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311. Oncolytic Adenovirus CG5757 Preferentially Infects Primary Human Tumor Tissues and Shows Strong Anti-Tumor Activity in Tumor Models Following Systemic Administration
CANCER-TARGETED GENE THERAPY: TARGETING OF NON-VIRAL VECTORS SRESPHP, with a 3000-fold increase in titer between round one and two. The selected phage showed a highly specific binding to the tumor after systemic administration, whereas binding to other organs such as lung, liver, kidney, and heart was reduced up to 90%. After tail vein injection, homing to the tumor was substantially reduced (to approximately 70%) in the presence of synthetic SRESPHP peptide, indicating that tumor phage interaction strictly depends on the displayed peptide. Immunohistochemical analysis of paraffin sections from mice tissues revealed the strongest staining within MTC cells and at the tumor invasion front, suggesting that the SRESPHP-peptide promotes phage internalization in vivo. Thus, the identified peptide is potentially useful to mediate selective gene and/or vector transfer to medullary thyroid tumors.
311. Oncolytic Adenovirus CG5757 Preferentially Infects Primary Human Tumor Tissues and Shows Strong Anti-Tumor Activity in Tumor Models Following Systemic Administration Yuanhao Li,1 Trini Arroyo,1 Stephen Thorne,2 Tony Reid,2 Natalie Nguyen,1 Neeraja Idamakanti,1 Melinda VanRoey,1 Gail Colbern,1 De-Chao Yu.1 1 Virotherapy, Cell Genesys, South San Francisco, CA; 2Stanford University, Stanford, CA. A human adenovirus serotype 5 based oncolytic virus CG5757 was designed to preferentially replicate in and kill cancer cells that have a defective retinoblastoma (Rb) pathway and overexpress telomerase. Tumor selectivity of CG5757 was examined using a newly developed ex vivo primary tumor tissue culture model. Primary human colorectal tumors were obtained after surgical resection and placed in Millicell insert with media containing hormones, which can keep tissue viable for about 5 days. The paired tumor or normal tissues from same tissue sample were used in a viral infection experiment to determine the tumor selectivity of CG5757. Cultured tissues were infected with CG5757 or wild type (wt) Ad5 for five days, and progeny viral production was determined by TCID50 assay. Results of the paired tissue cultures from five colon cancer patients indicate that wt Ad5 has similar viral productivity in tumor and normal tissues, whereas CG5757 has higher productivity in tumor tissues. In general, CG5757 produces 100- to 1,000-fold more virus in tumor tissue compared to normal tissue. Immunohistochemical staining for adenoviral E1A shows that CG5757 has positive infection only in the cancer tissue (colon), whereas no signal is detected in normal tissues of colon, pancreas or spleen. Similarly in vitro viral specificity characterization using a panel of tumor cell lines also suggest a strong tumor selectivity of CG5757. Anti-tumor efficacy of CG5757 following intravenous injection was examined in a subcutaneous tumor model. In this study, pre-established prostate cancer LNCaP xenografts in nude mice were treated with two intravenous injections of 4x1010 viral particles of CG5757 given at a 3-day interval. Significant anti-tumor efficacy was observed in CG5757-treated animals. Eight weeks after treatment, CG5757-treated animals exhibited approximately 72% inhibition of tumor growth. These studies demonstrate the potential therapeutic efficacy of such dual promoter-controlled oncolytic adenoviruses in cancers that are Rb-defective and telomerasepositive.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
312. Human Plasminogen Kringle 5Engineered Murine Mammary Cancer Cells Arrest Tumor Growth and Promote Long-Term Survival Sabrina R. Perri,1 Moïra François,2 Jacques Galipeau.1,2,3 1 Division of Experimental Medicine, Lady Davis Institute, McGill University, Montreal, QC, Canada; 2Lady Davis Institute, Montreal, QC, Canada; 3Hematology-Oncology, Lady Davis Institute, Jewish General Hospital, Montreal, QC, Canada. OBJECTIVE: Neoangiogenesis has been strongly correlated with aggressive breast tumor growth and metastasis. However, since tumor-associated neovasculature is not of malignant origin, it retains the ability to respond physiologically to growth arrest signals and is the proposed basis for breast tumor regression in vivo. The fifth kringle (K5) domain of human plasminogen is distinct from angiostatin (K1-4), and has been shown -on its own- to act as a potent suppressor of angiogenesis. We propose that the K5 domain may serve as a potent angiostatic agent and that it may act as a useful therapeutic transgene within a breast cancer gene therapy strategy. To test this hypothesis, we have developed a K5-expressing retroviral vector, gene-modified murine DA3 mammary cells to produce soluble human K5 protein and characterized the anti-tumor potency of the de novo produced K5 peptide in vivo. METHODS & RESULTS: The 381bp K5 domain cDNA Pro447–Asp546 of human plasminogen (InvivoGen, San Diego, CA) was His-tagged at the C terminus and cloned into a bicistronic retroviral vector comprising the enhanced green fluorescent protein reporter (GFP) gene. Upon transfection of the K5 retrovector plasmid into 293GPG retroviral