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.     

Identification of Lck-derived peptides capable of inducing HLA-A2-restricted and tumor-specific CTLs in cancer patients with distant metastases.

The Lck protein (p56(lck)), a src family tyrosine kinase essential for T cell development and function, is aberrantly expressed in various types of cancers. We revealed recently that Lck can be a tumor antigen recognized by HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes (CTLs) of cancer patients with metastases. In this study, we tried to identify Lck-derived epitopes capable of inducing HLA-A2-restricted and tumor-specific CTLs in cancer patients. The tumor-infiltrating lymphocytes (TILs) from 2 HLA-A2 cancer patients were found to respond to COS-7 cells when co-transfected with the lck gene and either HLA-A0201, -A0206, or A0207 cDNA. These TILs contained CTLs capable of recognizing either the Lck(61-69), the Lck(246-254), or the Lck(422-430) peptide among 24 different peptides, all of which were prepared based on the HLA-A2 binding motif. Importantly, in vitro sensitization with the latter 2 peptides induced tumor-specific CTLs in HLA-A2(+) cancer patients with metastases, but not in those without metastases. Overall, the Lck(246-254) and Lck(422-430) peptides could be useful for specific immunotherapy of HLA-A2(+) cancer patients, especially with distant metastases.     

Identification of SART3-derived peptides capable of inducing HLA-A2-restricted and tumor-specific CTLs in cancer patients with different HLA-A2 subtypes

We recently identified the SART3 antigen encoding shared tumor epitopes recognized by HLA-A2402-restricted and tumor-specific CTLs. Our study investigated whether the SART3 antigen encodes peptides recognized by the HLA-A2-restricted CTLs. The HLA-A2-restricted and tumor-specific CTL line recognized COS-7 cells co-transfected with the SART3 gene and either HLA-A0201, -A0206 or -A0207 cDNA but not those co-transfected with the SART3 gene and HLA-A2402 or -A2601 cDNA. The 2 SART3 peptides at positions 302 to 310 and 309 to 317 possessed the ability to induce HLA-A2-restricted and tumor-specific CTLs from peripheral blood mononuclear cells of cancer patients with various histological types and different HLA-A2 subtypes. Therefore, these 2 peptides could be useful for specific immunotherapy of a relatively large number of HLA-A2(+) cancer patients.     

Identification of a MAGE-2-encoded human leukocyte antigen-A24-binding synthetic peptide that induces specific antitumor cytotoxic T lymphocytes.

Because MAGE-2 gene is expressed in a wide variety of malignant tumors and HLA-A24 is the most common allele in the Japanese population and is also frequently present in Caucasians, the identification of MAGE-2-encoded peptide presented by HLA-A24 is, therefore, considered to be important in order to develop specific immunotherapy for malignant tumors using peptides as a vaccine. By using a MHC-binding assay, eight peptides derived from MAGE-2 were found to bind with sufficient affinity to the HLA-A24 molecule. When the induction of specific cytotoxic T lymphocytes (CTLs) was examined using a simplified method, the highest human lymphocyte antigen (HLA) binder (EYLQLVFGI) in these peptides was able to elicit CTLs from unseparated peripheral blood mononuclear cells in HLA-A24 healthy donors by stimulation with freshly isolated, peptide-pulsed peripheral blood mononuclear cells as antigen-presenting cells and also by using interleukin 7 and keyhole-limpet hemocyanin in a primary culture. The induced CTL could, thus, lyse HLA-A24 tumor cells expressing MAGE-2, as well as the peptide-pulsed target cells, with antigen specificity in a HLA class I-restricted manner. The identification of this peptide may, thus, be of therapeutic value in peptide-based vaccines for the treatment of several types of malignant tumors expressing MAGE-2.     

Identification of a gene coding for a protein possessing shared tumor epitopes capable of inducing HLA-A24-restricted cytotoxic T lymphocytes in cancer patients.

Genes encoding tumor epitopes that are capable of inducing CTLs against adenocarcinomas and squamous cell carcinomas, two major human cancers histologically observed in various organs, have rarely been identified. Here, we report a new gene from cDNA of esophageal cancer cells that encodes a shared tumor antigen recognized by HLA-A2402-restricted and tumor-specific CTLs. The sequence of this gene is almost identical to that of the KIAA0156 gene, which has been registered in GenBank with an unknown function. This gene encodes a Mr 140,000 protein that is expressed in the nucleus of all of the malignant tumor cell lines tested and the majority of cancer tissues with various histologies, including squamous cell carcinomas, adenocarcinomas, melanomas, and leukemia cells. However, this protein was undetectable in the nucleus of any cell lines of nonmalignant cells or normal tissues, except for the testis. Furthermore, this protein was expressed in the cytosol of all of the proliferating cells, including normal cells and malignant cells, but not in normal tissues, except for the testis and fetal liver. Two peptides of this protein were recognized by HLA-A2402-restricted CTLs and were able to induce HLA-A24-restricted and tumor-specific CTLs from peripheral blood mononuclear cells of most of HLA-A24+ cancer patients tested, but not from peripheral blood mononuclear cells of any healthy donors. These peptides may be useful in specific immunotherapy for HLA-A24+ cancer patients with various histological types.     

