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315. Therapeutic Interleukin-10 Gene Expression in Mammary Tumors Modulates Multiple Malignant Phenotypes and Reduces Metastatic Burden
CANCER IMMUNOTHERAPY particles containing human IFN-γ cDNA insert. Nine patients (7 CTCL, 2 CBCL) were enrolled in 3 successive cohorts at the following TG1042 doses: 3x10E9 total particles (tp), 3x10E10 tp and 3x10E11 tp. Patients received intratumoral injections of TG1042 into the designated lesions on days 1, 8, and 15 with no injection in the fourth week (1 treatment cycle) and thereafter up to 4 cycles, if there was no evidence of progressive disease (PD). The injected lesion was biopsied at baseline and after 3 injections and assessed for changes in lesion’s morphology, immunohistochemical changes of T and B-cell (CD3, 4, 8, 20, 21, 79a), NK (CD56), cytotoxic (TIA-1), and dendritic cell markers (CD1a) as well as for the expression of coxsackie-adenovirus receptor (CAR) and HLA class I molecules. In addition, the lesions were evaluated for the expression of transgene-derived IFN-γ as well as for total cytokine production (IFN-γ, IL-2, IL-13, IL-10) with respect to CD4 and CD8 T cells population by quantitative PCR. Clinical responses and stabilizations were observed in the majority of the patients. Injection site reaction was the most commonly observed adverse event. Following TG1042 injections, histology demonstrated pronounced changes in infiltrate pattern differing from initial lymphoma finding, with signs of vasculitis and increase in eosinophil and neutrophil numbers. Immunohistochemistry revealed increase in CD8 and TIA-1 immunoreactivity in various cell types. All lesions demonstrated clear up-regulation of CAR following injection of TG1042. Quantitative PCR showed decrease in CD4/CD8 ratio in 6/8 patients. Transgene-derived IFN-γ mRNA could be detected in injected lesions, implying successful IFN-γ gene transfer. Our results reveal for the first time the in situ immunological changes following the administration of adenoviral vector expressing IFN-γ in primary CTCL and CBCL, offering new insight and information of importance to the design of new gene delivery-based therapeutics in cancer. Principal investigator and consultant
314. Evaluation of Cytokine Gene Transfer Mediated by a Novel Cationic Liposome Preparation in an Orthotopic Bladder Cancer Model Qinghui Wu,1 Ratha Mahendran,1 Kesavan Esuvaranathan.1 1 Surgery, National University of Singapore, Singapore. Purpose: To assess cytokine gene expression by tumor cells in vitro and in vivo in an orthotopic mouse bladder cancer model after liposome-mediated gene transfer. To evaluate the treatment effect of cytokines on the growth of orthotopic tumor. Materials and Methods: The murine bladder cancer cell lines MB49 was maintained in RPMI 1640. 1 X 105 cells was instilled into the bladder of C57BL/6 mice after electric cautery to establish the tumor model. The plasmid vectors were constructed by inserting the coding sequences of IFN-α1 and GM-CSF into plasmid vector pBudCE4.1. Transient transfection was performed using a cationic lipid DOTAP and methyl-b-cyclodextrin solubilized cholesterol (MBC). The expression level of IFN-α1 and GM-CSF in culture medium was checked by ELISA .The effects of cytokine gene transfer on tumor cell proliferation and some surface marker expression such as MHC-I and II were evaluated. The presence of the tumors was confirmed by histological examination and MRI scan of the mouse bladders. The expression of the transgene in situ was confirmed by immunohistochemistry and x-gal staining. Four groups of animals with orthotopic bladder tumor were treated with control vector, GM-CSF, interferon-alpha or both cytokines respectively twice a week by intravesical instillation of the gene tranfer mixture. Results: Superficial bladder tumors were consistently established by intravesical instillation of MB49 cells. The tumors were detectable by MRI scan. The expression levels of both cytokines in transfected cell lines were increased significantly. The inhibition of in vitro tumor cells proliferation and up-regulation of some surface markers S124
were observed. In situ gene transfer to bladder tumors was accomplished via intravesical instillation of plasmid DNA/DOTAP/ MBC beta-galactosidase (b-gal) after a single 2h in situ transfection. Using immunohistochemistry, it could be clearly seen that cytokine was produced by the urothelial cells. The survival of animals in treatment group was increased as compare to control group, some tumors were even cured. Conclusion: The orthotopic bladder cancer model is very useful tool to study the feasibility of potential cytokine gene therapy. We demonstrated here that the orthotopic mouse bladder cancer can be transfected with a single instillation of liposome-DNA complexes. The results also suggest that our liposome-mediated cytokine transfection system appears to be a potential promising non-viral gene treatment modality to bladder cancer in vivo.
