WT1 is a tumor-associated antigen in colon cancer that can be recognized by in vitro stimulated cytotoxic T cells

The Wilms' tumor suppressor gene (WT1) has been shown to be overexpressed in acute and chronic leukemias and in a variety of solid human malignancies, including cancers of the breast and lung. In our present study, we investigated the potential role of WT1 gene in human colon cancer. WT1 mRNA and protein expression was analyzed in a panel of human colon cancer cell lines and primary colon carcinomas by RT-PCR and Western blot analysis, respectively. A mutational screen of WT1' zinc-finger region was carried out by sequence analysis. Finally, using peptide-stimulated cytotoxic T cells it was investigated whether WT1-expressing colon tumor cells are a potential target for antigen-specific immunotherapy. Medium to high abundant levels of WT1 mRNA were detected by RT-PCR in 10 of 12 (83%) colon cell lines and by quantitative, real-time RT-PCR in 13 of 15 (87%) primary tumors, whereas only very low levels of expression were found in 2 primary tumors. Interestingly, however, low levels of WT1 mRNA were also detected in all samples derived from normal colon mucosa. When RT-PCR products were examined by sequence analysis, both +KTS and -KTS splice isoforms but no zinc-finger mutations were found, suggesting that the wild-type form of the WT1 gene is expressed. To determine whether the WT1 protein can serve as a target antigen for immunotherapy, 2 HLA-A2.1-restricted WT1 peptides (Db126 and WH187) were used for the in vitro induction of WT1-specific cytotoxic T lymphocytes (CTLs). The WH187-specific CTLs not only lysed target cells pulsed exogenously with cognate peptide but also WT1-expressing colon tumor cells in a HLA-restricted manner. These findings identify the WT1 protein as an attractive target for the development of antigen-specific immunotherapy in human colon cancer.     

An HLA-A24-restricted cytotoxic T lymphocyte epitope of a tumor-associated protein, survivin.

To date an increasing number of T-cell epitopes derived from various tumor-associated antigens have been reported, and they proved to play significant roles for tumor rejection both in vivo and in vitro. Survivin was originally identified as a member of the inhibitor of apoptosis protein family. Expression of this gene is developmentally regulated. Although survivin is expressed during normal fetal development, the expression is barely detected in terminally differentiated adult tissues except for testis, thymus, and placenta. In contrast, it is abundantly expressed in a wide variety of malignant tissues. We examined the expression of survivin and the two splicing variants survivin-2B and survivin-DeltaEx3 in various cancer cells, immortalized cells, and normal adult tissues. It was demonstrated that two splicing variants were detected in various types of cancer cells as well as survivin, and their expression was more restricted to cancer cells as compared with survivin expression. To identify HLA-A24-restricted T-cell epitopes from survivin and the variant proteins, three peptides were selected from amino acid sequence of these proteins, based on the HLA-A24-binding motif. Peptide binding assay to HLA-A24 revealed that only one peptide designated as survivin-2B80-88 (AYACNTSTL) was capable of binding to HLA-A24. By stimulating peripheral blood lymphocytes with the peptide-pulsed antigen-presenting cells, CTLs were successfully induced in vitro from five of five HLA-A24-positive cancer patients. The CTLs showed significant cytotoxicity against HLA-A24-positive survivin-2B-positive cancer cells. These data suggest that survivin-2B80-88 may be a potent T-cell epitope eliciting CTL response against a splicing variant survivin-2B, which is specifically expressed in many kinds of cancer cells.     

