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770. Selective Elimination of Alloreactive GVHD-Mediating Donor T Cells by Ex Vivo sFasL Treatment
CANCER-TARGETED GENE THERAPY: PRECLINICAL STUDIES II Functions of targeted genes were assigned using a novel standardized task.We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity when compared to randomly generated data. Similar data was obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells might allow a prediction also for the in vivo situation, where target cells are scarce.
770. Selective Elimination of Alloreactive GVHD-Mediating Donor T Cells by Ex Vivo sFasL Treatment Osnat Bohana-Kashtan,1 Hyam I. Levitsky,1 Curt I. Civin.1 1 Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD. Graft-versus-host disease (GVHD), the major cause of morbidity and mortality after allogeneic blood and marrow transplantation, is mediated mainly by donor T cells. However, when all or most T cells are removed from donor allografts in an attempt to prevent GVHD, problems with graft failure, reduced immune reconstitution, and tumor relapse have emerged. Our goal was to develop a clinically applicable strategy to selectively deplete from the donor allograft the anti-recipient T cells that mediate GVHD, without ablating the remainder of the T cells that mediate immune responses against tumor antigens and infectious agents. Previous work done in our lab, using mouse models, has shown that recombinant human soluble Fas ligand (sFasL) can efficiently delete alloreactive donor T cells during their activation in response to irradiated recipient cells in a mixed lymphocyte reaction (MLR). This ex vivo sFasL treatment abrogated GVHD while sparing donor T cells with anti-tumor reactivity (Georgantas et al, submitted). The present work was performed in a human model system, in order to extend our work toward clinical application. In our human model system, peripheral blood mononuclear cells (PBMCs: 1st party; responder cells) from a given human donor were activated in vitro in response to irradiated PBMCs from a second donor (2nd party; stimulator cells), in the presence (or absence) of sFasL. Analyses of day 1-7 MLR cultures showed that recipient alloantigen-activated responder (donor) T cells began to up-regulate Fas expression on day 2 and that indirect (non-specific) activation of the 2nd party stimulator cells was detectable by day 5. Based on these findings, the sFasL treatment was limited to 2-3 days. Secondary MLRs, performed on cells harvested after the 2-3-day sFasL treatment during the first MLR, showed a marked depletion of anti-2nd party alloreactivity, but preservation of competence to different non-alloreactive antigens, such as cytomegalovirus (CMV), and 3rd party stimulator cells. In addition, sFasL treatment efficiently deleted or prevented the development of CD25+CFSEhigh, CD25+CFSElow, and CD25-CFSElow subsets of activated responder cells.
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CANCER-TARGETED GENE THERAPY: PRECLINICAL STUDIES II 771. Human mda-7/Interleukin 24 (IL-24) Protein Kills Breast Cancer Cells Via the IL-20 Receptor and Is Antagonized by IL-10 Minghong Zheng,1 Dora Bocangel,1 Abner Mhashilkar,1 Rajagopal Ramesh,2 Kelly K. Hunt,3 Suhendan Ekmekcioglu,4 Nancy Poindexter,4 Elizabeth A. Grimm,4 Sunil Chada.1 1 Research and Development, Introgen Therapeutics Inc, Houston, TX; 2Thoracic and Cardiovascular Surgery, MD Anderson Cancer Center, Houston, TX; 3Surgical Oncology, MD Anderson Cancer Center, Houston, TX; 4Experimental Therapeutics, MD Anderson Cancer Center, Houston, TX. The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted human IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of breast cancer cells results in G2/M phase cell-cycle arrest and killing, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 killing. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Exogenous IL-24 killed breast cancer cells by induction of apoptosis and this effect was abolished by addition of anti-IL-24 antibody or anti-IL20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Furthermore, IL-10 treatment inhibits killing mediated by IL-24. In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptormediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity. Sunil Chada, Minghong Zheng, and Dora Bocangel are Introgen employees.
