Keywords:Cancer testis antigens Published by Elsevier B.V. All rights reserved. IntroductionThe search for tumor-associated antigens (TAA) capable to induce a tumor-directed immune response and the development of cancer vaccines targeting these TAA have been a major effort for the tumor immunology community in the past decades.

Endoglin/CD105 is well acknowledged as being the most reliable marker of proliferation of endothelial cells, and it is overexpressed on tumour neovasculature. Our current knowledge of its structure, physiological role, and tissue distribution suggests that targeting of endoglin/CD105 is a novel and powerful diagnostic and therapeutic strategy in human malignancies, through the imaging of tumour-associated angiogenesis and the inhibition of endothelial cell functions related to tumour angiogenesis. Among biotherapeutic agents, monoclonal antibodies have shown a major impact on the clinical course of human malignancies of different histotypes. Along this line, the potential efficacy of anti-endoglin/CD105 antibodies and their derivatives for clinical purposes in cancer is supported by a large body of available pre-clinical in vitro and in vivo data. In this review, the main findings supporting the translation of antibody-based endoglin/CD105 targeting from pre-clinical studies to clinical applications in human cancer are summarized and discussed.

In the last three decades huge efforts have been made to characterize genetic defects responsible for cancer development and progression, leading to the comprehensive identification of distinct cellular pathways affected by the alteration of specific genes. Despite the undoubtable role of genetic mechanisms in triggering neoplastic cell transformation, epigenetic modifications (i.e., heritable changes of gene expression that do not derive from alterations of the nucleotide sequence of DNA) are rapidly emerging as frequent alterations that often occur in the early phases of tumorigenesis and that play an important role in tumor development and progression. Epigenetic alterations, such as modifications in DNA methylation patterns and post-translational modifications of histone tails, behave extremely different from genetic modifications, being readily revertable by ''epigenetic drugs'' such as inhibitors of DNA methyl transferases and inhibitors of histone deacetylases. Since epigenetic alterations in cancer cells affect virtually all cellular pathways that have been associated to tumorigenesis, it is not surprising that epigenetic drugs display pleiotropic activities, being able to concomitantly restore the defective expression of genes involved in cell cycle control, apoptosis, cell signaling, tumor cell invasion and metastasis, angiogenesis and immune recognition. Prompted by this emerging clinical relevance of epigenetic drugs, this review will focus on the large amount of available data, deriving both from in vitro experimentations and in vivo pre-clinical and clinical studies, which clearly indicate epigenetic drugs as effective modifiers of cancer phenotype and as positive regulators of tumor cell biology with a relevant therapeutic potential in cancer patients.

Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide. Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition. In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions. In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients. On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects.

Purpose:To investigate the potential of the DNA hypomethylating agent 5-aza-2 ¶-deoxycytidine (5-aza-CdR) to improve the effectiveness of immunotherapeutic approaches against melanocyte differentiation antigens. Experimental Design: The effect of 5-aza-CdR on the constitutive expression of gp100 was investigated in 11human melanoma cell lines by real-time reverse transcription-PCR and indirect immunofluorescence (IIF) analyses. 5-aza-CdR^mediated changes in the levels of expression of human leukocyte antigen (HLA) class I antigens and HLA-A2 allospecificity, intercellular adhesion molecule-1 (ICAM-1), and leukocyte-function^associated antigen-3 were investigated by IIF analysis on melanoma cells under study. The recognition of gp100-positive Mel 275 melanoma cells, treated or not with 5-aza-CdR, by HLA-A2^restricted gp100 (209^217) -specific CTL was investigated by 51 Cr-release assays, IFN-g release and IFN-g ELISPOT assays.Results: The constitutive expression of gp100 was not affected by 5-aza-CdR on all melanoma cells investigated. Compared with untreated cells, the exposure of Mel 275 melanoma cells to 5-aza-CdR significantly (P < 0.05) up-regulated their expression of HLA class I antigens and of ICAM-1. These phenotypic changes significantly (P < 0.05) increased the lysis of 5-aza-CdRt reated Mel 275 melanoma cells by gp100-specific CTL and increased their IFN-g release. 5-aza-CdR treatment of Mel 275 cells also induced a higher number of gp100-specific CTL to secrete IFN-g. Conclusions: Treatment with 5-aza-CdR improves the recognition of melanoma cells by gp100-specific CTL through the up-regulation of HLA class I antigens expression; ICAM-1 also contributes to this phenomenon. These findings highlight a broader range of therapeutic implications of 5-aza-CdR when used in association with active or adoptive immunotherapeutic approaches against a variety of melanoma-associated antigens.Human leukocyte antigen (HLA) class I antigens play a crucial role in antitumor cellular immune responses (1, 2), and levels of expression of both HLA class I antigens and tumor-associated antigen (TAA) contribute to determine the extent of TAA-specific CTL recognition of neoplastic cells (3, 4). Thus, the identification of new strategies potentially able to up-regulate the expression of HLA class I antigens and/or of therapeutic TAA on neoplastic cells may improve the clinical effectiveness of T cell -based immunotherapeutic approaches in cancer patients.Along this line, we have shown that the DNA hypomethylating agent 5-aza-2 ¶-deoxycytidine (5-aza-CdR, Dacogen) up-regulates the constitutive cell surface expression of HLA class I antigens and of HLA class I allospecificities and concomitantly induces or up-regulates that of different cancer testis antigens (CTA) in cultured human melanomas (5). Furthermore, 5-aza-CdR administration has also proven to induce a persistent up-regulation of HLA class I antigens, along with a long-lasting induction and up-regulation of CTA expression, in human melanomas grafted into ...

The multifaceted immunomodulatory activity of DNA hypomethylating agents improves immunogenicity and immune recognition of neoplastic cells; thus, we predicted they could be utilized to design new immunotherapeutic combinations in cancer. Testing this hypothesis, the antitumor efficacy of the DNA hypomethylating agent 5-aza-2 0 -deoxycytidine (5-AZA-CdR) combined with the anti-CTLA-4 monoclonal antibody (mAb) 9H10 in syngeneic transplantable murine models was investigated. Murine mammary carcinoma TS/A or mesothelioma AB1 cells were injected in BALB/c, athymic nude, and SCID/Beige mice that were treated with 5-AZA-CdR, mAb 9H10, or their combination. Tumor volumes were captured at different time-points; molecular and immunohistochemical assays investigated changes in neoplastic and normal tissues. A significant antitumor effect of 5-AZA-CdR combined with mAb 9H10 was found: compared to controls, a 77% (p < 0.01), 54% (p < 0.01) and 33% (p D 0.2) decrease in TS/A tumor growth was induced by 5-AZA-CdR combined with mAb 9H10, 5-AZA-CdR or mAb 9H10, respectively. These antitumor activities were confirmed utilizing the AB1 model. 5-AZA-CdR-based regimens induced a promoter-demethylationsustained tumor expression of cancer testis antigens. MHC class I expression was up-regulated by 5-AZA-CdR. Antitumor efficacy of 5-AZA-CdR in athymic nude and SCID/Beige mice was not increased by mAb 9H10. In BALB/c mice, combined treatment induced the highest tumor infiltration by CD3 C lymphocytes, which included both CD8 C and CD4 C T cells; no such infiltrates were observed in normal tissues. This significant immune-related antitumor activity of 5-AZA-CdR combined with CTLA-4 blockade, demonstrated in highly aggressive mouse tumor models, provides a strong scientific rationale to implement epigenetically-based immunotherapies in cancer patients.

Our findings show that SGI-110 is a highly attractive therapeutic agent to comprehensively increase immunogenicity and immune recognition of neoplastic cells, and provide the scientific rationale for its clinical development to design novel chemo-immunotherapeutic approaches in cancer patients.

Quantitative evaluation of T cell responses of patients receiving antitumoral vaccination with a protein is difficult because of the large number of possible HLA-peptide combinations that could be targeted by the response. To evaluate the responses of patients vaccinated with protein MAGE-3, we have developed an approach that involves overnight stimulation of blood T cells with autologous dendritic cells loaded with the protein, sorting by flow cytometry of the T cells that produce IFN-γ, cloning of these cells, and evaluation of the number of T cell clones that secrete IFN-γ upon stimulation with the Ag. An important criterion is that T cell clones must recognize not only stimulator cells loaded with the protein, but also stimulator cells transduced with the MAGE-3 gene, so as to exclude the T cells that recognize contaminants generated by the protein production system. Using this approach it is possible to measure T cell frequencies as low as 10−6. We analyzed the frequencies of anti-vaccine CD4 T cells in five metastatic melanoma patients who had been injected with a MAGE-3 protein without adjuvant and showed evidence of tumor regression. Anti-MAGE-3 CD4 T cells were detected in one of the five patients. The frequency of the anti-MAGE-3 CD4 T cells was estimated at 1/60,000 of the CD4 T cells in postvaccination blood samples, representing at least an 80-fold increase in the frequency found before immunization. The frequencies of one anti-MAGE-3 CD4 T cell clonotype were confirmed by PCR analysis on blood lymphocytes. The 13 anti-MAGE-3 clones, which corresponded to five different TCR clonotypes, recognized the same peptide presented by HLA-DR1.