Spontaneous CD4+ T cell responses against TRAG-3 in patients with melanoma and breast cancers

The taxol resistance gene TRAG-3 was initially isolated from cancer cell lines that became resistant to taxol in vitro. TRAG-3 is a cancer germline Ag expressed by tumors of different histological types including the majority of melanoma, breast, and lung cancers. In the present study, we report that patients with stage IV melanoma and breast cancers developed spontaneous IFN-gamma-producing CD4+ T cell responses against a single immunodominant and promiscuous peptide epitope from TRAG-3 presented in the context of multiple HLA-DR molecules. The TRAG-3-specific CD4+ T cells and clones were expanded in vitro and recognized not only peptide pulsed APCs but also autologous dendritic cells (DCs) loaded with the TRAG-3 protein. All stage IV melanoma patients with TRAG-3-expressing tumors developed spontaneous CD4+ T cell responses against TRAG-3, demonstrating its strong immunogenicity. None of these patients had detectable IgG Ab responses against TRAG-3. TCRbeta gene usage studies of TRAG-3-specific CD4+ T cell clones from a melanoma patient and a normal donor suggested a restricted TCR repertoire in patients with TRAG-3-expressing tumors. Altogether, our data define a novel profile of spontaneous immune responses to cancer germline Ag-expressing tumors, showing that spontaneous TRAG-3-specific CD4+ T cells are directed against a single immunodominant epitope and exist independently of Ab responses. Because of its immunodominance, peptide TRAG-3(34-48) is of particular interest for the monitoring of spontaneous immune responses in patients with TRAG-3-expressing tumors and for the development of cancer vaccines.     

TRAG-3, a novel gene, isolated from a taxol-resistant ovarian carcinoma cell line

The mechanisms responsible for the development of the taxol resistance phenotype are unclear, and are likely explained by multiple mechanisms. To understand the molecular changes associated with drug resistance more fully, a taxol-resistant subline, derived from the human ovarian cancer cell line SKOV-3, was established through selection by culture in incrementally increasing taxol concentrations. Comparison of SKOV-3 to SKOV-3TR by differential display identifies a new gene, TRAG-3 (Taxol Resistance Associated Gene- 3). In comparison to the parental line, SKOV-3, TRAG-3 mRNA is overexpressed in the taxol-resistant cell line SKOV-3TR. The nucleotide sequence of the TRAG-3 cDNA contains an open reading frame of 333bp that predicts for a protein product of 110 amino acids. A GenBank search identifies a cosmid clone containing a genomic sequence corresponding to that of TRAG-3. DNA and protein analysis reveals that TRAG-3 has no homology to any known cDNAs or proteins. Northern analysis demonstrates that TRAG-3 is overexpressed in the taxol-resistant breast cancer cell line MDA 435TR as well as the doxorubicin-resistant multiple myeloma cell lines 8226/DOX40 and 8226/MDR10V. A survey of normal tissue shows minimal or absent TRAG-3 mRNA expression. Screening of a wide variety of cancer cell lines demonstrates TRAG-3 expression in many cell lines derived from different tissue types. In summary, TRAG-3 is a novel gene whose expression is associated with the chemotherapy-resistant and neoplastic phenotype.     

Coordinated expression of clustered cancer/testis genes encoded in a large inverted repeat DNA structure

Cancer/testis antigens (CTA) are expressed in cancers and testis or placenta only and, therefore are considered promising targets for cancer immunotherapy and diagnosis. One family of CTA is the MAGEA family which comprises 13 members and was shown to be expressed synchronously with members from the CSAG (TRAG-3) family of CTA. The MAGEA genes are arranged in 4 subclusters located on the X chromosome. Subcluster III exposes a remarkable gene organization with an inverted repeat (IR) DNA structure of a triplicated couplet of a MAGEA gene and a CSAG gene. Analyzing the mRNA expression pattern of all genes of the MAGEA and CSAG family of cancer/testis genes, we show that the MAGEA and CSAG genes encoded in the large IR are expressed coordinately and independent from the MAGEAs encoded outside the IR. These results reinforce our hypothesis that the large MAGEA/CSAG-IR DNA structure has an impact on the regulation of gene expression.     

Spontaneous T-cell responses against peptides derived from the Taxol resistance–associated gene-3 (TRAG-3) protein in cancer patients

Expression of the cancer-testis antigen Taxol resistance–associated gene-3 (TRAG-3) protein is associated with acquired paclitaxel (Taxol) resistance, and is expressed in various cancer types; e.g., breast cancer, leukemia, and melanoma. Thus, TRAG-3 represents an attractive target for immunotherapy of cancer. To identify HLA-A*02.01–restricted epitopes from TRAG-3, we screened cancer patients for spontaneous cytotoxic T-cell responses against TRAG-3–derived peptides. The TRAG-3 protein sequence was screened for 9mer and 10mer peptides possessing HLA-A*02.01–binding motifs. Of 12 potential binders, 9 peptides were indeed capable of binding to the HLA-A*02.01 molecule, with binding affinities ranging from strong to weak binders. Subsequently, lymphocytes from cancer patients (9 breast cancer patients, 12 melanoma patients, and 13 patients with hematopoietic malignancies) were analyzed for spontaneous reactivity against the panel of peptides by ELISpot assay. Spontaneous immune responses were detected against 8 epitope candidates in 7 of 9 breast cancer patients, 7 of 12 melanoma patients, and 5 of 13 patients with hematopoietic malignancies. In several cases, TRAG-3–specific CTL responses were scattered over several epitopes. Hence, no immunodominance of any single peptide was observed. Furthermore, single-peptide responses were detected in 2 of 12 healthy HLA-A2⁺ donors, but no responses were detectable in 9 HLA-A2^– healthy donors or 4 HLA-A2^– melanoma patients. The identified HLA-A*02.01–restricted TRAG-3–derived epitopes are targets for spontaneous immune responses in breast cancer, hematopoietic cancer, and melanoma patients. Hence, these epitopes represent potential target structures for future therapeutic vaccinations against cancer, possibly appropriate for strategies that combine vaccination and chemotherapy; i.e., paclitaxel treatment.     

The role of the cancer testis antigen PRAME in tumorigenesis and immunotherapy in human cancer

Preferentially expressed antigen in melanoma (PRAME), which belongs to the cancer/testis antigen (CTA) gene family, plays a pivotal role in multiple cellular processes and immunotherapy response in human cancers. PRAME is highly expressed in different types of cancers and is involved in cell proliferation, apoptosis, differentiation and metastasis as well as the outcomes of patients with cancer. In this review article, we discuss the potential roles and physiological functions of PRAME in various types of cancers. Moreover, this review highlights immunotherapeutic strategies that target PRAME in human malignancies. Therefore, the modulation of PRAME might be useful for the treatment of patients with cancer.     

Effective immunotherapy of cancer by DNA vaccination.

Direct injection of naked plasmid DNA either intramuscularly or intradermally induces strong, long-lived cell-mediated and humoral immune responses to the antigen encoded by the gene vaccine. In the present study, we used gene vaccination with naked plasmid DNA to induce prophylactic immune responses to tumor associated antigens. MAGE-1 (melanoma antigen 1) is an ideal candidate for cancer vaccines because it belongs to a family of genes that are expressed in a number of human tumors of various histological types but not in normal adult tissues except for the testis, and because both humoral and cell-mediated immune responses against MAGE-1 antigen were detected in tumor patients. Intradermal administration of plasmid DNA encoding MAGE-1 (pcMAGE1) induced anti-MAGE-1-specific antibody in BALB/c mice. In contrast, no detectable level of anti-MAGE-1 antibody was induced by intramuscular injection of pcMAGE1. Also, intradermal injection of pcMAGE1 was capable of generating CTLs reactive with MAGE-1-transfected murine tumor cells, M-MSV-MAGE1. Most of the mice (8 out of 10) immunized with pcMAGE1 rejected the challenge of M-MSV-MAGE1 tumor cells, compared with control animals most of which developed tumors. This suggests that intradermal DNA vaccination could provide a novel immunotherapy of cancer.     

肿瘤-睾丸-脑抗原家族新成员PNMA5的克隆和表达谱分析

综合运用几种生物信息学数据库 ,分析了人类X染色体上可能的未知基因 ,筛选获得一个新的肿瘤抗原基因并克隆鉴定了它的全长cDNA .该基因全长为 3194bp ,编码一个 4 48个氨基酸残基的蛋白质 ,它与肿瘤 睾丸 脑抗原家族成员PNMA1、PNMA2及PNMA3有很高的同源性 ,命名为PNMA5 .在 16种人正常组织中 ,PNMA5仅在睾丸与脑中表达 ;而在所检测的肝癌、胃癌、肠癌、肺癌、乳腺癌与头颈部肿瘤 ,PNMA5在肠癌中表达 .PNMA5是肿瘤 睾丸 脑抗原家族的一个新成员 .认识这些肿瘤 睾丸 脑抗原的意义在于它们在肿瘤组织的表达可能是肿瘤破坏机体免疫耐受的一种机制 ,其相关神经副肿瘤综合症可能是一些隐匿肿瘤的早期表现 .     

The mouse Mageb18 gene encodes a ubiquitously expressed type I MAGE protein and regulates cell proliferation and apoptosis in melanoma B16-F0 cells

Although many cancer vaccines have been developed against type?I MAGE (melanoma antigen) genes owing to their shared tumour-specific expression properties, studies about their expression and functions are relatively limited. In the present study, we first identify a non-testis-specific type?I MAGE gene, Mageb18 (melanoma antigen family B 18). Mouse Mageb18 is also expressed in digestion- and immune-related tissues as well as testis, and its expression in testis is age-dependent. Mageb18 is expressed in many mouse-derived cell lines, and DNA demethylation and histone acetylation mediate the reactivation of Mageb18 in Mageb18-negtive H22 and C6 cells. We also show that mouse Mageb18 encodes a 46?kDa protein which is predominantly localized in the cytoplasm. In testis, the endogenous MAGEB18 protein is mainly expressed in proliferative spermatogonia and primary and secondary spermatocytes, but less so in spermatids. Finally, we demonstrate that knockdown of MAGEB18 inhibits the growth of B16-F0 cells and induces apoptosis, which correlates with increased levels of TP53 (tumour protein 53), p21, Bax and caspase 3. The results of the present study thus uncover an important phenomenon that the expression of certain type?I MAGE genes, at least for Mageb18, is non-testis-specific. Although they can regulate various malignant phenotypes of cancer cells, it is necessary to study further their expression pattern in normal tissues before using them to develop more effective and safer cancer vaccines.     

黑色素瘤抗原编码基因家族新成员MAGE-D1的组织表达谱研究

MAGE D1是黑色素瘤抗原编码基因家族 (MAGE)中MAGE D亚家族的新成员 .为了研究该基因的性质及其可能功能 ,采用Northernblot和Dotblot杂交技术研究了其组织表达谱 .结果发现 ,该基因在多种肿瘤组织和正常组织中均广泛表达 .在所检测的 4 8种肿瘤组织中 ,经与对应正常组织进行比较发现 ,该基因在 13种肿瘤组织中的表达显著增高 ,而在 7种肿瘤组织中的表达则显著降低 .进一步分析提示该基因在多种胚胎组织中的表达高于成年组织 .由于MAGE A、 B、 C亚家族均具有在肿瘤组织 睾丸中特异表达的特点 ,而作为MAGE D亚家族成员的MAGE D1并非在肿瘤组织中特异表达 ,提示需要对MAGE基因家族进行深入的功能研究 .     

MAGE-F1, a novel ubiquitously expressed member of the MAGE superfamily

Most known members of the MAGE superfamily are expressed in tumors, testis and fetal tissues, which has been described as a cancer/testis or "CT" expression pattern. We have identified a novel member of this superfamily, MAGE-F1, which is expressed in all adult and fetal tissues tested. In addition to normal tissues, MAGE-F1 is expressed in many tumor types including ovarian, breast, cervical, melanoma and leukemia. MAGE-F1 is encoded on chromosome 3, identifying a sixth chromosomal location for a MAGE superfamily gene. The coding region of MAGE-F1 is contained within a single exon and includes a microsatellite repeat. Sequence analysis and expression profiles define a new class of ubiquitously expressed MAGE superfamily genes that includes MAGE-F1, MAGE-D1, MAGE-D2/JCL-1 and NDN. The finding that several MAGE genes are ubiquitously expressed suggests a role for MAGE encoded proteins in normal cell physiology. Furthermore, potential cross-reactivity to these ubiquitously expressed MAGE gene products should be considered in the design of MAGE-targeted immunotherapies for cancer.     

Expression of SSX-2 and SSX-4 genes in neuroblastoma

The SSX genes are members of the family of cancer/testis antigens that encode tumor-associated antigens recognizable by autologous cytolytic T lymphocytes. Their expression is common in tumors of diverse lineages and absent in normal tissues except testis and thyroid. In this study, sixty-seven neuroblastomas (NB) (12 stage 1, 13 stage 2, 12 stage 3, 12 stage 4S and 13 stage 4) were examined by RT-PCR and a sensitive chemiluminescent detection method for SSX-2 and SSX-4 expression. Seventy-two percent (13/18) of stage 4 NB expressed SSX-2 and 67% (12/18) expressed SSX-4. SSX-2 and SSX-4 positivity correlated with metastatic NB stage 4 (p=0.02 and p=0.006, respectively). Sensitivity experiments showed SSX-2 detection was one tumor cell in 10(6) normal cells, and one in 10(4) for SSX-4. All normal tissues (n=6), with the exception of testis, normal bone marrow (BM, n=12) and normal peripheral blood (PBL, n=10) were negative for SSX-2 and SSX-4 expression. Thirty-two BM and 14 PBL obtained from 35 stage 4 NB patients at 24 months from their diagnosis were evaluated for SSX-2 expression. Unlike another cancer/testis antigen, GAGE, only one BM sample was positive, and no prognostic utility could be established. Further investigation of SSX expression at other relevant time points is warranted.     

Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer

Immunotherapy is emerging as a supplement to conventional cancer treatment, and identifying antigen targets for specific types of cancer is critical to optimizing therapeutic efficacy. Cancer/testis antigens are highly promising targets for immunotherapy due to their cancer-specific expression and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many other CT antigens, such as MAGE-A proteins. Surprisingly, we detected no ADAM2, CALR3 and SAGE1 in the 67 lung cancers (mainly non-small lung cancer) and 189 breast cancers, while MAGE-A proteins were present in 15% and 7–16% of these tumor types, respectively. Treatment with DNA methyltransferase inhibitors has been proposed as an attractive strategy to increase the expression of cancer/testis antigens in tumors before immunotargeting; however, neither ADAM2, CALR3 nor SAGE1 could be significantly induced in lung and breast cancer cell lines using this strategy. Our results suggest that ADAM2, CALR3 and SAGE1 cancer/testis antigens are not promising targets for immunotherapy of breast and lung cancer.     

Analysis of the antibody repertoire of lymphoma patients

Cancer testis or cancer germline antigens (CGA) are promising vaccine candidates because they are expressed only in malignant but not in normal tissues, except for germ cells in the testis. Since non-Hodgkin's lymphomas (NHL) express the known CGA at low frequencies, we aimed at increasing the number of CGA with frequent expression in NHL by screening a cDNA expression library derived from normal testis for reactivity with high-titered IgG antibodies in the sera of lymphoma patients using SEREX, the serological identification of antigens by recombinant cDNA expression cloning. The analysis of 1.6x10(6) clones with the sera of 25 lymphoma patients revealed 42 clones which coded for 23 antigens, 12 of which had already been included in the SEREX databank. Four cDNA clones coded for unknown and 19 for known genes. Three antigens reacted only with the serum by which they had been detected, 9 antigens reacted with the sera of several NHL patients, but not with that of healthy controls, and 11 antigens reacted with both normal and NHL sera. Most of the antigens were ubiquitously expressed. Only HOM-NHL-6, HOM-NHL-8, HOM-NHL-21 and HOM-NHL-23 showed a restricted expression pattern. HOM-NHL-6 and HOM-NHL-8 were homologous to the previously described CGA NY-ESO-1 and HOM-TES-14/SCP-1, respectively. HOM-NHL-21 was expressed in rare cases of lymphomas, but not in normal tissues except for testis and brain, while HOM-NHL-23 appeared to be a testis-specific antigen. In summary, using the antibody repertoire of these 25 NHL patients, no new CGA were detected. The number of CGA detectable by the classical SEREX approach appears to be limited, and novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of NHL patients.     

Hodgkin’s lymphoma RNA-transfected dendritic cells induce cancer/testis antigen-specific immune responses

Cytotoxic T lymphocytes (CTL) can kill Hodgkin's lymphoma (HL) cells, and CTL have been used for the treatment of Epstein-Barr virus (EBV)-positive HL. For patients with EBV-negative HL, this strategy cannot be employed and alternative target structures have to be defined. In order to establish a system for the stimulation of HL-reactive T cells, we used dendritic cells (DC) as antigen-presenting cells for autologous T cells and transfected these DC with RNA from established HL cell lines. After stimulation of peripheral blood mononuclear cells (PBMC) with RNA-transfected DC, we analyzed the reactivity of primed PBMC by interferon gamma enzyme-linked immunospot. Our results suggest the presence of antigens with expression in HL cell lines and recognition of these antigens in combination with DC-derived human leukocyte antigen molecules. By the analysis of Gene Expression Omnibus microarray data sets from HL cell lines and primary HL samples in comparison with testis and other normal tissues, we identified HL-associated cancer testis antigens (CTA) including the preferentially expressed antigen in melanoma (PRAME). After stimulation of PBMC with RNA-transfected DC, we detected PRAME-reactive T cells. PRAME and other HL-associated CTA might be targets for HL-specific immune therapy or for the monitoring of HL-directed immune responses.     

Expression of cancer testis antigens in human BRCA-associated breast cancers: potential targets for immunoprevention?

Introduction

Novel breast cancer risk-reducing strategies for individuals with germline mutations of the BRCA1 and/or BRCA2 genes are urgently needed. Identification of antigenic targets that are expressed in early cancers, but absent in normal breast epithelium of these high-risk individuals, could provide the basis for the development of effective immunoprophylactic strategies. Cancer testis (CT) antigens are potential candidates because their expression is restricted to tumors, and accumulating data suggest that they play important roles in cellular proliferation, stem cell function, and carcinogenesis. The objective of this study was to examine the expression of CT antigens and their frequency in BRCA-associated breast cancers.

Methods

Archived breast cancer tissues (n?=?26) as well as morphologically normal breast tissues (n?=?7) from women carrying deleterious BRCA 1 and/or 2 mutations were obtained for antigen expression analysis by immunohistochemistry. Expression of the following CT antigens was examined: MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1.CT7, NY-ESO-1, MAGE-C2/CT10, and GAGE.

Results

CT antigens were expressed in 16/26 (61.5%, 95% CI 43?C80%) of BRCA-associated cancers, including in situ tumors. Thirteen of twenty-six (50%) breast cancers expressed two or more CT antigens; three cancers expressed all seven CT antigens. MAGE-A was expressed in 13/26 (50%) of cancers, NY-ESO-1 was expressed in 10/26 (38%) of tumors. In contrast, none of the CT antigens were expressed in adjacent or contralateral normal breast epithelium (P?=?0.003).

Conclusions

We report a high CT antigen expression rate in BRCA-associated breast cancer as well as the lack of expression of these antigens in benign breast tissue of carriers, identifying CT antigens as potential vaccine targets for breast cancer prevention in these high-risk individuals.     

Molecular and immunological evaluation of the expression of cancer/testis gene products in human colorectal cancer

Tumor-specific gene products, such as cancer/testis (CT) antigens, constitute promising targets for the development of T cell vaccines. Whereas CT antigens are frequently expressed in melanoma, their expression in colorectal cancers (CRC) remains poorly characterized. Here, we have studied the expression of the CT antigens MAGE-A3, MAGE-A4, MAGE-A10, NY-ESO-1 and SSX2 in CRC because of the presence of well-described HLA-A2-restricted epitopes in their sequences. Our analyses of 41 primary CRC and 14 metastatic liver lesions confirmed the low frequency of expression of these CT antigens. No increased expression frequencies were observed in metastatic tumors compared to primary tumors. Histological analyses of CRC samples revealed heterogeneous expression of individual CT antigens. Finally, evidence of a naturally acquired CT antigen-specific CD8⁺ T cell response could be demonstrated. These results show that the expression of CT antigens in a subset of CRC patients induces readily detectable T cell responses.