in 76%, 45%, and 38% of the GBM primary cell lines, respectively. HLA-A1-restricted epitope specific for MAGE-1 peptide (EADPTGHSY) CTL clone B07 and HLA-A2-restricted epitope specific for HER-2 peptide (KIFGSLAFL) CTL clone A05 and gp100 peptide (ITDQVPFSV) CTL clone CK3H6 were used in this study. The specificity of CTL clone was verified by HLA/peptide tetramer staining. Three CTL clones could efficiently recognize GBM tumor cells in an antigen-specific and MHC class I-restricted manner. IFN-gamma treatment can dramatically increase MHC class I expression of GBM tumor cells and significantly increase CTL recognition of tumor cells. Treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the mRNA expression of MAGE-1 and epitope presentation by autologous MHC. These data indicate that HER-2, gp100, and MAGE-1 could be used as tumor antigen targets for surrogate assays for antigen-specific CTLs or to develop antigen-specific active immunotherapy strategies for glioma patients.     

Immunotherapy of bladder cancer using autologous dendritic cells pulsed with human lymphocyte antigen-A24-specific MAGE-3 peptide.

Recent investigations have demonstrated the efficacy of autologous dendritic cells (DCs) pulsed with tumor antigens to generate tumor-specific CTLs against cancer cells. Melanoma antigens (MAGE) are a family of tumor-specific antigens shown to be expressed in various tumors, including bladder cancers and melanoma, but not in normal tissues except for the testis. Because invasive bladder cancers are frequently reported to express MAGE, we explored the possibility of establishing a new immunotherapeutic modality against advanced bladder cancer using autologous DCs pulsed with one of the MAGE-3 epitope peptides (IMPKAGLLI), which is synthesized to bind specifically to HLA-A24. A MAGE-3-expressing bladder cancer cell line, FY, was newly established from a lymph node metastasis of bladder cancer in a HLA-A24+ patient. The FY cell-specific CTL response was significantly higher when CTL was induced by autologous DCs pulsed with IMPKAGLLI than by FY cells alone or by nonpulsed DCs in vitro. A total of four HLA-A24+ patients with advanced MAGE-3+ bladder cancers were treated with s.c. injections of autologous DCs pulsed with IMPKAGLLI every 2 weeks for a minimum of 6 and a maximum of 18 times. Three of four patients showed significant reductions in the size of lymph node metastases and/or liver metastasis. No significant untoward side effects were noted in these patients. This study indicated that, at sometime in the future, tumor-specific DC-based cancer immunotherapy may be useful as an additional treatment modality against advanced bladder cancer.     

Identification of peptide vaccine candidates sharing among HLA-A3+, -A11+, -A31+, and -A33+ cancer patients.

PURPOSE: Only a few studies have been reported on CTL epitope peptides restricted with alleles other than HLA-A2 and -A24. The HLA-A11, -A31, and -A33 alleles share similar binding motifs with HLA-A3 and -A68 alleles, and, thus, are classified as an HLA-A3 supertype. This study tried to identify CTL epitope peptides as vaccine candidates sharing by HLA-A3(+), -A11(+), -A31(+), and -A33(+) cancer patients. EXPERIMENTAL DESIGN: Seven peptides possessing the ability to induce HLA-A31-restricted and tumor-reactive CTLs were examined for their ability to induce HLA-A3-, -A11-, and -A33-restricted and tumor-reactive CTLs from peripheral blood mononuclear cells (PBMCs) of 18 epithelial cancer patients. The five reference peptides all have the ability to induce CTL activity restricted with one of the HLA-A3 supertypes, and, thus, were also examined as positive controls. RESULTS: Three peptides (2 from beta-tublin5- and 1 from CGI37-derived peptides) induced tumor-reactive CTLs in PBMCs of HLA-A3(+), -A11(+), and -A33(+) cancer patients with various frequencies (17-50%). One RLI- or KIAA0036-derived peptide induced tumor-reactive CTLs in PBMCs of HLA-A3(+) and -A11(+) or HLA-A11(+) and -A33(+) cancer patients also with various frequencies (22-67%), respectively, whereas the other peptide induced CTL activity in only HLA-A33(+) patients. Among the five reference peptides tested, one peptide, TRP2-197, induced CTL activity in both HLA-A11(+)- and -A33(+)-restricted manners. CONCLUSIONS: We identified new peptide vaccine candidates for HLA-A3, -A11, -A31, and -A33 positive cancer patients. This study may facilitate the development of both basic and clinical studies of peptide-based immunotherapy for cancer patients with other alleles of HLA-A2 and -A24.     

Generation of HER2 specific, HLA-A24 restricted CTLs derived from gastric cancer patients

Cancer vaccine therapy is one of the new treatment modalities for gastric cancer at the advanced stage. In this study, we have identified HER2 peptide epitopes restricted by HLA-A24, which is one of the most common alleles in Japanese. We generated immature DCs from PBMCs in a HLA-A24+, HER2+ gastric cancer patient. Immature DCs were co-incubated with irradiated PC-9 cell line, and then autologous PBMCs were co-incubated with PC-9-derived antigen-loaded DCs. As a result, we were able to generate HER2 reactive, HLA-A24-restricted CTL lines. The CTL's specificity was evaluated with ELISPOT analysis and cytotoxic assay. The CTLs specifically recognized cancer cells expressing HLA-A24 and HER2. We synthesized a set of HLA-A24 binding, HER2-derived peptides to identify HLA-A24 restricted peptide epitopes derived from HER2. In conclusion, we were able to identified HER2 peptide epitopes restricted by HLA-A24, suggesting that these epitopes will be new targets for cancer vaccine therapy.     

肿瘤抗原MAGE-12新的HLA-A2限制性细胞毒性T细胞表位的预测

目的预测黑色素瘤抗原MAGE-12的HLA—A2限制性CTL表位。方法采用SYFPEITHI超基序远程预测系统与量化基序多项式法、延展基序联合应用，筛选MAGE-12抗原HLA—A0201限制性CTL表位。结果共预测出5个MAGE-12抗原HLA—A2限制性CTL表位。结论超基序法与量化基序，延展基序联合应用可以提高预测效率，为实验方法探索MAGE-12的表位提供有用线索。     

Identification of a new MAGE-A10 antigenic peptide presented by HLA-A*0201 on tumor cells

MAGE-A antigens belong to cancer/testis (CT) antigens that are expressed in tumors but not in normal tissues with the exception of testis and placenta. Among MAGE-A antigens, MAGE-A10 is extensively expressed in various histological types of tumors, representing an attractive target for tumor immunotherapy. Cytotoxic T lymphocytes (CTLs) play a key role in anti-tumor immune responses, so the identification of CTL epitopes derived from MAGE-A10 would contribute a lot to the design of epitope-based vaccines for tumor patients. In this study, we predicted HLA-A*0201-restricted CTL epitope peptides of MAGE-A10, followed by peptide/HLA-A*0201 binding affinity and complex stability assays, and induced peptide-specific CTL immune responses. Of the selected three peptides (designated P1, P2 and P3), P1 (MAGE-A10310-318, SLLKFLAKV) could elicit peptide-specific CTLs both in vitro from HLA-A*0201-positive PBMCs and in HLA-A*0201/Kb transgenic mice. And, the induced CTLs could lyse MAGE-A10-expressing tumor cells in a HLA-A*0201-restricted fashion but not MAGE-A10-negative tumor cells. Our results demonstrate that the peptide MAGE-A10310-318 is a HLA-A*0201-restricted CTL epitope of MAGE-A10 and could serve as a target for therapeutic antitumoral vaccination.     

Cyclin D1-specific cytotoxic T lymphocytes are present in the repertoire of cancer patients: implications for cancer immunotherapy

PURPOSE: Cyclin D1, a key cell cycle regulator, is overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for cancer immunotherapy. Nevertheless, it had previously been suggested that efficient T cells recognizing cyclin D1-derived epitopes are absent from the repertoire because of thymic deletion. We attempted to induce autologous CTL from healthy donors and patients with cyclin D1-overexpressing tumors using a highly efficient T-cell expansion system based on CD40-activated B cells as antigen-presenting cells. EXPERIMENTAL DESIGN: Cyclin D1-derived, HLA-A*0201-restricted epitopes were predicted by multiple computer algorithms, screened in HLA-A2-binding assays, and used for T-cell stimulation. The generated CTL lines and clones were analyzed by IFN-gamma enzyme-linked immunosorbent spot assay or cytolysis assay. RESULTS: After screening, at least two naturally processed and presented HLA-A*0201-binding cyclin D1 epitopes were identified. CTL specific for these epitopes could be successfully generated from HLA-A2(+) donors. T cells efficiently recognized target cells pulsed with the cognate peptide and cyclin D1-expressing tumor cell lines in an HLA-A*0201-restricted manner. More importantly, HLA-A*0201-matched, primary cyclin D1(+) tumor cells were efficiently recognized by cyclin D1-specific CTL. These CTL could be generated from patients with mantle cell lymphoma and cyclin D1(+) colon cancer. CONCLUSIONS: These results underscore that cyclin D1 needs to be considered as a target for broad-based antitumor immunotherapy.     

Cancer immunotherapy in head and neck region

There is no one common immunotherapy for the treatment of head and neck cancer (H&N cancer). A streptococcal agent, OK-432, which is classified as a biological response modifier (BRM), is occasionally used by means of local administration for recurrent H&N cancer, and the response rate is approximately 18%. In regard to specific immunotherapy, a murine monoclonal antibody (named mAb 225) against the epidermal growth factor receptor (FGFR) that is frequently overexpressed in H&N cancer has been produced in the U.S.A. Furthermore, to obviate human anti-mouse antibody responses, a chimeric human-to-murine version of mAb 225 (C225) was developed by exchanging the constant regions of mAb 225 to counterparts in human immune globulin. Phase I clinical trials of C225 in the U.S.A. demonstrated that treatment with C225 was well tolerated and that C225 given in combination with cisplatin has biologic activity. On the other hand, many tumor antigens recognized by cytotoxic T lymphocytes (CTL) have been identified from a variety of malignant tumors and some of them, including the MAGE-3 antigen, are frequently expressed in H&N cancer. We identified an MAGE-3-derived epitope recognized by HLA-A24-restricted CTL from peripheral blood mononuclear cells (PBMC). In contrast we failed to generate CTL specific for MAGE-3+/HLA-A24+ tumors from PBMC in any of 5 HLA-A24+ cancer patients whose tumors expressed the MAGE-3 gene. Therefore, we did not apply MAGE-3-derived CTL epitope in clinical uses such as peptide vaccine and peptide pulsed dendritic cell infusion for H&N cancer.     

A wild-type sequence p53 peptide presented by HLA-A24 induces cytotoxic T lymphocytes that recognize squamous cell carcinomas of the head and neck.

Evidence has accumulated indicating that HLA-A2-restricted CTLs specific for human wild-type sequence p53 epitopes lyse tumor cells expressing mutant p53. To explore the possibility that wild-type sequence p53 peptides could also be used in vaccines for patients expressing HLA-A24 antigen, another frequent HLA class I allele, we investigated the induction of HLA-A24-restricted p53-specific CTLs from the peripheral blood lymphocytes of normal donors. Of six p53-derived peptides possessing an HLA-A24 binding motif, the p53 peptide 125-134 (p53(125-134)) was found to have a high binding capacity and induced peptide-specific CTLs from peripheral blood mononuclear cells, using peptide-pulsed autologous dendritic cells and subsequent cultivation with cytokines interleukin 2 and interleukin 7. Bulk CTL populations lysed peptide-pulsed HLA-A24+ targets as well as HLA-A24+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. However, IFN-gamma pretreatment of HLA-A24+ SCCHN cell lines was necessary for lysis, suggesting that a ligand density higher than that normally expressed by tumor cells is required for these CTLs to mediate lysis. Moreover, a cloned CTL, designated TH#99, isolated from the bulk population by limiting dilution, lysed HLA-A24+ SCCHN targets more efficiently than the bulk CTL population. Lysis was inhibited by anti-HLA class I monoclonal antibody but not by anti-HLA-DR monoclonal antibody. These results indicate that HLA-A24-restricted CTLs recognizing the wild-type sequence p53(125-134) can be generated using autologous dendritic cells from precursors present in peripheral blood lymphocytes obtained from normal HLA-A24+ donors. This finding suggests that vaccine strategies targeting wild-type sequence p53 epitopes can be extended to a wider range of cancer patients.     

Specific immunotherapy with cancer vaccines

With the recent progress in molecular biology and gene technology, many new cancer-specific antigens have been identified. Many studies have demonstrated the role of HLA class I-restricted cytotoxic T lymphocytes (CTLs) in cancer specific-immunotherapies. We have also established HLA-A24- and A26-restricted and cancer-specific CTLs from a patient with squamous cell carcinoma of the esophagus. Using CTLs, we identified a new gene SART-1 by cDNA-expression cloning and some SART-1-derived cancer rejection peptides were also identified. Further more, using the same approach, we identified a cyclophilin B gene that encodes antigenic epitopes recognized by HLA-A24-restricted and tumor-specific CTLs. Now we are performing phase I trials using these peptide vaccines and have found an increase in CTL precursor frequency in some cases in an in vitro study. However, other recent studies have reported that many tumors escape from CTL recognition by downregulation of HLA class I expression. Moreover, most cancer cells produce a suppressor agents against the immune system. Therefore, we must resolve these major problems to produce successful cancer vaccine therapy soon.     

Analysis of MAGE-3 derived synthetic peptide as a human lung cancer antigen recognized by cytotoxic T lymphocytes

BACKGROUND: The human MAGE-3 gene was originally discovered in melanoma cells that encode tumor antigens, and has been reported to be expressed in various types of tumors, including lung cancer, but not in normal tissues other than testis or placenta. Our aim in this study was to clarify whether HLA-A2 restricted MAGE-3 peptide (FLWGPRALV) could be a lung cancer antigen recognized by cytotoxic T lymphocytes (CTL). METHODS: MAGE-3-derived peptide-specific CTL were induced from the peripheral blood mononuclear cells (PBMC) of HLA-A0201-positive healthy donors and the regional lymph node lymphocytes (RLNL) of HLA-A2-positive patients with lung cancer by multiple stimulations with peptide-pulsed HLA-A0201-positive antigen-presenting cells. RESULTS: Lymphocytes stimulated with MAGE-3 peptide exhibited specific lysis of Epstein-Barr virus-transformed B cells (EBV-B) pulsed with MAGE-3 peptide, but not with control peptide derived from influenza matrix protein, erbB-2, or wild type p53. Specific activity for MAGE-3-presenting targets was found after the second stimulation, and increased depending on the number of stimulations. The peptide-specific activity was inhibited by the addition of monoclonal antibodies against MHC class I and HLA-A2. Such CTL also recognized tumor cell lines expressing both HLA-A2 and MAGE-3 in an MHC class I-restricted manner, but did not recognize tumor cell lines that did not express HLA-A2 or MAGE-3. CONCLUSION: These results suggested the MAGE-3 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with lung cancer.     

Identification of HLA-A*0201-restricted cytotoxic T lymphocyte epitope from TRAG-3 antigen.

PURPOSE: Identification of tumor antigen and subsequent identification of T-cell epitope from these antigens make specific immunotherapy for malignant tumor applicable. Because TRAG-3 antigen is expressed in most melanomas and 54% of non-small cell lung carcinomas and HLA-A2.1-expressing individuals cover >50% in the population of China, we aim at identifying TRAG-3-encoded peptide presented by HLA-A2.1. EXPERIMENTAL DESIGN: In our study, a HLA-A2.1-restricted CTL epitope was identified by using the following four-step procedure: (a) computer-based epitope prediction from the amino acid sequence of TRAG-3 antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward LB373-MEL cells expressing TRAG-3 antigen and HLA-A2.1. RESULTS: Of the four tested peptides, effectors induced by a peptide of TRAG-3 at residue position 58-66 lysed LB373-MEL cells expressing both TRAG-3 and HLA-A2.1. Our results indicate that peptide TRAG-3(58 approximately 66) (ILLRDAGLV) is a new HLA-A2.1-restricted CTL epitope capable of inducing TRAG-3 specific CTLs in vitro. CONCLUSIONS: Because TRAG-3 is a cancer/testis antigen expressed in most melanomas and half of non-small cell lung carcinomas, identification of the TRAG-3/HLA-A2.1 peptide ILLRDAGLV may facilitate peptide-based specific immunotherapy for various histological tumors.     

Identification of a SART-1-derived peptide capable of inducing HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes

We have described the SART-1 gene-encoding peptides recognized by HLA-A2601-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). We now have investigated whether SART-1 encodes peptides capable of inducing the HLA-A24-restricted CTLs. Among the 18 different peptides with HLA-A24-binding motifs, the SART-1(690-698) peptide (EYRGFTQDF) was most strongly recognized by the HLA-A24-restricted and tumor-specific CTLs established from an esophageal cancer patient. After a third stimulation in vitro, this peptide induced HLA-A24-restricted CTLs recognizing the SART-1(259)+ tumor cells in PBMCs of all HLA-A24 homozygous and the majority of HLA-A24 heterozygous cancer patients and healthy donors tested. A similar activity, induction of CTLs from PBMCs, was observed in the Saccharomyces cerevisiae-derived nonapeptide (EYRGFTPMF) that shares 7 amino acids with the SART-1(690-698) peptide. The SART-1(690-698) peptide-induced CTL activity was significantly higher in PBMCs of HLA-A24 homozygotes than in HLA-A24 heterozygotes. The CTL precursor frequency in PBMCs after a third stimulation in vitro with the SART-1(690-698) peptide was high (>1/200) in both cancer patients and healthy donors. The SART-1(690-698) peptide could thus be useful for specific immunotherapy of HLA-A24+ cancer patients.     

Peptides derived from human insulin-like growth factor-II mRNA binding protein 3 can induce human leukocyte antigen-A2-restricted cytotoxic T lymphocytes reactive to cancer cells

Insulin-like growth factor-II mRNA binding protein 3 (IMP-3) is an oncofetal protein expressed in various malignancies including lung cancer. This study aimed to identify immunogenic peptides derived from IMP-3 that can induce tumor-reactive and human leukocyte antigen (HLA)-A2 (A*02:01)-restricted cytotoxic T lymphocytes (CTL) for lung cancer immunotherapy. Forty human IMP-3-derived peptides predicted to bind to HLA-A2 were analyzed to determine their capacity to induce HLA-A2-restricted T cells in HLA-A2.1 (HHD) transgenic mice (Tgm). We found that three IMP-3 peptides primed HLA-A2-restricted CTL in the HLA-A2.1 Tgm. Among them, human CTL lines reactive to IMP-3 (515) NLSSAEVVV(523) were reproducibly established from HLA-A2-positive healthy donors and lung cancer patients. On the other hand, IMP-3 (199) RLLVPTQFV(207) reproducibly induced IMP-3-specific and HLA-A2-restricted CTL from healthy donors, but did not sensitize CTL in the HLA-A2.1 Tgm. Importantly, these two IMP-3 peptide-specific CTL generated from healthy donors and cancer patients effectively killed the cancer cells naturally expressing both IMP-3 and HLA-A2. Cytotoxicity was significantly inhibited by anti-HLA class I and anti-HLA-A2 monoclonal antibodies, but not by the anti-HLA-class II monoclonal antibody. In addition, natural processing of these two epitopes derived from the IMP-3 protein was confirmed by specific killing of HLA-A2-positive IMP-3-transfectants but not the parental IMP-negative cell line by peptide-induced CTL. This suggests that these two IMP-3-derived peptides represent highly immunogenic CTL epitopes that may be attractive targets for lung cancer immunotherapy.