Identification of tumor antigens is essential in advancing immune-based therapeutic interventions in cancer. Particularly attractive targets are those molecules that are selectively expressed by malignant cells and that are also essential for tumor progression. We have used a computer-based differential display analysis tool for mining of expressed sequence tag clusters in the human Unigene database and identified Brachyury as a novel tumor antigen. Brachyury, a member of the T-box transcription factor family, is a key player in mesoderm specification during embryonic development. Moreover, transcription factors that control mesoderm have been implicated in the epithelial-to-mesenchymal transition (EMT), which has been postulated to be a key step during tumor progression to metastasis. Reverse transcription-PCR analysis validated the in silico predictions and showed Brachyury expression in tumors of the small intestine, stomach, kidney, bladder, uterus, ovary, and testis, as well as in cell lines derived from lung, colon, and prostate carcinomas, but not in the vast majority of the normal tissues tested. An HLA-A0201epitope of human Brachyury was identified that was able to expand T lymphocytes from blood of cancer patients and normal donors with the ability to lyse Brachyury-expressing tumor cells. To our knowledge, this is the first demonstration that (a) a T-box transcription factor and (b) a molecule implicated in mesodermal development, i.e., EMT, can be a potential target for human T-cell-mediated cancer immunotherapy. Metastatic disease is responsible for the majority of human cancer deaths. Understanding the molecular mechanisms of metastasis is a major step in designing effective cancer therapeutics. We have shown that the T-box transcription factor Brachyury induces in tumor cells EMT, an important step in the progression of primary tumors toward metastasis. Overexpression of Brachyury in human carcinoma cells induced changes characteristic of EMT, including upregulation of mesenchymal markers, downregulation of epithelial markers, and an increase in cell migration and invasion. Conversely, inhibition of Brachyury resulted in downregulation of mesenchymal markers and loss of cell migration and invasion and diminished the ability of human tumor cells to form lung metastases in a xenograft model. Furthermore, we found Brachyury to be overexpressed in various human tumor tissues and tumor cell lines compared with normal tissues. The selective expression of Brachyury in tumor cells and its role in EMT and cancer progression suggest that Brachyury may be an attractive target for antitumor therapies. We have also evaluated Brachyury as a potential target for lung cancer therapy. Our results showed Brachyury protein expression in 41% of primary lung carcinomas, including 48% of adenocarcinomas and 25% of squamous cell carcinomas. With the exception of normal testis and some thyroid tissues, the majority of normal tissues evaluated in this study were negative for the expression of Brachyury protein. Brachyury-specific T cells could lyse Brachyury-positive tumors and the level of Brachyury corresponded to resistance of tumor cells to EGFR kinase inhibition. We hypothesize that the elimination of Brachyury-positive tumor cells may be able to prevent and/or diminish tumor dissemination and the establishment of metastases. The ability of Brachyury-specific T-cell lines to lyse Brachyury-positive tumor cells, in vitro, supports the development of Brachyury-based immunotherapeutic approaches for the treatment of lung cancer. Small molecule targeted therapeutics such as BCL-2 inhibitors are being examined as monotherapy in phase I/II clinical trials for several types of tumors. However, few data are available about the effect of BCL-2 inhibitors on immune function.