772. Inhibition of Ovarian Tumor Growth Following Treatment with an Oncolytic Vaccinia Virus Xiang Da (Eric) Dong,1 Mark E. O’Malley,1 Sri Chalikonda1,1 Zongsheng Guo,1 Herbert J. Zeh,1 David L. Bartlett.1 1 Division of Surgical Oncology, University of Pittsburgh, Pittsburgh, PA. Background: Recombinant vaccinia virus is currently being explored as a potential replicating oncolytic virus for cancer virotherapy due to its exceptional ability to replicate in tumor cells. Vaccinia tropism is not limited to tumor tissues, but also other tissues including ovarian follicles, possibly due to the presence of angiogenic factors such as vascular endothelial growth factor (VEGF). We therefore investigated the utility of vaccinia virus as an oncolytic therapy in a murine model of ovarian cancer. Methods: A murine ovarian surface epithelial cell (MOSEC) line was employed for this model. Intraperitoneal inoculation of the MOSEC cell line leads to a highly malignant neoplasm containing both carcinomatous and sarcomatous components as well as production of hemorrhagic ascitic fluid. These mice were treated with a thymidine kinase (TK) and vaccinia growth factor (VGF) double-deleted virus with the cytokine deaminase (CD) gene inserted Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
770. Selective Elimination of Alloreactive GVHD-Mediating Donor T Cells by Ex Vivo sFasL Treatment Osnat Bohana-Kashtan,1 Hyam I. Levitsky,1 Curt I. Civin.1 1 Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD. Graft-versus-host disease (GVHD), the major cause of morbidity and mortality after allogeneic blood and marrow transplantation, is mediated mainly by donor T cells. However, when all or most T cells are removed from donor allografts in an attempt to prevent GVHD, problems with graft failure, reduced immune reconstitution, and tumor relapse have emerged. Our goal was to develop a clinically applicable strategy to selectively deplete from the donor allograft the anti-recipient T cells that mediate GVHD, without ablating the remainder of the T cells that mediate immune responses against tumor antigens and infectious agents. Previous work done in our lab, using mouse models, has shown that recombinant human soluble Fas ligand (sFasL) can efficiently delete alloreactive donor T cells during their activation in response to irradiated recipient cells in a mixed lymphocyte reaction (MLR). This ex vivo sFasL treatment abrogated GVHD while sparing donor T cells with anti-tumor reactivity (Georgantas et al, submitted). The present work was performed in a human model system, in order to extend our work toward clinical application. In our human model system, peripheral blood mononuclear cells (PBMCs: 1st party; responder cells) from a given human donor were activated in vitro in response to irradiated PBMCs from a second donor (2nd party; stimulator cells), in the presence (or absence) of sFasL. Analyses of day 1-7 MLR cultures showed that recipient alloantigen-activated responder (donor) T cells began to up-regulate Fas expression on day 2 and that indirect (non-specific) activation of the 2nd party stimulator cells was detectable by day 5. Based on these findings, the sFasL treatment was limited to 2-3 days. Secondary MLRs, performed on cells harvested after the 2-3-day sFasL treatment during the first MLR, showed a marked depletion of anti-2nd party alloreactivity, but preservation of competence to different non-alloreactive antigens, such as cytomegalovirus (CMV), and 3rd party stimulator cells. In addition, sFasL treatment efficiently deleted or prevented the development of CD25+CFSEhigh, CD25+CFSElow, and CD25-CFSElow subsets of activated responder cells.
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CANCER-TARGETED GENE THERAPY: PRECLINICAL STUDIES II 771. Human mda-7/Interleukin 24 (IL-24) Protein Kills Breast Cancer Cells Via the IL-20 Receptor and Is Antagonized by IL-10 Minghong Zheng,1 Dora Bocangel,1 Abner Mhashilkar,1 Rajagopal Ramesh,2 Kelly K. Hunt,3 Suhendan Ekmekcioglu,4 Nancy Poindexter,4 Elizabeth A. Grimm,4 Sunil Chada.1 1 Research and Development, Introgen Therapeutics Inc, Houston, TX; 2Thoracic and Cardiovascular Surgery, MD Anderson Cancer Center, Houston, TX; 3Surgical Oncology, MD Anderson Cancer Center, Houston, TX; 4Experimental Therapeutics, MD Anderson Cancer Center, Houston, TX. The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted human IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of breast cancer cells results in G2/M phase cell-cycle arrest and killing, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 killing. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Exogenous IL-24 killed breast cancer cells by induction of apoptosis and this effect was abolished by addition of anti-IL-24 antibody or anti-IL20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Furthermore, IL-10 treatment inhibits killing mediated by IL-24. In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptormediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity. Sunil Chada, Minghong Zheng, and Dora Bocangel are Introgen employees.
772. Inhibition of Ovarian Tumor Growth Following Treatment with an Oncolytic Vaccinia Virus Xiang Da (Eric) Dong,1 Mark E. O’Malley,1 Sri Chalikonda1,1 Zongsheng Guo,1 Herbert J. Zeh,1 David L. Bartlett.1 1 Division of Surgical Oncology, University of Pittsburgh, Pittsburgh, PA. Background: Recombinant vaccinia virus is currently being explored as a potential replicating oncolytic virus for cancer virotherapy due to its exceptional ability to replicate in tumor cells. Vaccinia tropism is not limited to tumor tissues, but also other tissues including ovarian follicles, possibly due to the presence of angiogenic factors such as vascular endothelial growth factor (VEGF). We therefore investigated the utility of vaccinia virus as an oncolytic therapy in a murine model of ovarian cancer. Methods: A murine ovarian surface epithelial cell (MOSEC) line was employed for this model. Intraperitoneal inoculation of the MOSEC cell line leads to a highly malignant neoplasm containing both carcinomatous and sarcomatous components as well as production of hemorrhagic ascitic fluid. These mice were treated with a thymidine kinase (TK) and vaccinia growth factor (VGF) double-deleted virus with the cytokine deaminase (CD) gene inserted Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy