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Abstract Background Cancer-testis antigens (CTAs) and tumour-associated antigens (TAAs) are frequently expressed in hepatocellular carcinoma (HCC); however, the role of tumour-antigen-specific T cell immunity in HCC progression is poorly defined. We characterized CTA- and TAA-specific T cell responses in different HCC stages and investigated their alterations during HCC progression. Methods Fifty-eight HCC patients, 15 liver cirrhosis patients, 15 chronic hepatitis B patients and 10 heathy controls were enrolled in total. IFN-γ ELSPOT using CTAs, including MAGE-A1, MAGE-A3, NY-ESO-1, and SSX2, and two TAAs, SALL4 and AFP, was performed to characterize the T-cell immune response in the enrolled individuals. The functional phenotype of T cells and the responsive T cell populations were analyzed using short-term T-cell culture. Results T cell responses against CTAs and TAAs were specific to HCC. In early-stage HCC patients, the SALL4-specific response was the strongest, followed by MAGE-A3, NY-ESO-1, MAGE-A1 and SSX2. One-year recurrence-free survival after transcatheter arterial chemoembolization plus radiofrequency ablation treatment suggested the protective role of CTA-specific responses. The four CTA- and SALL4-specific T cell responses decreased with the progression of HCC, while the AFP-specific T cell response increased. A higher proportion of CD4+ T cells specific to CTA/SALL4 was observed than AFP-specific T cell responses. Conclusions The IFN-γ ELISPOT assay characterized distinct profiles of tumour-antigen-specific T cell responses in HCC patients. CTA- and SALL4-specific T cell responses may be important for controlling HCC in the early stage, whereas AFP-specific T cell responses might be a signature of malignant tumour status in the advanced stage. The application of immunotherapy at an early stage of HCC development should be considered.

Background: Cancer-testis antigens (CTAs) and tumour-associated antigens (TAAs) are frequently expressed in hepatocellular carcinoma (HCC); however, the role of tumour-antigen-specific T cell immunity in HCC progression is poorly defined. We characterized CTA- and TAA-specific T cell responses in different HCC stages and investigated their alterations during HCC progression.Methods: Fifty-eight HCC patients, 15 liver cirrhosis patients, 15 chronic hepatitis B patients and 10 heathy controls were enrolled in total. IFN-γ ELSPOT using CTAs, including MAGE-A1, MAGE-A3, NY-ESO-1, and SSX2, and two TAAs, SALL4 and AFP, was performed to characterize the T-cell immune response in the enrolled individuals. The functional phenotype of T cells and the responsive T cell populations were analyzed using short-term T-cell culture.Results: T cell responses against CTAs and TAAs were specific to HCC. In early-stage HCC patients, the SALL4-specific response was the strongest, followed by MAGE-A3, NY-ESO-1, MAGE-A1 and SSX2. One-year recurrence-free survival after transcatheter arterial chemoembolization plus radiofrequency ablation treatment suggested the protective role of CTA-specific responses. The four CTA- and SALL4-specific T cell responses decreased with the progression of HCC, while the AFP-specific T cell response increased. A higher proportion of CD4+ T cells specific to CTA/SALL4 was observed than AFP-specific T cell responses.Conclusions: The IFN-γ ELISPOT assay characterized distinct profiles of tumour-antigen-specific T cell responses in HCC patients. CTA- and SALL4-specific T cell responses may be important for controlling HCC in the early stage, whereas AFP-specific T cell responses might be a signature of malignant tumour status in the advanced stage. The application of immunotherapy at an early stage of HCC development should be considered. [ABSTRACT FROM AUTHOR]

Abstract: Background and Aims: Human telomerase reverse transcriptase is a catalytic enzyme involved in telomere elongation. It is expressed in many tumours, including hepatocellular carcinoma. The purpose of the present study was to identify major histocompatibility complex class II‐restricted helper T cell epitopes derived from human telomerase reverse transcriptase in patients with hepatocellular carcinoma. Methods: TEPITOPE software was used to predict helper T cell epitopes based on the entire amino acid sequence of human telomerase reverse transcriptase, and peptides were synthesized based on the predicted sequence. Interferon (IFN)‐γ enzyme linked immunospot assay was performed to examine the T cell response to each of the synthesized peptides in peripheral blood mononuclear cells. Furthermore, the peptides were labelled with fluorescein isothiocyanate to test their binding affinity for major histocompatibility complex class II molecules. Lastly, the association between patient characteristics and the level of immune response to these epitopes was examined. Results: Positive T cell response (>10% enzyme linked immunospot positivity) was detected against 4 of 10 peptides. Among all peptides, positive T cell response to the hTERT68 peptide was detected most frequently. While hTERT68 was HLA‐DRB1*0405‐restricted, it also bound to other MCH class II molecules. Positive helper T cell response was detected most frequently in hepatocellular carcinoma patients with a low serum alpha‐foetoprotein level. Several treatments for hepatocellular carcinoma enhanced the immune response against the peptides. Conclusion: Our findings indicate that helper T cell epitopes identified in the present study may be useful to investigate immune responses and for immunotherapy in hepatocellular carcinoma patients. [ABSTRACT FROM AUTHOR]

Summary: The entirety of human leukocyte antigen (HLA)‐presented peptides is referred to as the HLA ligandome of a cell or tissue, in tumours often termed immunopeptidome. Mapping the tumour immunopeptidome by mass spectrometry (MS) comprehensively views the pathophysiologically relevant antigenic signature of human malignancies. MS is an unbiased approach stringently filtering the candidates to be tested as opposed to epitope prediction algorithms. In the setting of peptide‐specific immunotherapies, MS‐based strategies significantly diminish the risk of lacking clinical benefit, as they yield highly enriched amounts of truly presented peptides. Early immunopeptidomic efforts were severely limited by technical sensitivity and manual spectra interpretation. The technological progress with development of orbitrap mass analysers and enhanced chromatographic performance led to vast improvements in mass accuracy, sensitivity, resolution, and speed. Concomitantly, bioinformatic tools were developed to process MS data, integrate sequencing results, and deconvolute multi‐allelic datasets. This enabled the immense advancement of tumour immunopeptidomics. Studying the HLA‐presented peptide repertoire bears high potential for both answering basic scientific questions and translational application. Mapping the tumour HLA ligandome has started to significantly contribute to target identification for the design of peptide‐specific cancer immunotherapies in clinical trials and compassionate need treatments. In contrast to prediction algorithms, rare HLA allotypes and HLA class II can be adequately addressed when choosing MS‐guided target identification platforms. Herein, we review the identification of tumour HLA ligands focusing on sources, methods, bioinformatic data analysis, translational application, and provide an outlook on future developments. [ABSTRACT FROM AUTHOR]

GTL001 is a bivalent therapeutic vaccine containing human papillomavirus (HPV) 16 and HPV18 E7 proteins inserted in the Bordetella pertussis adenylate cyclase (CyaA) vector intended to prevent cervical cancer in HPV-infected women with normal cervical cytology or mild abnormalities. To be effective, therapeutic cervical cancer vaccines should induce both a T cell-mediated effector response against HPV-infected cells and a robust CD8 + T-cell memory response to prevent potential later infection. We examined the ability of GTL001 and related bivalent CyaA-based vaccines to induce, in parallel, effector and memory CD8 + T-cell responses to both vaccine antigens. Intradermal vaccination of C57BL/6 mice with GTL001 adjuvanted with a TLR3 agonist (polyinosinic-polycytidylic acid) or a TLR7 agonist (topical 5% imiquimod cream) induced strong HPV16 E7-specific T-cell responses capable of eradicating HPV16 E7-expressing tumors. Tumor-free mice also had antigen-specific memory T-cell responses that protected them against a subsequent challenge with HPV18 E7-expressing tumor cells. In addition, vaccination with bivalent vaccines containing CyaA-HPV16 E7 and CyaA fused to a tumor-associated antigen (melanoma-specific antigen A3, MAGEA3) or to a non-viral, non-tumor antigen (ovalbumin) eradicated HPV16 E7-expressing tumors and protected against a later challenge with MAGEA3- and ovalbumin-expressing tumor cells, respectively. These results show that CyaA-based bivalent vaccines such as GTL001 can induce both therapeutic and prophylactic anti-tumor T-cell responses. The CyaA platform can be adapted to different antigens and adjuvants, and therefore may be useful for developing other therapeutic vaccines. [ABSTRACT FROM AUTHOR]

Purpose: Breast cancer is a leading cause of cancer deaths in women, but despite steady improvements in therapies, treatment is still suboptimal. Immunotherapy holds promise as a more effective therapy for breast cancer; supporting this, our prior study showed that patients possessing HER2-reactive CD8+ T cells in blood experience survival superior to patients without these cells. Here, we define a composite set of biomarkers that identify patients with T cell responses to tumour antigens. Methods: We assessed T cell responses following in vitro stimulation with the HER2, MUC1 and SUR tumour-associated antigens (TAA) by flow cytometry and intracellular cytokine staining in 50 breast cancer patients. We also measured HLA type, serum cytokines, tumour-infiltrating leukocytes and blood leukocyte populations. Results: We found few correlations between TAA-reactive T cells and HLA type, serum cytokines and tumour-infiltrating leukocytes, whereas blood leukocyte phenotypes broadly correlated with TAA responses. This showed monocytes, natural killer cells, dendritic cells and T cells to be inversely associated with both CD4+ and CD8+ T cells reactive to tumour antigens. Moreover, combining multiple parameters improved the accuracy in predicting patients with TAA-responsive T cells. Conclusion: This study therefore defines composite immune profiles that identify patients responding to TAAs which may allow better personalisation of cancer therapies. [ABSTRACT FROM AUTHOR]

Canine mammary gland tumour ( MGT) is the most common neoplasm in female dogs and has similar biological characteristics to human MGT. Spontaneous canine MGT is a more attractive clinical model in oncological research than that of the murine experimental model. Tumour-associated antigens ( TAAs), which are produced in tumour cells, are applied as tumour markers, tumour vaccine antigens and molecular targets of therapeutic drugs. In this study, we have primarily identified 13 different TAAs of canine MGT by serological immunoscreening of cDNA expression library. The results of serological mini-arrays of identified antigens showed that CCDC41 antigen specially reacted with 35% of sera from MGT-dogs and did not react with control sera. We also found that HSPH1 mRNA expression levels increased significantly in MGT tissues. These findings will contribute to the development of diagnostic technologies and translational target therapies for dogs. Clinical relevance: HSPH1, which is strongly expressed in the tumour tissue, will be a possible vaccine antigen of canine MGT. [ABSTRACT FROM AUTHOR]

The Wilms' tumour-1 ( WT1) protein is considered a prime target for cancer immunotherapy based on its presumptive immunogenicity and widespread expression across a variety of malignancies. However, little is known about the naturally occurring WT1-specific T-cell repertoire because self-derived antigens typically elicit low frequency responses that challenge the sensitivity limits of current detection techniques. In this study, we used highly efficient cell enrichment procedures based on CD137, CD154, and pHLA class I tetramer staining to conduct a detailed analysis of WT1-specific T cells from the peripheral blood. Remarkably, we detected WT1-specific CD4+ and CD8+ T-cell populations in the vast majority of healthy individuals. Memory responses specific for WT1 were commonly present in the CD4+ T-cell compartment, whereas WT1-specific CD8+ T cells almost universally displayed a naive phenotype. Moreover, memory CD4+ and naive CD8+ T cells with specificity for WT1 were found to coexist in some individuals. Collectively, these findings suggest a natural discrepancy between the CD4+ and CD8+ T-cell lineages with respect to memory formation in response to a self-derived antigen. Nonetheless, WT1-specific T cells from both lineages were readily activated ex vivo and expanded in vitro, supporting the use of strategies designed to exploit this expansive reservoir of self-reactive T cells for immunotherapeutic purposes. [ABSTRACT FROM AUTHOR]

The incidence of thyroid cancer has appeared as an increasing trend globally, especially in Asian countries. In this study, the expression of mucin-1 (MUC1) and Thomsen-Friedenreich antigen, Galβ1-3GalNAcα1-R (CD176) was investigated by immunohistochemistry in papillary thyroid carcinomas (PTCs), which accounts for approximately 80 % of all thyroid cancer. We found that 78 % of PTC overexpressed MUC1. Importantly, we observed firstly that CD176 was expressed in 63 % of PTC, but was faintly or not expressed in normal thyroid tissues and benign thyroid disease tissues, indicating that CD176 is also a tumour-associated antigen for PTCs. Moreover, expression of CD176 was strongly correlated with MUC1 by immunohistochemical staining in PTCs. Furthermore, we used the immunochemical method to confirm that MUC1 is a common and main carrier of CD176 in PTCs. Our data demonstrated that MUC1 and CD176 might be promising biomarkers for thyroid cancer. [ABSTRACT FROM AUTHOR]

Vaccines have classically been used for disease prevention. Modern clinical vaccines are continuously being developed for both traditional use as well as for new applications. Typically thought of in terms of infectious disease control, vaccination approaches can alternatively be adapted as a cancer therapy. Vaccines targeting cancer antigens can be used to induce anti-tumour immunity and have demonstrated therapeutic efficacy both pre-clinically and clinically. Various approaches now exist and further establish the tremendous potential and adaptability of anti-cancer vaccination. Classical strategies include ex vivo-loaded immune cells, RNA- or DNA-based vaccines and tumour cell lysates. Recent oncolytic virus development has resulted in a surge of novel viruses engineered to induce powerful tumour-specific immune responses. In addition to their use as cancer vaccines, oncolytic viruses have the added benefit of being directly cytolytic to cancer cells and thus promote antigen recognition within a highly immune-stimulating tumour microenvironment. While oncolytic viruses are perfectly equipped for efficient immunization, this complicates their use upon previous exposure. Indeed, the host’s anti-viral counter-attacks often impair multiple-dosing regimens. In this review we will focus on the use of oncolytic viruses for anti-tumour vaccination. We will explore different strategies as well as ways to circumvent some of their limitations.

Cyclin B1 is a checkpoint protein that regulates cell division from G2 to the M phase. Studies in mice have shown that cyclin B1 vaccine-induced immunity significantly delayed or prevented the spontaneous cancer development later in life. We hypothesized that if these results showing a protective effect of anti-cyclin B1 antibodies could be extrapolated to the human condition, cancer-free individuals should have higher levels of endogenous antibodies than patients with cancers characterized by the over-expression of this tumour-associated antigen. To test this hypothesis, we characterized a large (1739 subjects) number of multi-ethnic patients with breast cancer (which over-expresses cyclin B1) and matched controls for anti-cyclin B1 immunoglobulin (Ig) G antibodies. Multivariate analyses, after adjusting for the covariates, showed that cancer-free individuals had significantly higher levels of naturally occurring IgG antibodies to cyclin B1 than patients with breast cancer (mean ± standard deviation: 148·0 ± 73·6 versus 126·1 ± 67·8 arbitrary units per ml; P < 0·0001). These findings may have important implications for cyclin B1-based immunotherapy against breast cancer and many other cyclin B1-over-expressing malignancies. [ABSTRACT FROM AUTHOR]

The most common ovarian cancer is epithelial ovarian cancer (EOC) characterised by few early symptoms, widespread peritoneal dissemination and ascites at advanced stages that result in poor prognosis. Despite the recent progress in its management, including surgery and chemotherapy, EOC remains the most lethal gynaecological malignancy in women. Due to the limitations of current therapeutic approaches, many patients die of secondary disease (metastasis). MUC1 is associated with cellular transformation and tumorigenicity and is considered as an attractive therapeutic target for cancer therapy owning to its over-expression in most adenocarcinomas including EOC. Tumour-associated MUC1 plays an important role in EOC metastasis and progression. In neoplastic tissues, MUC1 is underglycosylated and reveals epitopes that are masked in the normal cells. This feature makes it possible to target tumour-associated MUC1 with antibodies, toxins or radionuclides or use a vaccine targeting tumour-associated MUC1 antigen. The shed tumour-associated MUC1 in blood can be used as a diagnostic biomarker for EOC detection and monitoring. Our recent results have shown that over-expression of MUC1 plays a very important role in EOC progression and MUC1 is an ideal target for targeted therapy to control metastatic and recurrent EOC. This review will summarize some important new findings supporting the role of MUC1 in EOC metastasis and progression and focus on the MUC1-based targeted therapy for control of metastatic and recurrent EOC. [ABSTRACT FROM AUTHOR]

Background & Aims Chondroitin-glucuronate C5-epimerase is an enzyme that converts D-glucuronic acid to L-iduronic acid residues in dermatan sulphate biosynthesis. It is also identified to be a tumour-associated antigen recognized by cytotoxic T cells ( CTLs) and its enhanced expression in many cancers has been reported. In the present study, we investigated the usefulness of this molecule as an immunotherapeutic target in hepatocellular carcinoma ( HCC). Methods The expression of chondroitin-glucuronate C5-epimerase in hepatoma cell lines and HCC tissues was confirmed by immunofluorescence and immunohistochemical analysis. CTL responses were investigated by several immunological techniques using peripheral blood mononuclear cells ( PBMCs) or tumour-infiltrating lymphocytes. To determine the safety of immunotherapy using chondroitin-glucuronate C5-epimerase-derived peptide, 12 patients with HCC were administered s.c. vaccinations of the peptides and analysed. Results Chondroitin-glucuronate C5-epimerase was expressed in HCC cell lines and human tissues including alpha-foetoprotein ( AFP)-negative individuals. Chondroitin-glucuronate C5-epimerase-specific CTLs could be generated by stimulating PBMCs of HCC patients with peptides and they showed cytotoxicity against HCC cells expressing the protein. The frequency of CTL precursors investigated by enzyme-linked immunospot ( ELISPOT) assay was 0-34 cells/3 × 105 PBMCs and the infiltration of interferon-gamma-producing CTLs into the tumour site was confirmed. In the vaccination study, no severe adverse events were observed and the peptide-specific CTLs were induced in 4 of 12 patients tested. Conclusions Chondroitin-glucuronate C5-epimerase is a potential candidate for tumour antigen with immunogenicity and the peptides derived from this antigen could be useful in HCC immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract: Malignant pleural mesothelioma (MPM) is resistant to conventional treatments. Novel, targeted treatments are hampered by the relative lack of MPM-associated tumour antigens. The aim of this study was to evaluate the level of expression and the relevance of 5T4 as a tumour-associated antigen in MPM. 5T4 expression was assessed by Western blotting, flow cytometry, immuno-cytochemistry and -histochemistry in 11 mesothelioma cell lines, 21 tumour biopsies, and ex vivo tumour cells obtained from the pleural fluid (PF) of 10 patients. 5T4 antibody levels were also determined in the plasma of patients and healthy donors. The susceptibility of MPM cells to 5T4-specific T-cell-mediated killing was determined using an HLA-A2+, CD8+ T-cell line, developed against the 5T417–25 peptide. We report here that cell surface 5T4 expression was detected in all mesothelioma cell lines and PF cell samples. Mesothelin and CD200, a suggested mesothelioma marker, were co-expressed with 5T4 on tumour cells in PF. Immunohistochemistry confirmed overexpression of 5T4, similar to mesothelin, on tumour cells but not on reactive stroma in all tissue sections tested. Median 5T4 antibody levels were 46% higher in patient than in healthy donor plasma, indicating immune recognition. Importantly, 5T4-specific CD8+ T-cells were able to kill four out of six HLA-A2+ MPM cell lines but not an HLA-A2− cell line, demonstrating immune recognition of MPM-associated 5T4 antigen at the effector T-cell level. We conclude that 5T4 is a potential new antigen for targeted therapies such as immunotherapy in MPM, as it is overexpressed on mesothelioma cells and recognised by 5T4-specific cytotoxic T-cells. Our findings have been translated into a Phase II clinical trial applying 5T4-targeted therapies in MPM patients. [Copyright &y& Elsevier]

Since their discovery, tumour-associated antigens (TAA) have provided highly inviting targets for cancer therapy, especially immunotherapy. Evidence now points to their involvement in the malignant phenotype of transformed cells and heightens their importance for being targeted by different treatments. TAA vary in their nature and pattern of expression and this influences the way therapy is directed towards them. While large numbers of these antigens have been isolated from solid tumours, fewer are linked with haematological malignancies. Those TAA found in this latter group of cancers, referred to as leukaemia-associated antigens (LAA), also appear to have significant potential for promoting the malignant phenotype and have been described in detail in terms of expression and therapy. Interestingly, the action of some of LAA in blood cancers, which are stem cell derived, could act as model for solid tumours, which are increasingly thought to be also derived from a cancer stem cell origin. In this review, TAA and their use in immunotherapy will be discussed. The nature and expression of these antigens will be described together with the events that provide tumours, including haematological cancers, with the ability to avoid immune deletion. [ABSTRACT FROM AUTHOR]

Background: Identification of tumour-associated antigens (TAAs) that induce cytotoxic T lymphocytes (CTLs) specific to cancer cells is critical for the development of anticancer immunotherapy. In this study, we aimed at identifying a novel TAA of pancreatic cancer for immunotherapy.Methods: On the basis of the genome-wide cDNA microarray analysis, we focused on KIF20A (also known as RAB6KIFL/MKlp2) as a candidate TAA in pancreatic cancer cells. The HLA-A2 (A*02:01)-restricted CTL epitopes of KIF20A were identified using HLA-A2 transgenic mice (Tgm) and the peptides were examined to check whether they could generate human CTLs exhibiting cytotoxic responses against KIF20A(+), HLA-A2(+) tumour cells in vitro.Results: KIF20A was overexpressed in pancreatic cancer and in some other malignancies, but not in their non-cancerous counterparts and many normal adult tissues. We found that KIF20A-2 (p12-20, LLSDDDVVV), KIF20A-8 (p809-817, CIAEQYHTV), and KIF20A-28 (p284-293, AQPDTAPLPV) peptides could induce HLA-A2-restricted CTLs in HLA-A2 Tgm without causing autoimmunity. Peptide-reactive human CTLs were generated from peripheral blood mononuclear cells of HLA-A2(+) healthy donors by in vitro stimulation with the three peptides, and those CTLs successfully exhibited cytotoxic responses to cancer cells expressing both KIF20A and HLA-A2.Conclusion: KIF20A is a novel promising candidate for anticancer immunotherapeutic target for pancreatic cancers. [ABSTRACT FROM AUTHOR]

Van Elssen C H M J, Frings P W H, Bot F J, Van de Vijver K K, Huls M B, Meek B, Hupperets P, Germeraad W T V & Bos G M J (2010) Histopathology 57, 597-606 Expression of aberrantly glycosylated Mucin-1 in ovarian cancer Aims: Mucin 1 (MUC1) is an important tumour-associated antigen (TAA), both overexpressed and aberrantly glycosylated in adenocarcinomas. The aim of this study was to examine the MUC1-glycosylation status of primary ovarian adenocarcinomas and metastatic lesions. Methods and results: Paraffin-embedded tissue sections of 37 primary ovarian adenocarcinomas representing all histotypes (22 serous, five mucinous, two clear-cell, eight endometrioid), four serous borderline tumours with intraepithelial carcinoma, seven sections of ovarian endometriosis and 13 metastatic lesions were analysed by immunohistochemistry. Non-neoplastic ovarian surface epithelium and serous cystadenomas were used as controls. All epithelia expressed MUC1 protein. Of primary tumours, 76% expressed the differentiation-dependent glycoform and 84% the cancer-associated glycoform (Tn/Sialyl-Tn-epitopes). In metastatic lesions this was 77% and 85%, respectively. Notably, in 57% of ovarian endometriosis and 75% of intraepithelial lesions, the cancer-associated MUC1 epitopes were expressed, whereas normal ovarian surface epithelium and serous cystadenomas did not express these epitopes. Conclusions: The underglycosylated MUC1 epitopes are expressed by all histotypes of primary ovarian adenocarcinomas, by the vast majority of metastatic lesions and by possible ovarian cancer precursor lesions, but not by normal ovarian tissue. These results indicate that MUC1-associated Tn/STn-epitopes are important targets for immunotherapy and diagnostic imaging in ovarian cancer patients. [ABSTRACT FROM AUTHOR]

Alloreaction is known to accumulate several theoretical advantages that can improve dendritic cell (DC)-based anti-infective or antitumour strategies. Allogeneic DC have already been tested in experimental and clinical studies, but their efficacy compared with their autologous counterparts was rarely investigated and conclusions diverge. This study compared antigen-specific T cell responses following priming with autologous versus allogeneic DC and examined the possibility of screening these responses in order to select allogeneic DC that lead to a great amplification. Allogeneic DC obtained from donors matched with the single HLA-A2 allele were efficient in generating in vitro peptide-specific T cell responses. When randomly chosen, allogeneic DC generated a broad range of antigen-specific T cell responses in comparison with autologous DC. When screened and selected, allogeneic DC markedly enhanced peptide-specific T cell priming and allowed a more efficient boosting of resulting T cells. These selected allogeneic DC provided a favourable cytokinic and cellular environment that can help concurrent antigen-specific responses. Ex vivo selected allogeneic DC provide adjuvant effects that lead to amplification of concomitant antigen-specific T cell responses. [ABSTRACT FROM AUTHOR]

Abstract: There is an important need to find relevant biomarkers that show high sensitivity and specificity for early diagnosis and prognosis of cancer. An immune response to cancer is elicited in humans, as demonstrated in part by the identification of autoantibodies against a number of tumor-associated antigens in sera from patients with different types of cancer. Identification of tumor-associated antigens and their cognate autoantibodies is a promising strategy for the discovery of relevant biomarkers. During the past few years, proteomic approaches, including SEREX, SERPA and, more recently, protein microarrays, have been the dominant strategies used to identify tumor-associated antigens and their cognate autoantibodies. In this review, we aim to describe advantages, drawbacks, and recent improvements of these approaches for the study of humoral responses. [Copyright &y& Elsevier]

T-cell-based antigen-specific immunotherapy targeting tumour-associated antigens offers the potential for cancer immunotherapy. However, the majority of identified tumour-associated antigens are also expressed at low levels in normal tissues and mechanisms of tolerance induction are likely to affect the quality of T-cell responses to such antigens. In this study a T-cell receptor transgenic model was developed to determine the magnitude of T-cell tolerance to the tumour-associated antigen murine double minute-2 (MDM2), a widely expressed protein that is found at elevated levels in many tumours. The analysis of transgenic mice showed that thymic deletion was responsible for purging large numbers of MDM2-specific T cells from the repertoire. However, some T cells with specificity for MDM2 were able to escape thymic deletion and persisted in the peripheral T-cell pool. Functional analysis revealed that these T cells displayed defects in antigen-driven expansion. This functional impairment of the MDM2-specific T cells was maintained following adoptive transfer of the T cells into hosts that were unable to present the T-cell-receptor-recognized antigen. This study demonstrates that thymic deletion and the functional impairment of T cells present in the periphery both operate to establish T-cell tolerance to the tumour-associated antigen MDM2. Furthermore, the tolerant phenotype was stable and did not require continuous MDM2 peptide presentation in normal tissues. [ABSTRACT FROM AUTHOR]

Prognosis for patients suffering from malignant glioma has not substantially improved. Specific immunotherapy as a novel treatment concept critically depends on target antigens, which are highly overexpressed in the majority of gliomas, but the number of such antigens is still very limited. SOX2 was identified by screening an expression database for transcripts that are overexpressed in malignant glioma, but display minimal expression in normal tissues. Expression of SOX2 mRNA was further investigated in tumour and normal tissues by real-time PCR. Compared to cDNA from pooled normal brain, SOX2 was overexpressed in almost all (9 out of 10) malignant glioma samples, whereas expression in other, non-malignant tissues was almost negligible. SOX2 protein expression in glioma cell lines and tumour tissues was verified by Western blot and immunofluorescence. Immunohistochemistry demonstrated SOX2 protein expression in all malignant glioma tissues investigated ranging from 6 to 66% stained tumour cells. Human leucocyte antigen-A*0201-restricted SOX2-derived peptides were tested for the activation of glioma-reactive CD8+ cytotoxic T lymphocytes (CTLs). Specific CTLs were raised against the peptide TLMKKDKYTL and were capable of lysing glioma cells. The abundant and glioma-restricted overexpression of SOX2 and the generation of SOX2-specific and tumour-reactive CTLs may recommend this antigen as target for T-cell-based immunotherapy of glioma.British Journal of Cancer (2007) 96, 1293–1301. doi:10.1038/sj.bjc.6603696 www.bjcancer.com Published online 20 March 2007 [ABSTRACT FROM AUTHOR]

In a phase II study, 43 renal cell carcinoma patients were treated with individualised doses of ABR-214936; a fusion of a Fab recognising the antigen 5T4, and Staphylococcal enterotoxin A. Drug was given intravenously on 4 consecutive days, treatment was repeated 1 month later. Treatment was associated with moderate fever and nausea, but well tolerated. Of 40 evaluable patients, 28 had disease control at 2 months, and at 4 months, one patient showed partial response (PR) and 16 patients stable disease. Median survival, with minimum follow-up of 26 months was 19.7 months with 13 patients alive to date. Stratification by the Motzer's prognostic criteria highlights prolonged survival compared to published expectation. Patients receiving higher drug exposure had greater disease control and lived almost twice as long as expected, whereas the low-exposure patients survived as expected. Sustained interleukin-2 (IL-2) production after a repeated injection appears to be a biomarker for clinical effect, as the induced-IL-2 level on the day 2 of treatment correlated with survival. The high degree of disease control and the prolonged survival suggest that this treatment can be effective. These findings will be used in the trial design for the next generation of drug, with reduced antigenicity and toxicity.British Journal of Cancer (2007) 96, 567–574. doi:10.1038/sj.bjc.6603567 www.bjcancer.com Published online 6 February 2007 [ABSTRACT FROM AUTHOR]

Abstract: Epitopes found on Mucin-1 are differentially expressed on tumour versus normal tissue. Such epitopes have also been shown to have a potential in immunotherapy and tumour detection. The major epitope explored in this context is located within the variable number of tandem repeats. It has however recently been demonstrated that this epitope exists in several sequence variants. The standard sequence is highly antigenic while the most common sequence variant is much less so. We have now explored routes employing defined synthetic antigens to ensure the development of human recombinant antibody specificities targeting both sequence variants of this epitope. These antibodies may serve as a platform for the development of human antibodies for efficient targeting of Mucin-1 in human disease. [Copyright &y& Elsevier]

5T4 is a surface glycoprotein expressed on placental trophoblasts and also on a wide range of human carcinomas. Its highly restricted expression on normal tissues and broad distribution on many carcinomas make 5T4 a promising target for cancer immunotherapy. In the current study, we set out to investigate whether a 5T4-specific cytotoxic T lymphocyte (CTL) repertoire exists in healthy individuals. CD4-depleted peripheral blood mononuclear cells (PBMCs) from blood donors were screened using an ex vivo interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay. A panel of overlapping peptides, spanning the full length of the 5T4 protein, was used as a source of antigen. In the process of screening, one out of 30 blood donors demonstrated a positive ex vivo IFN-γ ELISPOT response to a single 5T4 peptide. A polyclonal T-cell line was derived from this donor by culturing PBMCs with autologous peptide-pulsed dendritic cells (DCs). The resulting polyclonal T-cell line and clones were tested in a 51Cr-release assay and by ELISPOT and were shown to be peptide specific. Furthermore, antigen-presenting cells (APCs), infected with a viral vector expressing 5T4, were able to stimulate IFN-γ production by the peptide-specific T-cell clones. A minimal CD8 epitope, PLADLSPFA, has been identified and found to be restricted through human leucocyte antigen (HLA) Cw7. Subsequently, we have demonstrated that HLA-Cw7-positive colorectal cancer patients vaccinated with a recombinant vaccinia viral vector encoding 5T4 (TroVax) are capable of mounting a strong IFN-γ ELISPOT response to this novel CTL epitope. These findings have potential application in cancer immunotherapy in terms of subunit vaccine design and the monitoring of immune responses induced in patients by 5T4-based therapies. [ABSTRACT FROM AUTHOR]

Immunoscreening of an Ewing’s family of tumour (EFT)-derived cDNA library using formerly described EFT-specific antibodies led to the isolation of a 3.5 kb cDNA, named Ewing’s tumour-associated antigen 16 ( ETAA16). The ETAA16 cDNA shows no homology to any functionally characterised human gene. Only a bovine cDNA expressed in bovine testis and hepatocytes is functionally characterised as it encodes for a junction plaque associated protein and showed a homology of 69.9% at amino acid level to ETAA16. The human cDNA encodes for a 926 amino acid tumour antigen with a calculated molecular weight of 103 kDa. The epitope of the ETAA16-specific antibody, Ak16, covers the central region of the protein which is part of an extra cellular domain. The human ETAA16 gene locus has been assigned to chromosome 2p13-15 by FISH analyses and is confirmed by the human genome sequencing project. As demonstrated by flow cytometry, the cell surface expression of ETAA16 antigen is restricted to ET cell lines and not expressed on other small blue round cell tumours or other kind of tumour. RT-PCR analysis revealed a high expression of ETAA16 in brain, liver and kidney while lung and heart were negative. Immunohistochemistry showed an intracellular expression of ETAA16 in different kind of non-Ewing tumour tissues. These results suggest that ETAA16 may function as a tumour-specific cell surface antigen in EFTs. [ABSTRACT FROM AUTHOR]

Reports on the adjuvant therapy of renal cell carcinoma in patient selection and therapeutic options. Renal cell carcinoma, survival and prognostic factors; Postoperative surveillance protocol for patients with localized and locally advanced renal cell carcinoma; Risk group assessment and clinical outcome algorithm to predict the natural history of patients with surgically resected renal cell carcinoma; Correlations with tumor oxygen measurements and prognostic in locally advanced carcinoma of the cervix.

In the last few years, serological identification of tumour-associated antigens (TAAs) by recombinant cDNA expression cloning (SEREX) has enabled the mapping of humoral immune responses against TAAs in various types of cancer. The present paper describes the application of SEREX to Burkitt’s lymphoma (BL), a malignancy not previously characterized by SEREX. By using a cDNA library from a BL cell line that does not express IgG, technical difficulties related to background immunoglobulin clones were overcome. Screening with sera from three BL patients revealed immunoreactivity against seven different gene products, six of which represent known human genes. Five proteins had previously been characterized by SEREX in other malignancies or identified as targets of autoantibodies in autoimmune disease. Seroreactivity against ATF-2, a member of the AP-1 transcription factor family, was validated by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using recombinant ATF-2 protein. Autoantibody responses against ATF-2 were detected by ELISA in 6 of 8 BL patients, compared with 6 of 13 patients with T-cell non-Hodgkin’s lymphoma (T-NHL), 5 of 23 patients with follicular lymphoma and 2 of 27 diffuse large B-cell lymphoma patients. In contrast, reactivity was found in only 1 of 50 healthy volunteers. Next, we showed by immunohistochemistry that the activated form of ATF2 (ATF-2pp) was highly expressed in six different BL samples. We conclude that the SEREX approach with a B-cell cDNA source is applicable in NHL. Furthermore, we identified genes with possible involvement in the pathogenesis of BL using this technique. [ABSTRACT FROM AUTHOR]

Comparative evaluation of the prognostic value of MUC1, MUC2, sialyl-Lewisa and sialyl-Lewisx antigens in colorectal adenocarcinoma Aims: The significance of MUC1, MUC2 and sialylated Lewis blood group antigens as prognostic markers in colorectal adenocarcinoma was investigated in a large series of patients because previous investigations revealed inconsistent results due to unrelated tumour samples from different patient groups and methodological differences. Methods and results: Tissues from 243 patients with colorectal adenocarcinoma were stained immunohistochemically. MUC1 showed a strong immunoreactivity (in more than 35% of the tumour area) in 32.5%, MUC2 in 51.0%, sialyl-Lewisx in 67.9% and sialyl-Lewisa in 73.7% of the cases, respectively. MUC1 immunoreactivity displayed a significant correlation with tumour progression as reflected by advancing pTNM staging and poor differentiation. MUC2 expression was significantly stronger in mucinous adenocarcinomas. Sialyl-Lewisx immunostaining correlated with the extent of lymph node metastasis as well as low cytological differentiation. According to univariate and multivariate analysis (P < 0.0001) only MUC1 reactivity represented a marker of worse survival probability, opposed to the sialylated Lewis antigens that did not exert a predictive value. Conclusions: According to our data, MUC1 and sialyl-Lewisx immunoreactivity exhibit statistically significant correlations with established markers of tumour progression. However, only MUC1 presents as an independent prognostic factor of colorectal adenocarcinoma. [ABSTRACT FROM AUTHOR]

Introduction: Prostate cancer (PCa) is the third most prevalent malignancy in Malaysian men. Approximately half of these cases were detected late. While prostate-specific antigen (PSA) tests and digital rectal examinations are the gold standard for PCa screening, they cannot distinguish PCa from benign-prostatic hyperplasia (BPH). To this end, autoantibodies against tumour-associated antigens are emerging as promising biomarkers in the detection of cancers. This study aims to investigate serum autoantibody signatures as PCa specific biomarkers. Methods: A high-density protein microarray containing over 1600 immune-related human proteins to detect autoantibodies circulating in the sera of 30 patients with PCa and 30 controls with BPH was used. Putative markers were identified and ranked according to differential penetrance fold- change and high-frequency percentage between the two groups. Functional analysis for the top-ranked proteins was done using the feature selection method, unsupervised clustering approach, and functional annotation based on KEGG and gene ontology (GO). Results: A total of 27 positive interactions were detected in PCa patients’ sera, but none in controls’ sera. The protein-tyrosine kinase (PTK7), previously reported to be associated with increased PSA and Gleason scores, was expressed in most of the cases in this cohort. The protein function prediction analysis, which used machine learning algorithms, revealed a panel of 5 proteins, including PTK7, to be highly differentially expressed in PCa cases, with a ROC curve AUC of >.70. The current findings corroborate an earlier report on PTK7 overexpression as an independent predictor of PCa. Functional analysis of the top biomarkers revealed clusters related to RAS signalling, PI3k-Akt signalling pathway, and the MAPK signalling pathway. Conclusion: The preliminary findings of this study suggest that identifying autoantibodies could help distinguish PCa cases from BPH using a minimally invasive method, highlighting its potential role as a diagnostic and therapeutic target. [ABSTRACT FROM AUTHOR]

The tumour-associated antigen (TAA) GA733-2 is expressed as a non-secreted surface molecule on the majority of human colorectal carcinoma cells. The antigen has been used as a target for passive and active immunotherapy during the last decade. To determine the incidence of autoantibodies against this antigen, sera from 1068 patients with colorectal carcinoma were analysed for naturally occurring IgG antibodies against the baculovirus-produced GA733-2E protein. A total of 14.5% of the patients had IgG antibodies against the antigen. In 519 patients, sera were collected at the time of diagnosis and 15% of those patients had anti-GA733-2E IgG antibodies. There was a tendency to a higher frequency of patients with antibodies among those in the advanced Dukes stages: 11% in stage A and 32% in stage D respectively ( P = 0.06). Antibodies could be detected for up to 10 years after the diagnosis. Patients with Crohn's disease or colitis ulcerosa ( n = 20) did not elicit anti-GA733-2E antibodies. No healthy control donor ( n = 45) had detectable antibodies against the antigen. The specificity of GA733-2E-reactive serum IgG was indicated by significant inhibition of mAb17-1A (originally used to define GA733-2) binding to the GA733-2E antigen. Sera of positive patients bound to the GA733-2-expressing human colorectal carcinoma cell line, SW948. No significant correlation was found between the presence of antibodies and survival in the present patient population. However, the high incidence of autoantibodies against this tumour antigen in colorectal carcinoma patients confirms its antigenicity in humans and supports the use of the GA733-2 antigen as a target for immunotherapy. [ABSTRACT FROM AUTHOR]

Novel tumour-associated epitopes showing elevated levels in sera from patients with colon carcinoma were studied by means of monoclonal antibodies with respect to co-expression with the tumour-associated epitope CA50 on the cancer antigen (CanAg) glyco-conjugate complex. Co-expression of the different epitopes and CA50 was found on CanAg both from COLO 205 spent medium and sera from patients with colorectal cancer. In a few sera from patients with non-mucinous ovarian tumours, the CanAg complex was also found to express the CA125 epitope. The chemical characterisation showed that all of the novel epitopes on CanAg were of carbohydrate nature, and the results may suggest a structural relationship between several of them. Copyright © 1991 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We have reported the production of human monoclonal antibodies (mAb), by the fusion of lymph node lymphocytes from a primary carcinoma patient with murine myeloma cells. Seven heterohybridomas showed reactivity with class III antigens, and five hybridomas (1G12, 2D4, 4H5, 5D10 and 3B10) were reactive with class II antigens. One of these human mAbs (1G12) was intensively studied and results are presented here. 1G12 reacted strongly and specifically with five mammary carcinoma cell lines and showed no cross-reactivity with seven normal fibroblast cells. It continuously produced human mAbs (IgM) at a rate of 4.5-12.5 μg/ml over a period of 2 years. Human mAb 1G12 (IgM) was purified by either a combination of anion-exchange chromatography (ABx) and gel filtration (Superose 6) or affinity chromatography (agarose). Immunohistological analysis of frozen tissue sections was performed with biotinylated 1G12. All mammary carcinomas analysed ( n= 26) were positive, while the connective tissue of 36 different patients was completely negative with 1G12. In normal breast, endometrium and intestine only a weak or moderate staining of the epithelial cells was observed. Normal oesophagus, small bowel, cervix, uterus, lung and skin were completely negative. Partly purified tumour antigen recognized by 1G12 had a molecular mass of 1-2 MDa and showed strong binding with Ulex europaeus lectin I and Bauhina purpurea agglutinin, indicative for the glucoprotein nature of antigens. These results show that human mAb 1G12 may be useful for the analysis of tumour-associated antigen of mammary carcinoma patients. In further studies the therapeutic and diagnostic application of 1G12 should be analysed in more detail. [ABSTRACT FROM AUTHOR]

The humoral immune response against a tumour-associated antigen, polymorphic epithelial mucin (PEM, MUC1) in cancer patients was studied by isolating specific B cells primed for the antigen. Human B lymphocytes from tumour-draining lymph nodes, obtained from 12 patients with epithelial cancers, were immunoselected with magnetic beads coated with a 60mer synthetic peptide corresponding to three tandem repeats of the protein core of the MUC1 antigen. Short-term cultures of B cells were established utilizing interleukin-10 (IL-10), IL-4 and monoclonal antibody anti-CD40, and were maintained for a maximum of 3␣weeks. B cell culture supernatants contained human anti-MUC1 antibodies, as detected by enzyme-linked immunosorbent assay, in 6/12 of the patients tested. Five of these patients, all with early-stage cancer, also had high levels of circulating anti-MUC1 IgM antibodies in the serum. A significant correlation was found (two-tailed P = 0.041) between the presence of circulating anti-MUC1 antibodies and the ability to isolate PEM-specific B cells from tumour-draining lymph nodes. The technique proposed provides a useful method for the analysis of natural immunity against defined tumour antigens. [ABSTRACT FROM AUTHOR]

A monoclonal antibody 7A9 was raised against the tumour-associated glycoprotein TAG-12 purified from T47-D breast carcinoma cells. In immunoblots from cytosol of T47-D cells and from sera of breast cancer patients, antibody 7A9 detects the high molecular weight mucin-like TAG-12 antigen. A series of paraffin sections of normal, benign and malignant mammary tissues have been studied with monoclonal antibody 7A9 and the immunoalkaline phosphatase method. In resting gland, proliferating gland and fibroadenoma ducts, reactivity of 7A9 was mainly restricted to luminal membranes of epithelial cells and secretions. 77/79 primary breast carcinomas including ductal, lobular and various other carcinoma types showed cytoplasmic and/or membrane-associated staining with 7A9 in most tumour cells. Metastases (31/31) from different sites were also positive. Strong immunoreactivity with single tumour cells was noted in cytological preparations from freshly resected breast cancer tissue. Thus, monoclonal antibody 7A9 seems to be very useful for the targeting of breast carcinoma cells. [ABSTRACT FROM AUTHOR]

The number of identified tumour-associated antigens for cutaneous lymphoma is still very restricted, which limits the elucidation of the tumour immunology of these malignancies and the development of specific immunotherapies and immunodiagnostics. To identify new serologically defined antigens associated with cutaneous lymphoma. A phage expression library of the human testis transcriptom was established and immunoscreened with sera from 100 patients with cutaneous lymphoma and nine with parapsoriasis, and 81 age-matched control donors. Positive expression clones were sequenced to identify the respective antigen. The testis-specific protein 10 (TSGA10) was identified as an antigen recognized by sera of two patients with Mycosis fungoides but not by sera from healthy donors. By reverse transcription–polymerase chain reaction analysis, TSGA10 was found expressed in all cutaneous lymphoma samples tested, various tumour cell lines, testis, peripheral blood mononuclear cells, skin, isolated lymphocytes, keratinocytes and fibroblasts. TSGA10 overexpression had previously been reported for other cancers. TSGA10 is a new tumour-associated antigen of cutaneous lymphoma. [ABSTRACT FROM AUTHOR]