ABCE1, a member of ATP-binding cassette transporter gene, encodes peptides capable of inducing HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes in colon cancer patients

To provide a scientific basis for specific immunotherapy of colon cancer, this study focused on the identification of tumor-associated antigens recognized by HLA-A2-restricted and cancer-reactive cytotoxic T lymphocytes (CTLs) from tumor-infiltrating lymphocytes (TIL) of a colon cancer patient. We identified a gene coding a member of the ATP-binding cassette transporter (ABCE1), which is known as an inhibitor to the 2-5A/RNase L system, a central pathway of interferon action, and its gene expression is amplified in drug-resistant cells. The ABCE1 mRNA was ubiquitously expressed in tumor cell lines, as well as normal cells or tissues. Among 21 peptides with the HLA-A2 binding motifs, 2 ABCE1-derived peptides were recognized by the colon cancer-reactive CTLs in a dose-dependent manner. CTL precursors reactive to these 2 ABCE1 peptides were detected in the peripheral blood mononuclear cells (PBMCs) of the colon cancer patient, from whom the TILs had been obtained. In addition, these ABCE1 peptides had the potential to generate HLA-A2-restricted and colon cancer-reactive CTLs from the PBMCs of colon cancer patients, but not from those of healthy donors. Their cytotoxicity against colon cancer was mainly ascribed to peptide-specific and CD8+ CTLs. No definite cytotoxicity was observed against normal T cell blasts, irrespective of the expression of the ABCE1 mRNA. These results indicate that the ABCE1 and its peptides could be target molecules in specific immunotherapy for HLA-A2(+) colon cancer patients.     

Cytokines required for induction of histocompatibility leukocyte antigen-class I-restricted and tumor-specific cytotoxic T lymphocytes by a SART1-derived peptide.

Although there have been several reports on peptides of human tumor-rejection antigens capable of inducing histocompatibility leukocyte antigen (HLA)-class I-restricted and tumor-specific cytotoxic T lymphocytes (CTLs), it is not yet clear which cytokines are required for CTL induction. This study has investigated the cytokine combinations required for optimal induction of CTLs by SART1(690-698) peptide, which is capable of inducing HLA-A24-restricted and tumor-specific CTLs in peripheral blood mononuclear cells (PBMCs). Pretreatment of PBMCs as a source of antigen-presenting cells (APCs) with interferon (IFN)-gamma, or to some extent with IFN-alpha, but not with any of the other cytokines tested, augmented the peptide-induced CTL activity in HLA-A24 heterozygotes, but not in HLA-A24 homozygotes. This IFN-gamma-mediated augmentation was inhibited by either interleukin (IL)-4 or IL-10. IL-2 alone in culture, along with weekly stimulation by peptide-pulsed APCs, was sufficient for the differentiation and proliferation of CTLs for the initial several weeks of culture. This IL-2-mediated activation of CTLs was inhibited by the addition of IFN-gamma, IL-4, or IL-10 to the IL-2 culture. For further expansion of the CTLs, dendritic cells (DCs) induced from PBMCs with IL-4 and granulocyte macrophage colony-stimulating factor (GM-CSF) were required as APCs. These results indicate that IFN-gamma and IL-2 are important in the activation of APCs and CTLs, respectively, while GM-CSF and IL-4 are needed for the induction of DCs, which in turn are required for further expansion of mature CTLs. These results are important in allowing for a better understanding of the cellular and molecular basis of tumor-specific immunity, and also for the development of peptide-based specific immunotherapy.     