Identification of HLA-A2-restricted epitopes of the tumor-associated antigen MUC2 recognized by human cytotoxic T cells

Previous studies have shown that self-antigens overexpressed in malignant tissue can provide a basis for a tumor-specific immune response. The mucin MUC2 is strongly overexpressed in all mucinous tumors of colon, breast, ovary and pancreas. In the corresponding normal tissue it is either not expressed (breast, ovary, pancreas) or it is expressed at considerably lower levels than in the mucinous tumors (colon). We therefore investigated whether the MUC2 molecule comprises HLA-A2-binding epitopes recognized by human cytotoxic T cells. Four MUC2 peptides with high affinity and stable binding to HLA-A2 were identified. Those peptides and additionally 3 peptides with moderate binding to HLA-A2 were loaded onto dendritic cells, which were used for stimulation of autologous T cells from healthy donors. Two MUC2 peptides, which belonged to the group of stable binders, induced specific cytotoxic T-cell lines. Target cells loaded with these peptides were strongly lysed in a concentration-dependent and HLA-A2-restricted manner. Our data show that the tumor-associated mucin MUC2 has potential as a target antigen for cytotoxic T cells in patients with mucinous carcinomas. Int. J. Cancer 75:688–693, 1998.© 1998 Wiley-Liss, Inc.     

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.     

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 HLA-A3-restricted CD8+ T cell epitopes derived from mammaglobin-A,a tumor-associated antigen of human breast cancer

Mammaglobin-A is highly overexpressed in breast cancer cell lines and primary breast tumors. This pattern of expression is restricted to mammary epithelium and metastatic breast tumors. Thus, mammaglobin-A-specific T cell immune responses may provide an important approach for the design of breast cancer-specific immunotherapy. The purpose of our study was to define the T cell-mediated immune response to mammaglobin-A. We determined that the frequency of mammaglobin-A-reactive CD8+ and CD4+ T cells in breast cancer patients is significantly higher than that observed in healthy female controls using limiting dilution analyses (p = 0.026 and p = 0.02, respectively). We identified 8 mammaglobin-A-derived 9-mer peptides with the highest binding affinity for the HLA-A3 molecule (Mam-A3.1-8) using a computer-assisted analysis of the mammaglobin-A protein sequence. Subsequently, we determined that CD8+ T cells from breast cancer patients reacted to peptides Mam-A3.1 (23-31, PLLENVISK), Mam-A3.3 (2-10, KLLMVLMLA), Mam-A3.4 (55-63, TTNAIDELK) and Mam-A3.8 (58-66, AIDELKECF) using an IFN-gamma enzyme-linked immunospot assay. A CD8+ T cell line generated in vitro against HLA-A*0301-transfected TAP-deficient T2 cells loaded with these peptides showed significant cytotoxic activity against the Mam-A3.1 peptide. This CD8+ T cell line showed a significant HLA-A3-restricted cytotoxic activity against mammaglobin-A-positive but not mammaglobin-A-negative breast cancer cells. In summary, our study identified four HLA-A3-restricted mammaglobin-A-derived epitopes naturally expressed by breast cancer cells, indicating the immunotherapeutic potential of this novel antigen for the treatment and prevention of breast cancer.     

Expression of Tn, sialosyl-Tn, and T antigens in human colon cancer

Mucin glycoproteins are major secretory products of the colon and contain O-linked oligosaccharides synthesized on a polypeptide backbone. The initial step in the synthesis of O-linked oligosaccharides is the addition of N-acetylgalactosamine to serine or threonine residues forming the Tn antigen. This substance can then receive additional carbohydrate residues such as sialic acid to form sialosyl-Tn antigen, or galactose to form T antigen. In the colon, the T antigen is an oncodevelopmental cancer-associated antigen but little is known about Tn and sialosyl-Tn expression. The present comparative immunohistochemical study was performed to analyze the expression of these antigens in fetal, normal adult, and malignant colorectal tissues with an aim toward elucidating whether Tn and sialosyl-Tn are also oncodevelopmental colon cancer-associated antigens and to gain insight into the earliest steps of mucin glycosylation in colonocytes. We used three reagents to detect Tn antigen (two monoclonal antibodies ETn1.01 and CU-1, and one lectin Vicia villosa), two reagents to detect sialosyl-Tn (monoclonal antibodies TKH2 and B72.3) and one to detect T antigen (monoclonal antibody AH9-16). Except for occasional reactivity with VVA and CU-1, cells of normal colonic mucosa did not express Tn, sialosyl-Tn, or T antigens. However, in the transitional mucosa immediately adjacent to cancer, all three antigens were expressed (ranging from 35 to 67% of cases depending upon the reagent). In colon cancers, the percentage of cases expressing each antigen were as follows: Tn 72-81%, sialosyl-Tn 93-96%, and T 71%. Unlike T antigen, which was preferentially expressed by moderately well- and well-differentiated adenocarcinomas, both Tn and sialosyl-Tn antigens were expressed by most histological subsets of colon cancers, including poorly differentiated adenocarcinomas and mucinous (colloid and signet ring cell type) carcinomas. The majority of cancers expressed both Tn and sialosyl-Tn, usually in association with T antigen. Only one cancer lacked all three antigens. Fetal colonic mucosal cells expressed all three antigens, particularly in goblet cell mucin. These results indicate that like T antigen, Tn and sialosyl-Tn are oncodevelopmental cancer-associated antigens in the colon. Moreover, Tn and sialosyl-Tn antigens appear to be useful markers of poorly differentiated adenocarcinomas and mucinous carcinomas: two histological subsets that often fail to express other cancer-associated antigens and that are often associated with a poor clinical outcome.(ABSTRACT TRUNCATED AT 400 WORDS)     

A unique gene having homology with the kinesin family member 18A encodes a tumour-associated antigen recognised by cytotoxic T lymphocytes from HLA-A2+ colon cancer patients

Colon cancer is one of the major malignant tumours for which the development of a new treatment modality is needed. To provide the scientific basis for a specific immunotherapy for colon cancer, we looked for tumour-associated antigens recognised by cytotoxic T lymphocytes (CTLs) from human leukocyte antigen (HLA)-A2+ colon cancer patients. We report here a unique gene, 3362 base-pairs (bp) long, which has homology with the kinesin family member 18A. This gene was expressed at the mRNA level in the majority of tumour cells, but not in any normal tissues tested except for testis and lung. Two of 16 peptides with HLA-A2-binding motifs were recognised by tumour-reactive CTLs. In addition, these two peptides had the ability to induce HLA-A2-restricted and cancer-reactive CTLs from peripheral blood mononuclear cells (PBMCs) of colon cancer patients with several HLA-A2 subtypes. Overall, this study provides new information about a colon cancer-related antigen that might be an appropriate target for specific immunotherapy in HLA-A2+ colon cancer patients.     

Small cell lung carcinomas express shared and private tumor antigens presented by HLA-A1 or HLA-A2.

Tumor-derived peptides presented by MHC class I molecules are targets for tumor rejection by CD8+ CTLs. MHC-restricted CD8+ CTLs are required also for the identification and characterization of tumor antigens that will be useful for immune therapy. For many human solid tumors, however, tumor antigens remain undefined because of the difficulty of generating MHC-restricted, tumor-specific CTLs required for their analysis. CD8+ CTL responses are modulated by CD4+ helper T cells and by antigen-presenting cells. In this study, highly purified CD8+ T cells were mixed with tumor cells in primary cultures in the absence of any other cells to reduce the complexity of CTL generation. Tumor cells were transfected with HLA-A1 or HLA-A2 and used to stimulate partly matched HLA-A1- or HLA-A2-positive CD8+ T cells. Partial MHC class I matching of tumor and CD8+ T cells and omission of other cells in primary culture was highly effective in generating MHC class I-restricted CTL to poorly immunogenic small cell lung carcinomas (SCLCs). Cytotoxicity was further enhanced by cotransfection of tumor cells with B7.1 (CD80). ICAM-1 (CD54) was not as effective as costimulation. SCLC cells presented tumor-specific peptides with HLA-A1 and HLA-A2 and were lysed by A1- or A2-restricted CD8+ CTLs. A1- and A2-restricted CD8+ CTLs detected shared tumor antigens on unrelated SCLC tumor lines in addition to private antigens. The use of direct antigen presentation by MHC class I-transfected tumors to MHC class I-matched CD8+ T cells is an effective way to generate MHC class I-restricted CTLs toward poorly immunogenic tumors in vitro, permitting the molecular identification of their tumor antigens.     

Two new tumor-specific antigenic peptides encoded by gene MAGE-C2 and presented to cytolytic T lymphocytes by HLA-A2

We have identified 2 antigens recognized by several melanoma-specific cytolytic T lymphocyte clones isolated from a melanoma patient with a clinical history of tumor regression after immunotherapy. Both antigens are presented by HLA-A2 and encoded by gene MAGE-C2, a cancer-germline gene shown previously to be silent in normal somatic tissues and expressed in 40% of melanomas and in other tumor types. One antigen corresponds to peptide ALKDVEERV(336-344), whereas the other corresponds to peptide LLFGLALIEV(191-200). The CTL clones recognizing these 2 peptides also recognized allogeneic tumor cell lines expressing MAGE-C2 and HLA-A2. These 2 new peptides are the first known MAGE-C antigens and represent promising targets for cancer immunotherapy.     

结肠癌患者化疗/热化疗前后CD4+CD25+T细胞和CTL变化

背景与目的:化疗是内科治疗结肠癌的主要方法之一,热化疗改善患者机体免疫状态的机制尚不清楚.本研究通过观察结肠癌患者化疗/热化疗前后CD4+CD25+调节性T(Treg)细胞及细胞毒T淋巴细胞功能的变化,探讨其临床意义.方法:入选结肠癌Ⅲ期术后患者60例,其中热化疗组30例,以单纯化疗组30例为对照组,收集患者治疗前后外周血,采用流式细胞术检测治疗前后外周血淋巴细胞中CD4+CD25+Treg细胞的表达,并检测患者CTL细胞杀伤功能.结果:(1)结肠癌患者单纯化疗组化疗前CD4+CD25+Treg细胞表达为19±9,化疗后为19±7,差异无统计学意义(F=0.026,P＞0.05);热化疗组化疗前CD4+CD25+Treg细胞表达为19±5,化疗后为14±4,差异有统计学意义(F=2.198,P＜0.05);(2)结肠癌患者单纯化疗组化疗前细胞毒T淋巴细胞(cytotoxic T lymphocytes,CTL)为10±3,化疗后为15±5,差异有统计学意义(F=187.801,P＜0.05);热化疗组化疗前CTL为12±3,化疗后CTL为18±5,两组比较差异有统计学意义(F=236.097,P＜0.05).即化疗组治疗前后CD4+CD25+Treg细胞变化不明显,而化疗联合热疗组治疗后CD4+CD25+Treg细胞水平显著降低.两组结肠癌患者治疗后CTL细胞杀伤功能均明显增强,以热化疗组增强更明显.结论:热化疗可明显改善结肠癌患者的免疫抑制状态,增强患者的细胞毒T淋巴细胞的功能.化疗联合热疗可提高患者疗效和预后.     

Serine proteinase inhibitor 9 can be recognized by cytotoxic T lymphocytes of epithelial cancer patients.

Serine proteinase inhibitor 9 (PI-9) inhibits granzyme B-mediated apoptosis and interleukin-1beta-converting enzyme activity. In this study, we report that the PI-9 gene encodes antigenic epitopes recognized by the HLA-A24-restricted and tumor-reactive cytotoxic T lymphocytes (CTLs) of epithelial cancer patients. Screening of an autologous cDNA library using a CTL line recognizing HLA-A24+ tumor cells resulted in the isolation of a cDNA, which had an identical coding region to the previously described PI-9 genes. PI-9 gene was expressed in approximately three-fourths of epithelial cancer cell lines and all leukemic cell lines tested. It was also expressed in normal peripheral blood mononuclear cells (PBMCs), but not in a normal fibroblast cell line. CTL sublines contained T cells capable of recognizing the PI-9(292-300) and PI-9(348-356) peptides among 13 different peptides having the HLA-A24 binding motifs. These two peptides were recognized by the CTL line in a dose-dependent and HLA class-I-restricted manner, and also possessed the ability to induce HLA class I-restricted and tumor-reactive CTLs in PBMCs from HLA-A24+ cancer patients. These results demonstrate that PI-9 is recognized by HLA class I-restricted and tumor-reactive CTLs of epithelial cancer patients.     

The in vitro generation of Ph1+ ALL-specific HLA-A24-restricted cytotoxic T lymphocytes using a synthetic 16 mer minor bcr-abl peptide

Sixteen mer peptide, which spans the junctional region of the acute lymphoid leukemia (ALL)-specific minor bcr-abl fusion protein and contains a motif that can bind to human leukocyte antigen (HLA)-A24, was constructed. We tried to generate Philadelphia chromosome 1 (Ph1) positive ALL-specific cytotoxic T lymphocytes (CTLs) from eight normal HLA-A24+ individuals with peptide-pulsed autologous dendritic cells (DCs). CTLs could be generated from the mononuclear cells (MNCs) of a single donor, which could kill peptide-pulsed autologous DCs and two A24+ ALL lines, while an HLA-A24+ CML line was only weakly killed and unpulsed DCs or the control lines Daudi or K562 were not recognized. Those CTLs consisted predominantly of CD8+ T cells whose cytotoxicity could be neutralized by monoclonal antibodies to HLA-class I or HLA-A24, and also produced interferon (IFN)-gamma after being stimulated with peptide-pulsed DCs.     

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.     

The polycomb group proteins, BMI-1 and EZH2, are tumour-associated antigens

We used SEREX technology to identify novel tumour-associated antigens in patients with primary hepatocellular carcinoma and found serological responses to the polycomb group (PcG) protein BMI-1, which is overexpressed in a range of different tumour types. Further studies identified T-cell responses to both BMI-1 and another PcG protein, EZH2, in cancer patients and at relatively lower levels in some normal donors. We next identified several CD8+ T-cell epitopes derived from BMI-1 and EZH2 and demonstrated that EZH2-derived peptides elicited more significant interferon-gamma (IFN-gamma) release than BMI-1-derived peptides. That CD8(+) T cells were responsible for the observed responses was confirmed for EZH2 by both IFN-gamma capture assays and tetramer staining using an HLA-A0201-restricted, EZH2-derived YMSCSFLFNL (aa 666-674) epitope. The ability of YMSCSFLFNL (aa 666-674) to stimulate the in vitro expansion of specific T cells from peripheral blood lymphocytes was greatly enhanced when the CD25(+) T-cell population was depleted. EZH2-specific cytotoxic T lymphocyte clones specific for two HLA-A0201 epitopes were generated and found to recognise endogenously processed EZH2 in both HLA-matched fibroblasts and tumour cell lines. Given the widespread overexpression of PcG proteins in cancer and their critical role in oncogenesis, these data suggest that they may be useful targets for cancer immunotherapy.     

Preoperative chemoradiation may not always be needed for patients with T3 and T2N+ rectal cancer

BACKGROUND:

Preoperative chemoradiation is becoming the standard treatment for patients with locally advanced rectal cancer. However, since the introduction of total mesorectal excision (TME), local recurrence rates have been reduced significantly, and some patients can be spared from potentially toxic over treatment. The current study was designed to assess the factors that predict recurrence in an institutional series of patients with rectal cancer who had clinical T2 lymph node‐positive (cT2N+) tumors or cT3N0/N+ tumors and underwent radical surgery without receiving preoperative chemoradiation.

METHODS:

Between November 1997 and November 2008, the authors' multidisciplinary group preoperatively staged 398 patients with rectal cancer by using endorectal ultrasonography and/or magnetic resonance imaging. The analysis included 152 consecutive patients with cT2N+, cT3N0, or cT3N+ rectal cancer who underwent TME without receiving preoperative chemoradiation. Macroscopic assessment of the mesorectal excision and circumferential resection margins were determined. Factors potentially related to local recurrence (LR), disease‐free survival (DFS) and cancer‐specific survival (CSS) were analyzed.

RESULTS:

After a median follow‐up of 39 months, the 5‐year actuarial LR, DFS, and CSS rates were 9.5%, 65.4%, and 77.8%, respectively, for the whole group. Threatened mesorectal fascia at preoperative staging was the only independent preoperative factor that predicted a higher risk for LR (P = .007), shorter DFS (P = .007), and shorter CSS (P = .05). In particular, the 5‐year LR rates for patients with and without preoperative threatened circumferential resection margins were 19.4% and 5.4%, respectively.

CONCLUSIONS:

The current results suggested that patients with rectal cancer clinically staged as T3N0/N+ or T2N+ with a free margin >2 mm from mesorectal fascia may undergo TME alone, avoiding over treatment with preoperative chemoradiation. Cancer 2011. © 2011 American Cancer Society.     

Generation and phenotypic characterization of new human ovarian cancer cell lines with the identification of antigens potentially recognizable by HLA-restricted cytotoxic T cells

This study describes a simple method for long-term establishment of human ovarian tumor lines and prediction of T-cell epitopes that could be potentially useful in the generation of tumor-specific cytotoxic T lymphocytes (CTLs). Nine ovarian tumor lines (INT.Ov) were generated from solid primary or metastatic tumors as well as from ascitic fluid. Notably all lines expressed HLA class I, intercellular adhesion molecule-1 (ICAM-1), polymorphic epithelial mucin (PEM) and cytokeratin (CK), but not HLA class II, B7.1 (CD80) or BAGE. While of the 9 lines tested 4 (INT.Ov1, 2, 5 and 6) expressed the folate receptor (FR-α) and 6 (INT.Ov1, 2, 5, 6, 7 and 9) expressed the epidermal growth factor receptor (EGFR); MAGE-1 and p185^HER-2/neu were only found in 2 lines (INT.Ov1 and 2) and GAGE-1 expression in 1 line (INT.Ov2). The identification of class I MHC ligands and T-cell epitopes within protein antigens was achieved by applying several theoretical methods including: 1) similarity or homology searches to MHCPEP; 2) BIMAS and 3) artificial neural network-based predictions of proteins MAGE, GAGE, EGFR, p185^HER-2/neu and FR-α expressed in INT.Ov lines. Because of the high frequency of expression of some of these proteins in ovarian cancer and the ability to determine HLA binding peptides efficiently, it is expected that after appropriate screening, a large cohort of ovarian cancer patients may become candidates to receive peptide-based vaccines. Int. J. Cancer 73:143–150, 1997. © 1997 Wiley-Liss, Inc.     

Ran, a small GTPase gene, encodes cytotoxic T lymphocyte (CTL) epitopes capable of inducing HLA-A33-restricted and tumor-reactive CTLs in cancer patients.

PURPOSE: The purpose is to identify a gene coding for tumor-associated antigen and peptide capable of inducing CTLs reactive to tumor cells with a HLA-A33-restricted fashion to provide scientific basis for specific immunotherapy to HLA-A33+ cancer patients. EXPERIMENTAL DESIGN: An expression gene-cloning method was used to identify the tumor-associated antigen gene. Northern blot analysis and immunohistochemistry were used to examine the mRNA and protein expression levels in various cells and tissues, respectively. Synthetic peptides were examined for their ability to induce HLA-A33+ tumor-reactive CTLs in peripheral blood mononuclear cells from cancer patients. RESULT: A gene of small GTPase, Ran, which controls the cell cycle through the regulation of nucleocytoplasmic transport, mitotic spindle organization, and nuclear envelope formation, was found to encode epitopes recognized by the HLA-A33-restricted CTLs established from T cells infiltrating into gastric adenocarcinoma. The expression of the Ran gene was increased in most cancer cell lines and cancer tissues at both the mRNA and protein levels. However, it was not enhanced in the surrounding normal cells or tissues. It was also undetectable in normal tissues as far as tested. Ran-derived peptides at positions 48-56 and 87-95 could induce CD8+ peptide-specific CTLs reactive to tumor cells from HLA-A33+ epithelial cancer patients in a HLA class I-restricted manner. CONCLUSIONS: Because of its increased expression in cancer cells and involvement in malignant transformation and/or the enhanced proliferation of cancer cells, the two Ran-directed peptides could be potent candidates in use for specific immunotherapy against HLA-A33+ epithelial cancers.