315. Therapeutic Interleukin-10 Gene Expression in Mammary Tumors Modulates Multiple Malignant Phenotypes and Reduces Metastatic Burden Van Tsai,1 Anja Muller,2 Albert Zlotnick,2 Brian Helmich,1 Iqbal Ahmed,1 Robert Ralston,1 Daniel Maneval,1 Drake LaFace.1 1 Canji, Inc., a Division of Schering-Plough, San Diego, CA; 2 DNAX Research Institute, Palo Alto, CA. We report here the use of recombinant adenoviral (rAd) vectors to induce expression of human IL-10 in established tumors to examine the capacity for suppressing tumor growth and spontaneous metastasis in a murine mammary carcinoma model. Prior reports of the effect of IL-10 expression on the metastatic phenotype have relied on transfection or pre-transduction prior to tumor implantation. Thus IL-10 expression had preceded the tissue responses required for establishing a solid tumor and promoting metastasis. Treatment of established tumors with rAd-empty control vector resulted in modest tumor growth inhibition, whereas, tumor regression was observed in the rAd/IL-10 treated mice. In Addition, spontaneous metastasis to axillary lymph nodes and lungs was markedly inhibited in rAd/IL-10 treated mice. Immunohistochemical analysis revealed markedly reduced CD31 expression in rAd/IL-10 treated tumors but not in rAd/empty vector control treated tumors. Moreover, rIL10 inhibited endothelial cell migration induced by activated macrophages. These results support the hypothesis that IL-10 can inhibit tumor metastasis by suppressing macrophage-mediated induction of angiogenesis. Additional mechanisms for inhibition of tumor growth and metastasis associated with expression of IL-10 were elucidated using a TaqMan RT-PCR based molecular profiling method. We performed an analysis of differential expression profiles of 121 genes for interdependent expression profiles associated with IL-10 expression that correlated with the capacity to suppress metastasis. Treatment with rAd/empty control vector induced very few changes in gene expression, whereas, rAd/IL-10 modulated expression of numerous genes that regulate tissue remodeling, angiogenesis and immune responses. Importantly, IL-10 suppressed gene expression of several key metalloproteinases and pro-metastatic cytokines that have been demonstrated to promote tumor progression and metastasis in breast carcinomas. The capacity of rAd/IL-10 to modulate expression of multiple mediators of this complex multistep progression may circumvent the conundrum of redundant molecular mechanisms promoting malignant phenotypes.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
314. Evaluation of Cytokine Gene Transfer Mediated by a Novel Cationic Liposome Preparation in an Orthotopic Bladder Cancer Model Qinghui Wu,1 Ratha Mahendran,1 Kesavan Esuvaranathan.1 1 Surgery, National University of Singapore, Singapore. Purpose: To assess cytokine gene expression by tumor cells in vitro and in vivo in an orthotopic mouse bladder cancer model after liposome-mediated gene transfer. To evaluate the treatment effect of cytokines on the growth of orthotopic tumor. Materials and Methods: The murine bladder cancer cell lines MB49 was maintained in RPMI 1640. 1 X 105 cells was instilled into the bladder of C57BL/6 mice after electric cautery to establish the tumor model. The plasmid vectors were constructed by inserting the coding sequences of IFN-α1 and GM-CSF into plasmid vector pBudCE4.1. Transient transfection was performed using a cationic lipid DOTAP and methyl-b-cyclodextrin solubilized cholesterol (MBC). The expression level of IFN-α1 and GM-CSF in culture medium was checked by ELISA .The effects of cytokine gene transfer on tumor cell proliferation and some surface marker expression such as MHC-I and II were evaluated. The presence of the tumors was confirmed by histological examination and MRI scan of the mouse bladders. The expression of the transgene in situ was confirmed by immunohistochemistry and x-gal staining. Four groups of animals with orthotopic bladder tumor were treated with control vector, GM-CSF, interferon-alpha or both cytokines respectively twice a week by intravesical instillation of the gene tranfer mixture. Results: Superficial bladder tumors were consistently established by intravesical instillation of MB49 cells. The tumors were detectable by MRI scan. The expression levels of both cytokines in transfected cell lines were increased significantly. The inhibition of in vitro tumor cells proliferation and up-regulation of some surface markers S124