Human leukocyte antigen-A2-restricted CTL responses to mutated BRAF peptides in melanoma patients

Mutated BRAF (BRAF(V600E)) is a potential immunotherapeutic target for melanoma because of its tumor specificity and expression in the majority of these lesions derived from different patients. BRAF(V600E) is expressed intracellularly and not on the cell surface, therefore providing a target for T cells but not B cells. Demonstration of patients' T cell responses to BRAF(V600E) would suggest the feasibility of active specific immunotherapy targeting the mutation in these patients. In the present study, BRAF(V600E) peptides with putative binding sites for human leukocyte antigen (HLA)-A2 were used to stimulate T lymphocytes of HLA-A2-positive melanoma patients. Four of five patients with BRAF(V600E)-positive lesions showed lymphoproliferative responses to BRAF(V600E) peptide stimulation. These responses were specific for the mutated epitope and HLA-A2 was restricted in three patients. Lymphocytes from these three patients were cytotoxic against HLA-A2-matched BRAF(V600E)-positive melanoma cells. None of the four patients with BRAF(V600E)-negative lesions and none of five healthy donors had lymphoproliferative responses specific for the mutated epitope. The high prevalence (approximately 50%) of HLA-A2 among melanoma patients renders HLA-A2-restricted BRAF(V600E) peptides attractive candidate vaccines for these 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.     

Erythropoietin-producing hepatocyte B6 variant-derived peptides with the ability to induce glioma-reactive cytotoxic T lymphocytes in human leukocyte antigen-A2+ glioma patients

We recently cloned a variant form of erythropoietin-producing hepatocyte (Eph)B6, a member of the Eph receptor tyrosine kinase family. In the present study, we examined the expression of the EphB6 variant (EphB6v) in a panel of brain tumor cell lines and glioblastoma tissues and we found that EphB6v was preferentially expressed in malignant brain tumors, such as glioblastomas and anaplastic astrocytomas. The EphB6v has a unique 54 amino acid sequence at the C-terminal that is not found in normal EphB6. Therefore, we attempted to identify antigenic peptides unique to EphB6v for immunotherapy. The two EphB6v-derived peptides exhibited the ability to bind to human leukocyte antigen (HLA)-A0201 molecules, and each of them was able to induce cytotoxic T lymphocytes in vitro in the peripheral blood mononuclear cells of HLA-A2⁺ glioma patients. The cytotoxicity was mediated by peptide-specific CD8⁺ T cells in an HLA-A2-restricted manner. The expression of EphB6v was also observed in different types of cancer (e.g. lung, colon, stomach, liver and pancreatic) cells. Taken together, the two peptides derived from EphB6v might be appropriate targets for peptide-based specific immunotherapy for HLA-A2⁺ patients with various cancers. ( Cancer Sci 2008; 99: 1656–1662)     

Kinesin superfamily protein-derived peptides with the ability to induce glioma-reactive cytotoxic T lymphocytes in human leukocyte antigen-A24+ glioma patients

One promising modality in the treatment of malignant glioma is specific immunotherapy. However, this modality requires information about target antigens and their epitope peptides that are recognized by T cells. In this study, we searched for new target candidates in specific immunotherapy for malignant glioma by utilizing cDNA microarray technology to compare gene expressions in malignant glioma tissues to those in benign glioma and a panel of normal tissues. The selected genes included three members of the kinesin superfamily proteins (KIFs): KIF1C, KIF3C, and KIF21B. RT-PCR showed that these three genes were expressed in the majority of glioma cell lines. These antigen-derived 25 peptides, which had the ability to bind to human leukocyte antigen (HLA)-A24 molecules, were first screened for their ability to be recognized by the immunoglobulin G of glioma patients, and then tested for their potential to induce peptide-specific and glioma-reactive cytotoxic T lymphocytes (CTLs) from the peripheral blood mononuclear cells of HLA-A24+ glioma patients. The results showed that the KIF1C149-158 and KIF3C512-520 peptides efficiently induced HLA-A24-restricted and glioma-reactive CD8+ T cells. These results suggest the existence of KIF-reactive CTL precursors in glioma patients, and should facilitate the development of specific immunotherapies for malignant glioma.     

Identification of parathyroid hormone-related protein-derived peptides immunogenic in human histocompatibility leukocyte antigen-A24+ prostate cancer patients

Parathyroid hormone-related protein (PTHrP) is a key factor in the development of bone metastases, which are a major barrier in treating prostate cancer patients. In this study, we attempted to identify PTHrP-derived peptides immunogenic in human histocompatibility leukocyte antigen (HLA)-A24(+) prostate cancer patients. Among four different PTHrP peptides carrying the HLA-A24 binding motif, both the PTHrP(36-44) and PTHrP(102-111) peptides efficiently induced peptide-specific cytotoxic T lymphocytes from peripheral blood mononuclear cells (PBMCs) of HLA-A24(+) prostate cancer patients. Peptide-stimulated PBMCs showed cytotoxicity against prostate cancer cells in an HLA-A24-restricted manner. Experiments using antibodies and cold inhibition targets confirmed that their cytotoxicity was dependent on PTHrP peptide-specific and CD8(+) T cells. Immunoglobulin G reactive to the PTHrP(102-111) or PTHrP(110-119) peptide was frequently detected in the plasma of prostate cancer patients, suggesting that the PTHrP(102-111) peptide is able to elicit cellular and humoral immune responses in cancer patients. These results indicate that the PTHrP could be a promising target molecule for specific immunotherapy of HLA-A24(+) prostate cancer patients with metastases.     

Identification of an epitope derived from CML66, a novel tumor-associated antigen expressed broadly in human leukemia, recognized by human leukocyte antigen-A*2402-restricted cytotoxic T lymphocytes

CML66 is a newly identified differentiation antigen that is expressed broadly in human leukemia and solid tumors, but its physiological function remains unknown. In the present study, to clarify the feasibility of CML66-targeted cancer immunotherapy, we attempted to identify cytotoxic T lymphocyte (CTL) epitopes derived from CML66. An immunogenic CML66-derived epitope (amino acid residues 76–84; YYIDTLGRI) capable of inducing human leukocyte antigen (HLA)-A*2402-restricted CTL specific for this peptide was identified. CML66-derived peptide-specific CTL efficiently lysed human leukemia cells, but not normal cells, in a HLA-A*2402-restricted fashion. Quantitative real-time polymerase chain reaction revealed that CML66 mRNA is expressed abundantly in primary acute myeloid leukemia cells, acute lymphoid leukemia cells, and chronic myelogenous leukemia cells in advanced phase, and that the expression level of CML66 mRNA in normal cells is low compared with that in leukemia cells. CML66-specific CTL precursors were detected in the peripheral blood of patients with acute leukemia. These data indicate that the CML66-derived epitope identified in the present study is a new target antigen for cellular immunotherapy of human leukemia. ( Cancer Sci 2008; 99: 1414–1419)     

Generation of cytotoxic T lymphocytes against native and altered peptides of human leukocyte antigen-A*0201 restricted epitopes from the human epithelial cell adhesion molecule.

A growing number of human tumor antigens have been described that can be recognized by CTLs in a MHC class I restricted fashion. The epithelial cell adhesion molecule (Ep-CAM) is expressed in a variety of human tumors and has attracted attention as a therapeutic target for monoclonal antibody serotherapy. We have identified immunogenic peptides derived from Ep-CAM, that bind to human leukocyte antigen-A*0201 and elicit strong peptide-specific human CTL responses, demonstrating that there is an effective T-cell repertoire against these Ep-CAM-derived peptides that can be recruited. Alterations to these peptides were made to increase their binding affinity to MHC class I molecules. The use of such "heteroclitic" peptides allowed generation of cytotoxic T cells that demonstrated increased killing of target cells pulsed not only with the heteroclitic but also with the native peptide. Most important, CTL cell lines that are generated against these peptides specifically lyse epithelial tumor cells expressing Ep-CAM but not normal hematopoietic or bronchial epithelial cells.     

Gene and peptide analyses of newly defined lung cancer antigens recognized by HLA-A2402-restricted tumor-specific cytotoxic T lymphocytes

We investigated tumor antigens recognized by HLA-A2402-restricted CTLs established from T cells infiltrating into lung adenocarcinoma. We report here three newly identified tumor antigen genes, including one unreported gene, temporarily referred to as clone 83, and two known genes, BTB domain containing 2 (BTBD2) and hairpin-binding protein. These genes were preferentially expressed in most of the cell lines of lung cancer and also of ovarian cancer and renal cell carcinoma at the mRNA level. The expression of these genes was confirmed in lung and other cancer tissue specimens. In normal tissues, clone 83 was expressed only in the colon, and hairpin-binding protein was not expressed at all, whereas BTBD2 was ubiquitously expressed. Clone 83, BTBD2, and hairpin-binding protein encoded two, one, and one epitope peptides that can be recognized by HLA-A2402-restricted CTLs, respectively. These epitope peptides possessed the ability to induce HLA-A24-restricted tumor-specific CTLs after in vitro stimulation in a culture of peripheral blood mononuclear cells from patients with lung cancer. These results suggest that these genes and peptides are potential candidates for cancer vaccines in HLA-A24(+) patients with lung cancer.     

Identification of Cyclophilin B-derived Peptides Capable of Inducing Histocompatibility Leukocyte Antigen-A2-restricted and Tumor-specific Cytotoxic T Lymphocytes

We recently suggested that cyclophilin B (Cyp-B) is a tumor antigen recognized by histocompatibility leukocyte antigen (HLA)-A24-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). In this study, we tried to identify Cyp-B-derived epitopes, which can induce HLA-A2-restricted and tumor-specific CTLs in cancer patients. The tumor-infiltrating lymphocytes (TILs) from an HLA-A0207 patient with colon cancer were found to respond to COS-7 cells when co-transfected with the Cyp-B gene and either HLA-A0201, -A0206 , or - A0207 cDNA. These TILs contained CTLs capable of recognizing either the Cyp-B_129–138 or the Cyp-B_172–179 peptide among 28 different peptides, all of which were prepared based on the HLA-A2 binding motif. Both Cyp-B peptides possessed the ability to induce tumor-specific CTLs in HLA-A2⁺ cancer patients. Cyp-B_{172–180 (v)}, which is a 9-mer peptide with valine added at the C terminus, showed no clear superiority over the parental Cyp-B_172-179 peptide in an in vitro sensitization experiment. In vitro -sensitized T cells with these peptides responded to cancer cells in an HLA-A2-restricted manner. These two Cyp-B peptides could be useful for specific immunotherapy of HLA-A2⁺ cancer patients.     

In vitro generation of cytotoxic T lymphocytes against HLA-A2.1-restricted peptides derived from human thymidylate synthase.

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.     

New peptides of the polycomb group protein enhancer of zeste homolog 2 with the potential to induce cancer-reactive cytotoxic T lymphocytes in human leukocyte antigen-A2+ prostate cancer patients

The polycomb group protein enhancer of zeste homolog 2 (EZH2) is linked to aggressive prostate cancer and could be an appropriate target in specific immunotherapy. In this study, we attempted to identify EZH2-derived peptides that have the potential to generate cancer-reactive cytotoxic T lymphocytes (CTLs) in human leukocyte antigen (HLA)-A2+ prostate cancer patients. Twelve EZH2-derived peptides were prepared based on the HLA-A2 binding motif. These peptide candidates were screened first by their ability to be recognized by immunoglobulin G (IgG), and then by their ability to induce peptide-specific cytotoxic T lymphocytes (CTLs). As a result, five EZH2 peptides recognized by IgG (EZH2 120-128, EZH2 165-174, EZH2 569-577, EZH2 665-674, and EZH2 699-708) were frequently detected in the plasma of prostate cancer patients. Among them, the EZH2 120-128 and EZH2 165-174 peptides effectively induced HLA-A2-restricted and cancer-reactive CTLs from prostate cancer patients. The cytotoxicity was mainly dependent on EZH2 peptide-specific and HLA-A2-restricted CD8+ T cells. These results indicate that these EZH2 120-128 and EZH2 165-174 peptides could be promising candidates in peptide-based immunotherapy for HLA-A2+ prostate cancer patients.     

Immediate early response gene X-1, a stress-inducible antiapoptotic gene, encodes cytotoxic T-lymphocyte (CTL) epitopes capable of inducing human leukocyte antigen-A33-restricted and tumor-reactive CTLs in gastric cancer patients

Peptide-based vaccine therapy, which is designed to elicit T-cell immunity against tumors, is an attractive approach for the treatment of cancer patients. To provide a scientific basis for peptide therapy, an increasing number of CTL-directed peptides have been identified, and some of them have been tried as antigen-specific immunotherapy in the past decade. Only a few studies, however, have been performed on such peptides restricted with alleles other than HLA-A2 and -A24. In the present study, we show that immediate early response gene X-1 (IEX-1), a stress-inducible protein associated with the regulation of cell proliferation and apoptosis, produces antigenic epitopes recognized by 850B-CTLs, HLA-A33-restricted CTLs newly established from T cells infiltrating into gastric adenocarcinoma. The IEX-1 gene was highly expressed in most cell lines and tissues from various types of cancer at both the mRNA and protein levels. However, it was not expressed at the protein level in any normal epithelium or connective tissues tested. Three IEX-1-derived peptides at positions 47-56, 61-69, and 65-73, which were recognized by the 850B-CTLs, could induce CD8(+) peptide-specific CTL reaction to tumor cells from HLA-A33(+) gastric cancer patients and other epithelial cancer patients, but not from healthy donors, in an HLA class I-restricted manner. Because increased expression of IEX-1 is suggested to be involved in the resistance to apoptosis and in the proliferation of cancer cells, these antigenic peptides could be potent candidates for peptide-based specific immunotherapy against HLA-A33(+) gastric cancer and other epithelial cancers.