The aims of our study were to investigate the effect of a small molecule BCL-2 inhibitor on immune function and determine the most effective way of combining this inhibitor with a recombinant vaccine to treat tumors. The therapeutic efficacy of such sequential therapy was measured as a reduction of pulmonary tumor nodules. Activated mature CD8 T lymphocytes were more resistant to GX15-070 as compared to early-activated cells. Regulatory T cell (Treg) function was significantly decreased after treatment with the BCL-2 inhibitor. In vivo, GX15-070 was given after vaccination so as to not negatively impact the induction of vaccine-mediated immunity, resulting in increased intratumoral activated CD8:Treg ratio and significant reduction of pulmonary tumor nodules. Our study is the first to show the effect of a small molecule BCL-2 inhibitor on the immune system and following a vaccine. It is also the first to demonstrate the efficacy of this sequence in reducing tumors in mouse models, providing a rationale for the design of combinational clinical studies. We hypothesized that two vaccine platforms targeting the same antigen might induce shared and distinct antigen-specific T-cell populations, and examined the possibility that two distinct vaccines could be used concomitantly. Using recombinant poxvirus and yeast vaccines, we compared the T-cell populations induced by these two platforms in terms of serum cytokine response, T-cell gene expression, T-cell receptor phenotype, antigen-specific cytokine expression, T-cell avidity, and T-cell antigen-specific tumor cell lysis. These studies demonstrated for the first time that vaccination with a recombinant poxvirus platform (rV/F-CEA/TRICOM) or a heat-killed yeast vaccine platform (yeast-CEA) elicits T-cell populations with both shared and unique phenotypic and functional characteristics. Furthermore, both the antigen and the vector play a role in the induction of distinct T-cell populations. In this study, we demonstrated that concurrent administration of two vaccines targeting the same antigen induces a more diverse T-cell population that leads to enhanced antitumor efficacy. These studies provided the rationale for future clinical studies investigating concurrent administration of vaccine platforms targeting a single antigen to enhance the antigen-specific immune response. Inhibition of TGF-beta1 signaling promotes central memory T cell differentiation: This study affirmed that isolated CD8+ T cells express mRNA and produce TGF-beta following cognate peptide recognition. Blockage of endogenous TGF-beta with either a TGF-beta-blocking Ab or a small molecule inhibitor of TGF-betaRI enhances the generation of CD62Lhigh/CD44high central memory CD8+ T cells accompanied with a robust recall response. Interestingly, the augmentation within the central memory T cell pool occurs in lieu of cellular proliferation or activation, but with the expected increase in the ratio of the Eomesoderm/T-bet transcriptional factors. Yet, the signal transduction pathway(s) seems to be noncanonical, independent of SMAD or mammalian target of rapamycin signaling. Enhancement of central memory generation by TGF-beta blockade is also confirmed in human peripheral blood mononuclear cells. The findings underscore the role(s) that autocrine TGF-beta plays in T cell homeostasis and, in particular, the balance of effector/memory and central/memory T cells. These results may provide a rationale to targeting TGF-beta signaling to enhance antigen-specific CD8+ T cell memory against a lethal infection or cancer.
|Palena, Claudia; Roselli, Mario; Litzinger, Mary T et al. (2014) Overexpression of the EMT driver brachyury in breast carcinomas: association with poor prognosis. J Natl Cancer Inst 106:|
|Kim, Peter S; Jochems, Caroline; Grenga, Italia et al. (2014) Pan-Bcl-2 inhibitor, GX15-070 (obatoclax), decreases human T regulatory lymphocytes while preserving effector T lymphocytes: a rationale for its use in combination immunotherapy. J Immunol 192:2622-33|
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|Ardiani, Andressa; Gameiro, Sofia R; Palena, Claudia et al. (2014) Vaccine-mediated immunotherapy directed against a transcription factor driving the metastatic process. Cancer Res 74:1945-57|
|Schlom, Jeffrey; Jochems, Caroline; Gulley, James L et al. (2013) The role of soluble CD40L in immunosuppression. Oncoimmunology 2:e22546|
|Takai, Shinji; Schlom, Jeffrey; Tucker, Joanne et al. (2013) Inhibition of TGF-*1 signaling promotes central memory T cell differentiation. J Immunol 191:2299-307|
|Zaharoff, David A; Hoffman, Benjamin S; Hooper, H Brooks et al. (2009) Intravesical immunotherapy of superficial bladder cancer with chitosan/interleukin-12. Cancer Res 69:6192-9|
|Hance, Kenneth W; Rogers, Connie J; Zaharoff, David A et al. (2009) The antitumor and immunoadjuvant effects of IFN-alpha in combination with recombinant poxvirus vaccines. Clin Cancer Res 15:2387-96|
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