were observed. In situ gene transfer to bladder tumors was accomplished via intravesical instillation of plasmid DNA/DOTAP/ MBC beta-galactosidase (b-gal) after a single 2h in situ transfection. Using immunohistochemistry, it could be clearly seen that cytokine was produced by the urothelial cells. The survival of animals in treatment group was increased as compare to control group, some tumors were even cured. Conclusion: The orthotopic bladder cancer model is very useful tool to study the feasibility of potential cytokine gene therapy. We demonstrated here that the orthotopic mouse bladder cancer can be transfected with a single instillation of liposome-DNA complexes. The results also suggest that our liposome-mediated cytokine transfection system appears to be a potential promising non-viral gene treatment modality to bladder cancer in vivo.
315. Therapeutic Interleukin-10 Gene Expression in Mammary Tumors Modulates Multiple Malignant Phenotypes and Reduces Metastatic Burden Van Tsai,1 Anja Muller,2 Albert Zlotnick,2 Brian Helmich,1 Iqbal Ahmed,1 Robert Ralston,1 Daniel Maneval,1 Drake LaFace.1 1 Canji, Inc., a Division of Schering-Plough, San Diego, CA; 2 DNAX Research Institute, Palo Alto, CA. We report here the use of recombinant adenoviral (rAd) vectors to induce expression of human IL-10 in established tumors to examine the capacity for suppressing tumor growth and spontaneous metastasis in a murine mammary carcinoma model. Prior reports of the effect of IL-10 expression on the metastatic phenotype have relied on transfection or pre-transduction prior to tumor implantation. Thus IL-10 expression had preceded the tissue responses required for establishing a solid tumor and promoting metastasis. Treatment of established tumors with rAd-empty control vector resulted in modest tumor growth inhibition, whereas, tumor regression was observed in the rAd/IL-10 treated mice. In Addition, spontaneous metastasis to axillary lymph nodes and lungs was markedly inhibited in rAd/IL-10 treated mice. Immunohistochemical analysis revealed markedly reduced CD31 expression in rAd/IL-10 treated tumors but not in rAd/empty vector control treated tumors. Moreover, rIL10 inhibited endothelial cell migration induced by activated macrophages. These results support the hypothesis that IL-10 can inhibit tumor metastasis by suppressing macrophage-mediated induction of angiogenesis. Additional mechanisms for inhibition of tumor growth and metastasis associated with expression of IL-10 were elucidated using a TaqMan RT-PCR based molecular profiling method. We performed an analysis of differential expression profiles of 121 genes for interdependent expression profiles associated with IL-10 expression that correlated with the capacity to suppress metastasis. Treatment with rAd/empty control vector induced very few changes in gene expression, whereas, rAd/IL-10 modulated expression of numerous genes that regulate tissue remodeling, angiogenesis and immune responses. Importantly, IL-10 suppressed gene expression of several key metalloproteinases and pro-metastatic cytokines that have been demonstrated to promote tumor progression and metastasis in breast carcinomas. The capacity of rAd/IL-10 to modulate expression of multiple mediators of this complex multistep progression may circumvent the conundrum of redundant molecular mechanisms promoting malignant phenotypes.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy