RESUMO
Specific targets of cellular immunity in human premalignancy are largely unknown. Monoclonal gammopathy of undetermined significance (MGUS) represents a precursor lesion to myeloma (MM). We show that antigenic targets of spontaneous immunity in MGUS differ from MM. MGUS patients frequently mount a humoral and cellular immune response against SOX2, a gene critical for self-renewal in embryonal stem cells. Intranuclear expression of SOX2 marks the clonogenic CD138(-) compartment in MGUS. SOX2 expression is also detected in a proportion of CD138(+) cells in MM patients. However, these patients lack anti-SOX2 immunity. Cellular immunity to SOX2 inhibits the clonogenic growth of MGUS cells in vitro. Detection of anti-SOX2 T cells predicts favorable clinical outcome in patients with asymptomatic plasmaproliferative disorders. Harnessing immunity to antigens expressed by tumor progenitor cells may be critical for prevention and therapy of human cancer.
Assuntos
Células-Tronco Embrionárias/imunologia , Proteínas HMGB/imunologia , Paraproteinemias/imunologia , Paraproteinemias/metabolismo , Fatores de Transcrição/imunologia , Biomarcadores , Proliferação de Células , Células Cultivadas , Progressão da Doença , Proteínas HMGB/metabolismo , Saúde , Humanos , Imunoglobulina G/imunologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Paraproteinemias/patologia , Paraproteinemias/terapia , Fatores de Transcrição SOXB1 , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Resultado do TratamentoRESUMO
BACKGROUND: We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients. METHODS: The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification. RESULTS: By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein. CONCLUSION: We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/química , Anticorpos Monoclonais/química , Antígenos de Neoplasias/química , Autoanticorpos/química , Regulação Neoplásica da Expressão Gênica , Autoimunidade , Neoplasias da Mama , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citosol/metabolismo , Humanos , Imuno-Histoquímica/métodos , Cinética , Estrutura Terciária de ProteínaRESUMO
Ectopic gene expression in tumors versus normal somatic tissues provides opportunities for the specific immunotargeting of cancer cells. SSX gene products are expressed in tumors of different histological types and can be recognized by tumor-reactive CTLs from cancer patients. Here, we report the identification of an SSX-2-derived immunodominant T cell epitope recognized by CD4(+) T cells from melanoma patients in association with HLA-DR. The epitope maps to the 37-58 region of the protein, encompassing the sequence of the previously defined HLA-A2-restricted immunodominant epitope SSX-2(41-49). SSX-2(37-58)-specific CD4(+) T cells were detected among circulating lymphocytes from the majority of melanoma patients analyzed and among tumor-infiltrating lymphocytes, but not in healthy donors. Together, our data suggest a dominant role of the 37-58 sequence in the induction of cellular CD4(+) T cell responses against SSX antigens and will be instrumental for both the onset and the monitoring of upcoming cancer-vaccine trials using SSX-derived immunogens.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T , Antígenos HLA-DR/imunologia , Epitopos Imunodominantes , Proteínas de Neoplasias/imunologia , Proteínas Repressoras/imunologia , Sequência de Aminoácidos , Cadeias HLA-DRB1 , Humanos , Melanoma/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologiaRESUMO
To identify novel, tissue-restricted cell surface proteins in cancer which can serve as targets for antibody-based diagnostics and therapeutics, a translated version of the expressed sequence tag database (tblastn) was mined for transcripts with similarity to the glycoprotein A33 (GPA33) colon cancer antigen. A novel human transcript, termed A34, was identified which encoded a putative cell surface protein, GPA34, which is approximately 30% identical to GPA33 and other members of the junctional adhesion molecule (JAM) family. Conventional end-point and quantitative real-time RT-PCR showed that A34 mRNA expression is highly tissue-restricted, as it is expressed predominantly in stomach and testis. A34 mRNA was also detected in 6/19 (31%) gastric cancers, 8/16 (50%) esophageal carcinomas, and 4/17 (23%) ovarian cancers, but not in lung, breast or colon carcinomas. A murine monoclonal antibody (mAb A34) was generated to the extracellular domain of the A34 protein and used to biochemically and immunohistochemically characterize the A34 antigenic system. The mAb A34 specifically recognized glycoproteins ranging in apparent size from 55-70 kDa, present in normal gastric mucosa and in COS-7 cells transfected with A34 cDNA. Of 31 different normal tissues examined by immunohistochemistry, GPA34 protein expression was detected primarily in normal stomach mucosa and testicular germ cells, and in the tumor cells of 5/17 (29%) gastric cancers, 7/11 (63%) esophageal cancers, and 2/21 (9%) ovarian cancers, in agreement with gene expression results. The A34 antigen and monoclonal antibody may be of considerable value for immunotherapy of different types of cancer.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Imunoterapia , Glicoproteínas de Membrana/imunologia , Neoplasias/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/uso terapêutico , Reações Antígeno-Anticorpo , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Humanos , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Neoplasias/metabolismo , Neoplasias/terapia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Monitoring human antibody recognition of tumor antigens could have potential diagnostic and prognostic significance. Serological analysis of recombinant cDNA expression libraries of human cancer has identified a number of immunogenic tumor antigens. To identify colon cancer antigens associated with a cancer-related serum IgG response, serum samples from 74 patients with colon cancer and 75 normal blood donors were screened for antibody reactivity to 77 serologically defined tumor antigens. The following 13 antigens reacted exclusively with sera from the colon cancer patients and not with sera from normal blood donors: p53, MAGEA3, SSX2, NY-ESO-1, HDAC5, MBD2, TRIP4, NY-CO-45, KNSL6, HIP1R, Seb4D, KIAA1416, and LMNA. Serum samples from 34 of 74 (46%) colon cancer patients detected 1 or more of these 13 antigens. Fifty-three of 74 colon cancer patients were of known clinicopathological stage. Analysis of antibody frequency showed that 5 of 7 (71%) stage I colon cancer patients, 4 of 11 (36%) stage II patients, 2 of 14 (14%) stage III patients, and 11 of 21 (52%) stage IV patients had serum IgG antibody that reacted with 1 or more of the 13 antigens. The mRNA expression patterns of 8 of these 13 antigens were altered in cancer. Three of the 13 antigens were cancer/testis antigens (MAGEA3, SSX2, and NY-ESO-1), which are expressed exclusively in normal gametogenic tissues and aberrantly expressed in a broad range of cancer types. Quantitative real-time reverse transcription-PCR showed that the mRNA expression levels of 2 antigens, HDAC5 and Seb4B, were down-regulated in colon cancer, 2 other antigens, KNSL6 and KIAA1416, were up-regulated, and another antigen, NY-CO-45, showed a variable level of mRNA expression in colon cancer. With regard to KNSL6 mRNA expression, only trace levels were detected in 15 different normal tissues with the exception of testis, which showed a high level of KNSL6 mRNA expression. In contrast, 9 of 9 colon cancer specimens showed overexpression of KNSL6 mRNA, ranging from 5 to 44 times the level detected in normal colon tissue, indicating that this antigen could also be a valuable therapeutic target.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias do Colo/imunologia , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/genética , Neoplasias do Colo/sangue , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Estudos Soroepidemiológicos , Testes SorológicosRESUMO
Cancer-testis (CT) antigens are expressed in a variety of cancers, but not in normal adult tissues, except for germ cells of the testis, and hence appear to be ideal targets for immunotherapy. In an effort to examine the potential of NY-ESO-1 and LAGE-1 CT antigens for immunotherapy in epithelial ovarian cancer (EOC), we examined the expression of these antigens by reverse transcription-PCR (RT-PCR) and immunohistochemistry (IHC) in a large panel of EOC tissues and cell lines. Sera from a subgroup of the patients were tested for NY-ESO-1/LAGE-1 antibody by ELISA. The data indicated that four ovarian cancer cell lines were positive for one or both CT antigens. Expression of NY-ESO-1 in EOC was demonstrated by RT-PCR and/or IHC in 82 of 190 (43%) specimens. NY-ESO-1 expression by IHC ranged from homogeneous to heterogeneous pattern. LAGE-1 mRNA expression was present in 22 of 107 (21%) tumor tissues. Overall, the expression of either NY-ESO-1 or LAGE-1 mRNA was present in 42 of 107 (40%) EOC specimens and coexpression of both antigens was demonstrated in 11% of specimens. Antibody to NY-ESO-1/LAGE-1 was present in 11 of 37 (30%) patients whose tumors expressed either NY-ESO-1 or LAGE-1. Detectable antibodies were present for up to 3 years after initial diagnosis. Although there was no statistically significant relation between expression of NY-ESO-1/LAGE-1 antigen and survival, the data showed aberrant expression of NY-ESO-1 and LAGE-1 by IHC/RT-PCR in a significant proportion of EOC patients. These findings indicate that NY-ESO-1 and LAGE-1 are attractive targets for antigen-specific immunotherapy in EOC.
Assuntos
Antígenos de Neoplasias , Proteínas de Membrana , Neoplasias Ovarianas/imunologia , Proteínas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/sangue , Antígenos de Superfície , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/terapia , Prognóstico , Biossíntese de Proteínas , Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
NY-ESO-1 is a cancer/testis antigen expressed in normal adult tissues solely in the testicular germ cells of normal adults and in various cancers. It induces specific humoral and cellular immunity in patients with NY-ESO-1-expressing cancer. The aim of this study was to determine the frequency of NY-ESO-1 mRNA and protein expression in malignant and benign breast tumors. NY-ESO-1 mRNA expression was detected by conventional reverse transcription-PCR and real-time PCR, and that of the protein expression by immunohistochemistry and Western blot analysis. Expression of NY-ESO-1 mRNA was detected in 37 of 88 (42%) cancer specimens, whereas that of the NY-ESO-1 protein was detected only in 1 mRNA-positive specimen. In the latter case, expression level of NY-ESO-1 mRNA relative to that in the testis was relatively high (75% of testicular expression) and to the other among breast cancer specimens. In benign breast lesions, 21 of 31 (68%) specimens expressed low levels of NY-ESO-1 mRNA. In 1 case of fibroadenoma, NY-ESO-1 mRNA was 8% of the testicular level, and protein was detected by Western blot analysis. Only 1 breast cancer patient had detectable antibody at time of surgery, which disappeared within 2 years. Tumor specimen from this patient was both NY-ESO-1 mRNA and protein positive, and NY-ESO-1-specific CD8 T cells were detected in this patient by IFN-gamma enzyme-linked immunospot assay using NY-ESO-1 recombinant adeno and vaccinia virus. A higher rate of NY-ESO-1 expression was noted in breast cancer with high histological grade and negative hormone receptor status, suggesting NY-ESO-1 as a potential tumor antigen for immunotherapy in patients with breast cancer and poor prognosis.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Adenoviridae/genética , Adulto , Anticorpos Antineoplásicos/sangue , Linfócitos T CD8-Positivos , Carcinoma Ductal/genética , Carcinoma Ductal/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Depleção Linfocítica , Masculino , Invasividade Neoplásica , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Vaccinia virus/genéticaRESUMO
Breast cancer accounts for 30-40% of all deaths from cancers in females. In an effort to identify tumor associated antigens that may be useful for immunotherapy, we utilized serological analysis of recombinant cDNA expression libraries (SEREX) technique to identify breast cancer-associated antigens. SEREX screening of cDNA expression libraries derived from 3 breast cancer patients identified a total of 88 positive clones (bcg-1 to bcg-88), including 27 hitherto unknown sequences. The cDNA sequences and mRNA expression patterns were characterized. Seroreactivity of the SEREX clones were determined in sera from 75 breast cancer patients, 75 colon cancer patients, and 25 healthy donors. Expression analysis on a cDNA panel from 17 different normal tissues by reverse transcription-PCR (RT-PCR) revealed tissue restricted mRNA expression of 2 of the 27 unknown antigens. Bcg-72 is expressed only in breast, prostate and thymus, while bcg-84 is expressed at moderate levels in testis, spleen and breast. The other 25 unknown antigens were expressed in most other tissues. Serologic assay revealed that 7 out of the 88 clones showed reactivity to at least one serum from either 75 breast or 75 colon cancer patients. These clones did not react with sera from a panel of 25 healthy adult individuals. Our results demonstrate the utility of the SEREX approach for the identification of potential tumor associated antigens in human breast cancer.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/sangue , Neoplasias da Mama/imunologia , Soros Imunes/sangue , Soros Imunes/imunologia , Antígenos de Neoplasias/genética , Neoplasias da Mama/genética , DNA Complementar/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Neoplásico/análise , RNA Neoplásico/genéticaRESUMO
SEREX (serological analysis of recombinant tumor cDNA expression libraries) is a technique designed to isolate tumor antigens that have elicited high-titer IgG responses in human hosts. This is an immunoscreening method for gene cloning, with two key features that distinguish it from earlier immunoscreenings used to identify targets in autoimmune diseases. First, the assay was designed, at last originally, to analyze autologous immunological responses to cancer, that is, the tumor cDNA library and the screening serum were obtained from the same patient. Second, the screening is performed with serum samples at high dilution (1:1000-1:100), and the secondary antibody used was specific for human IgG. This ensures that only antigens eliciting high-titer IgG responses will be isolated. This latter feature is important in that a key purpose of SEREX is to identify tumor antigens that have elicited both humoral and cell-mediated immune responses in cancer patients. Several tumor antigens identified by SEREX on various types of cancer indeed showed such characteristics and these antigens are being tested as targets for therapeutic cancer vaccines.
Assuntos
DNA Complementar/genética , Biblioteca Gênica , Vacinas Anticâncer , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia/métodos , Indicadores e Reagentes , Plasmídeos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificaçãoRESUMO
The NY-ESO-1 gene, located on chromosome Xq28, encodes a cancer/testis antigen in human. Normally expressed only in germ cells, NY-ESO-1 is activated in a wide range of human tumors, eliciting both antibody and cell-mediated immune responses in cancer patients. Clinical immunotherapeutic trials NY-ESO-1 gene products are now ongoing. A closely related gene, LAGE-1, was subsequently identified and shares similar biological features. By database search, we have identified a third member of the human NY-ESO-1 gene family. This gene, designated ESO3, is also located on the X chromosome, clustered with two exact copies of ESO1(NY-ESO-1) and one copy of ESO2(LAGE-1), within a approximately 400 kb segment. The exon-intron structures are conserved among ESO1-3. While ESO1 and ESO2(LAGE-1) share >80% protein sequence identity, homology between ESO3 and the other two members is lower (<50%). ESO3 is also distinctive in that, unlike ESO1 and 2 that are normally expressed only in testis, ESO3 messenger RNA (mRNA) is ubiquitously expressed in somatic tissues. In addition to ESO3, an intronless pseudogene highly homologous to ESO3 was found on chromosome 9, designated psiESO3.A search of the rodent databases identified mouse and rat counterparts of ESO3, named mESO3 and rESO3. mESO3 is similarly located on mouse X chromosome, with conserved exon-intron junctions. Protein sequence of mESO3 is 54% identical to ESO3 (70% identical at the conserved carboxyl end), and 32% to ESO1. mESO3 mRNA is also ubiquitously expressed in somatic tissues, as is rESO3. In addition, an intronless and presumably non-coding, copy of the mESO3, psimESO3, was identified on mouse chromosome 15. No counterpart of the ESO1 or 2 genes was found in rodents.
Assuntos
Sequência Conservada/genética , Evolução Molecular , Proteínas de Membrana , Proteínas/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Antígenos de Superfície , Sequência de Bases , Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Pseudogenes/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Testículo/metabolismoRESUMO
Cancer/testis (CT) antigens are immunogenic in cancer patients, exhibit highly tissue-restricted expression, and are considered promising target molecules for cancer vaccines. To date, 44 CT gene families have been identified and their expression studied in numerous cancer types. For example, bladder cancer, non-small cell lung cancer, and melanoma are high CT gene expressors, with 11/20 (55%), 17/33 (51%) and 17/32 (53%) of the CT transcripts examined by RT-PCR detected in 20% or more of the specimens examined, respectively. Breast and prostate cancer can be considered moderate CT gene expressors, with 12/32 (37%) and 6/20 (30%) CT transcripts having an expression frequency >20%, respectively, while renal and colon cancer are low CT gene expressors, with only 3/33 (9%) and 4/25 (16%) CT transcripts having an expression frequency >20%, respectively. In normal tissues, standardized RT-PCR experiments showed that 19/43 CT genes were testis-restricted, 10/43 CT genes were tissue-restricted (mRNA detected in 2 or fewer non-gametogenic tissues), 9/43 CT genes were differentially expressed (mRNA detected in 3-6 non-gametogenic tissues), and 5/43 CT genes were ubiquitously expressed. With the exception of testis-restricted CT transcripts, all remaining CT transcripts were expressed in normal pancreas. In terms of immunogenicity, 14/29 testis/tissue-restricted CT gene families have been shown to induce a cellular and/or humoral immune response in humans. In view of the expanding list of CT genes, a CT gene database was created to standardize CT nomenclature and accumulate relevant data regarding their expression profiles, immunogenicity, function (where known), gene structure and location, and orthologous groups.
Assuntos
Antígenos de Neoplasias/genética , Bases de Dados Genéticas/normas , Genes Neoplásicos/genética , Testículo , Antígenos de Neoplasias/classificação , Bases de Dados Genéticas/classificação , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Testículo/química , Testículo/metabolismoRESUMO
SCP-1 is a novel tumor antigen that belongs to the growing family of cancer/testis (CT) antigens, and it is a potential target for immunotherapy. In an effort to determine the expression of SCP-1 in epithelial ovarian cancer (EOC), one-step RT-PCR was performed with RNA from epithelial ovarian tumor tissues and with two normal ovarian surface epithelial cell lines. We used immunohistochemistry (IHC) to investigate SCP-1 expression in paraffin-fixed EOC samples and ELISA to test sera from a subgroup of patients for SCP-1 antibody. SCP-1 was expressed in 15 out of 100 (15%) primary tumors, as determined by RT-PCR. The normal ovarian surface epithelial cell lines were negative for SCP-1 expression, as were a panel of other normal tissues. None of the patients whose tumors were determined to be SCP-1 positive by RT-PCR expressed the antigen by IHC or demonstrated a humoral immune response by ELISA. Tumors that expressed SCP-1 mRNA tended to have a higher grade than those that did not (P = 0.03). There was a significant decrease in survival time (P = 0.004) for patients with SCP-1 mRNA-positive tumors compared to those with SCP-1 mRNA-negative tumors [median 25 mo, 95% confidence interval (CI) 0-56 mo; and median 97 mo, CI 32-162 mo, respectively]. The present study shows that SCP-1 mRNA expression in patients with EOC is associated with a poorer chance of survival. These findings imply that further evaluation of SCP-1 as a potential target for vaccine therapy in EOC is warranted.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias Ovarianas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/imunologiaRESUMO
Serological analysis of recombinant cDNA expression libraries (SEREX) has led to the identification of several categories of new tumor antigens. We analyzed a testicular cDNA expression library with serum obtained from a breast cancer patient and isolated 13 genes designated NW-BR-1 through NW-BR-13. Of these, 3 showed tumor-restricted expression (NW-BR-1, -2 and -3), the others being expressed ubiquitously. NW-BR-3, representing 9 of 24 primary clones, showed tissue-restricted mRNA expression, being expressed in normal testis but not in 15 other normal tissues tested by Northern blotting. RT-PCR analysis showed strong NW-BR-3 expression in normal testis, weak expression in brain, kidney, trachea, uterus and normal prostate, and was negative in liver, heart, lung, colon, small intestine, bone marrow, breast, thymus, muscle, spleen, and stomach. NW-BR-3 mRNA expression was found in different tumor tissues and tumor cell lines by RT-PCR, thus showing a 'cancer/testis' (CT)-like mRNA expression pattern. NW-BR-3 shares 71% nucleotide and amino acid homology to a mouse gene cloned from mouse testicular tissue. Based on the mRNA expression pattern, NW-BR-3 represents a new candidate target gene for cancer immunotherapy. NW-BR-1 and NW-BR-2 also showed tumor-restricted mRNA expression. NW-BR-1 is a partial clone of the breast differentiation antigen NY-BR-1 previously identified by SEREX. NY-BR-1 is expressed in normal breast, testis and 80% of breast cancers. NW-BR-2 is identical to the CT antigen SCP-1, initially isolated by SEREX analysis of renal cancer. This study provides further evidence that SEREX is a powerful tool to identify new tumor antigens potentially relevant for immunotherapy approaches.
Assuntos
Antígenos de Neoplasias/genética , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Testículo/metabolismo , Northern Blotting , Neoplasias da Mama/sangue , Clonagem Molecular/métodos , DNA Complementar/química , DNA Complementar/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Testículo/imunologia , Células Tumorais CultivadasRESUMO
Learning to identify tumor and tumor-associated antigens in patients with squamous cell carcinoma of the head and neck (HNSCC) may bring about better diagnostic and prognostic evaluations of the disease, innovative therapies based on immunological approaches, and a better understanding of the biology of tumorigenesis. Serological analysis of tumor antigens by recombinant cDNA expression cloning (SEREX) has been used to identify antigens in head and neck cancer to which patients have produced high-titered IgG antibodies. Four cDNA expression libraries have been screened with sera from 6 head and neck cancer patients. Thirty-seven individual gene products were identified. Thirty-one previously characterized proteins and 6 genes coding for molecules that are only partially characterized or novel were isolated. Tissue expression was evaluated by Northern blot analysis, RT-PCR, and in one case, quantitative real-time PCR (qPCR) using Taqman technology. Clone AU-HN-15 encoded a protein highly expressed in HNSCC tissues and cell lines. Tissue adjacent to the tumor had negligible expression. There was low or negligible expression in normal tissues, except for the brain and thymus. AU-HN-15 is identical to KIAA0530; it is an uncharacterized protein previously cloned from brain tissue and has a zinc finger domain. The cDNA encoding this protein has also been isolated in SEREX screens of testicular cancer, breast cancer, and colorectal cancer. Whether AU-HN-15 represents a tumor-antigen target suitable for prognostic or therapeutic purposes is still being analyzed.
Assuntos
Antígenos de Neoplasias/imunologia , Carcinoma de Células Escamosas/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Formação de Anticorpos/imunologia , Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/genética , Northern Blotting , Vacinas Anticâncer/imunologia , Clonagem Molecular , DNA Complementar/genética , Feminino , Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Cabeça e Pescoço/genética , Humanos , Masculino , Biblioteca de Peptídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Serological analysis of recombinant cDNA expression libraries (SEREX) has led to the identification of many of the antigens recognized by the immune system of cancer patients, which are collectively referred to as the cancer immunome. We used SEREX to screen a testicular cDNA expression library with sera obtained from non-small cell lung cancer patients and isolated cDNA clones for 82 antigens. These included a total of 31 antigens previously identified by SEREX, and 51 that did not match entries in the Cancer Immunome Database and were considered newly identified antigens. Overall, the antigens comprised 62 known proteins and 20 uncharacterized gene products. Six antigens (NY-TLU-6, -37, -39, -57, -70, -75) were identified as putative cell surface proteins that are potential targets for monoclonal antibody-based immunotherapy. Of these, the gonad-specific anion transport protein SLCO6A1 (NY-TLU-57) was shown to be tissue-restricted. RT-PCR showed it to be expressed strongly only in normal testis, and weakly in spleen, brain, fetal brain, and placenta. In addition, NY-TLU-57 mRNA was found in lung tumor samples (5/10) and lung cancer cell lines (6/11), as well as bladder (5/12) and esophageal (5/12) tumor samples. These data suggest that SLCO6A1 is a putative cancer/testis (CT) cell surface antigen of potential utility as a target for antibody-based therapy for a variety of tumor types. The analysis also permits us to estimate the eventual size of the SEREX-defined cancer immunome at around 4000 genes. This emphasizes the importance of continued SEREX screening to define the cancer immunome.
Assuntos
Proteínas de Transporte de Ânions/imunologia , Antígenos de Neoplasias/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Pulmonares/imunologia , Transportadores de Ânions Orgânicos/imunologia , Proteínas de Transporte de Ânions/biossíntese , Proteínas de Transporte de Ânions/genética , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Biblioteca Gênica , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Masculino , Transportadores de Ânions Orgânicos/biossíntese , Transportadores de Ânions Orgânicos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Testículo/imunologiaRESUMO
Cancer/testis (CT) antigens are considered promising candidates for vaccine-based immunotherapy. The aim of this study was to investigate which CT antigens should be targeted in immunotherapy of Japanese lung cancer. To determine the expression of 12 CT antigens in Japanese primary lung cancers and cell lines, a reverse-transcription polymerase chain reaction (RT-PCR) analysis was performed. Among 46 primary lung cancers, high expression rates were found for MAGE-3 (41%, 19/46), and SSX-4 (35%, 16/46). A similar pattern of CT antigen expression was observed in 29 lung cancer cell lines. The expression frequency of a certain CT antigen, namely NY-ESO-1, in Japanese cases was drastically different from that in Caucasians. Polyvalent CT antigen vaccine may be effective to increase the number of lung cancer patients eligible for cancer-specific immunotherapy. Vaccination with MAGE-3 and SSX-1 would cover 57% of all patients, with three antigens, MAGE-3, SSX-1, and MAGE-4, would cover 65%, and with four antigens, MAGE-3, SSX-1, MAGE-4 and SSX-4, would cover 70%. Simultaneous expression of two or more CT antigens was observed in 25/46 (54%) primary lung cancers and 18/29 (62%) lung cancer cell lines. Polyvalent CT antigen vaccines may be also effective to reduce a chance of emergence of antigen loss variants, thus preventing tumors from escaping from the immune system. For this purpose, vaccination with combinations of MAGE-3 with MAGE-6, SSX-4, MAGE-1 or BAGE may be effective for a quarter of Japanese lung cancer patients. In addition, in silico surveys of dbEST database were used for identification of new CT antigens. We identified a novel gene, TES101RP, expressed only in some small cell lung cancers (SCLC) and in testis, as confirmed by RT-PCR analysis.
Assuntos
Antígenos de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas de Membrana , Vacinas Anticâncer/genética , Humanos , Masculino , Antígenos Específicos de Melanoma , Proteínas de Neoplasias/genética , Proteínas/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/imunologia , Células Tumorais CultivadasRESUMO
The structural definition of human tumor antigens recognized by the autologous host has been a long-standing challenge in tumor immunology. The growing list of human tumor antigens recognized by autologous antibodies or cytotoxic T lymphocytes provides convincing evidence for immune recognition of cancer by the host of origin, as well as attractive targets for vaccine-based approaches to cancer therapy. In this regard, an approach called SEREX (serological analysis of recombinant cDNA expression libraries) has broad applicability to the analysis of the humoral immune response to cancer antigens. This method involves immunoscreening cDNA libraries prepared from tumor specimens with sera from cancer patients in order to identify gene products recognized by IgG antibody. Clones identified by SEREX can be directly sequenced, providing immediate structural definition of the antigenic target, and their expression profiles can be readily determined, providing information regarding their tissue distribution. Application of this technique has led to the discovery of a number of provocative tumor antigens.
Assuntos
Antígenos de Neoplasias/análise , Neoplasias/imunologia , Anticorpos Antineoplásicos , DNA Complementar , Biblioteca Gênica , Humanos , Soros Imunes , Proteínas RecombinantesRESUMO
Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.
Assuntos
Antígenos de Neoplasias/genética , Cromossomos Humanos X/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , Testículo/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Biologia Computacional , Primers do DNA , DNA Complementar/genética , Bases de Dados de Ácidos Nucleicos , Humanos , Masculino , Dados de Sequência Molecular , Família Multigênica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA/métodosRESUMO
Cancer/testis (CT) antigens are a category of tumor antigens with normal expression restricted to male germ cells in the testis but not in adult somatic tissues. In some cases, CT antigens are also expressed in ovary and in trophoblast. In malignancy, this gene regulation is disrupted, resulting in CT antigen expression in a proportion of tumors of various types. Since their initial identification by T-cell epitope cloning, the list of CT antigens has been greatly expanded through serological expression cloning (SEREX) and differential mRNA expression analysis, and approximately 20 CT antigens or antigen families have been identified to date. Characteristics commonly shared by CT antigens, aside from the highly tissue-restricted expression profile, include existence as multigene families, frequent mapping to chromosome X, heterogeneous protein expression in cancer, likely correlation with tumor progression, induction of expression by hypomethylation and/or histone acetylation, and immunogenicity in cancer patients. Spontaneous humoral and cell-mediated immune responses have been demonstrated against several CT antigens, including NY-ESO-1, MAGE-A, and SSX antigens. Since CT antigens are immunogenic and highly restricted to tumors, their discovery has led directly to the development of antigen-specific cancer vaccines, and clinical trials with MAGE-A and NY-ESO-1 are in progress.
Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia , Neoplasias/imunologia , Testículo/imunologia , Antígenos de Neoplasias/classificação , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Clonagem Molecular , Feminino , Expressão Gênica , Humanos , Imunoterapia Ativa , Masculino , Neoplasias/terapia , Especificidade de Órgãos , Ovário/imunologia , Trofoblastos/imunologiaRESUMO
Human SSX was first identified as the gene involved in the t(X;18) translocation in synovial sarcoma. SSX is a multigene family, with 9 complete genes on chromosome Xp11. Normally expressed almost exclusively in testis, SSX mRNA is expressed in various human tumors, defining SSX as a cancer/testis antigen. We have now cloned the mouse ortholog of SSX. Mouse SSX genes can be divided into Ssxa and Ssxb subfamilies based on sequence homology. Ssxa has only one member, whereas 12 Ssxb genes, Ssxb1 to Ssxb12, were identified by cDNA cloning from mouse testis and mouse tumors. Both Ssxa and Ssxb are located on chromosome X and show tissue-restricted mRNA expression to testis among normal tissues. All putative human and mouse SSX proteins share conserved KRAB and SSX-RD domains. Mouse tumors were found to express some, but not all, Ssxb genes, similar to the SSX activation in human tumors.