packaging cells, single clones were drug selected and characterized. Stable K5-expressing 293GPG cells were utilized to transduce murine DA3 mammary cancer cells. Genemodified polyclonal DA3-K5-GFP cells were GFP positive as assessed by flow cytometry analysis and capable of secreting soluble K5 protein as detected by anti-His immunoblot analysis. Upon subcutaneous implantation of one million DA3-K5-GFP cells in immunocompetent BALB/c mice, tumor growth was strongly suppressed as early as 7 days post-implantation (P = 0.01 by t test) as compared to DA3 control implanted mice and the anti-tumor effect remained sustained for over 3 months. Three months postimplantation 100% of DA3-K5-GFP implanted mice are tumorfree (possessing tumors ≤15mm3) as compared to 20% in the control group (P = 0.0002 by log-rank). As a result, the DA3-K5-GFP implanted mice possessed a clear survival advantage with 100% of mice surviving over 3 months as compared to 20% in the control cohort (P = 0.0003 by log-rank). CONCLUSION: We provide compelling evidence that soluble K5 protein expressed by retrovirally-engineered mammary cancer cells possesses potent anti-tumor properties which induces longterm tumor regression in immunocompetent mice. These findings demonstrate for the first time that soluble K5 protein holds promise in breast cancer gene therapy and further supports its potency as an anti-angiogenic agent. RELEVANCE: This study focuses on the development of an innovative gene therapy for breast cancer and exploits the use of a potent endogenous soluble protein to inhibit blood vessel formation in vivo in a clinically relevant animal model. Findings will ultimately provide further understanding of the biology of breast cancer angiogenesis.
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311. Oncolytic Adenovirus CG5757 Preferentially Infects Primary Human Tumor Tissues and Shows Strong Anti-Tumor Activity in Tumor Models Following Systemic Administration Yuanhao Li,1 Trini Arroyo,1 Stephen Thorne,2 Tony Reid,2 Natalie Nguyen,1 Neeraja Idamakanti,1 Melinda VanRoey,1 Gail Colbern,1 De-Chao Yu.1 1 Virotherapy, Cell Genesys, South San Francisco, CA; 2Stanford University, Stanford, CA. A human adenovirus serotype 5 based oncolytic virus CG5757 was designed to preferentially replicate in and kill cancer cells that have a defective retinoblastoma (Rb) pathway and overexpress telomerase. Tumor selectivity of CG5757 was examined using a newly developed ex vivo primary tumor tissue culture model. Primary human colorectal tumors were obtained after surgical resection and placed in Millicell insert with media containing hormones, which can keep tissue viable for about 5 days. The paired tumor or normal tissues from same tissue sample were used in a viral infection experiment to determine the tumor selectivity of CG5757. Cultured tissues were infected with CG5757 or wild type (wt) Ad5 for five days, and progeny viral production was determined by TCID50 assay. Results of the paired tissue cultures from five colon cancer patients indicate that wt Ad5 has similar viral productivity in tumor and normal tissues, whereas CG5757 has higher productivity in tumor tissues. In general, CG5757 produces 100- to 1,000-fold more virus in tumor tissue compared to normal tissue. Immunohistochemical staining for adenoviral E1A shows that CG5757 has positive infection only in the cancer tissue (colon), whereas no signal is detected in normal tissues of colon, pancreas or spleen. Similarly in vitro viral specificity characterization using a panel of tumor cell lines also suggest a strong tumor selectivity of CG5757. Anti-tumor efficacy of CG5757 following intravenous injection was examined in a subcutaneous tumor model. In this study, pre-established prostate cancer LNCaP xenografts in nude mice were treated with two intravenous injections of 4x1010 viral particles of CG5757 given at a 3-day interval. Significant anti-tumor efficacy was observed in CG5757-treated animals. Eight weeks after treatment, CG5757-treated animals exhibited approximately 72% inhibition of tumor growth. These studies demonstrate the potential therapeutic efficacy of such dual promoter-controlled oncolytic adenoviruses in cancers that are Rb-defective and telomerasepositive.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
312. Human Plasminogen Kringle 5Engineered Murine Mammary Cancer Cells Arrest Tumor Growth and Promote Long-Term Survival Sabrina R. Perri,1 Moïra François,2 Jacques Galipeau.1,2,3 1 Division of Experimental Medicine, Lady Davis Institute, McGill University, Montreal, QC, Canada; 2Lady Davis Institute, Montreal, QC, Canada; 3Hematology-Oncology, Lady Davis Institute, Jewish General Hospital, Montreal, QC, Canada. OBJECTIVE: Neoangiogenesis has been strongly correlated with aggressive breast tumor growth and metastasis. However, since tumor-associated neovasculature is not of malignant origin, it retains the ability to respond physiologically to growth arrest signals and is the proposed basis for breast tumor regression in vivo. The fifth kringle (K5) domain of human plasminogen is distinct from angiostatin (K1-4), and has been shown -on its own- to act as a potent suppressor of angiogenesis. We propose that the K5 domain may serve as a potent angiostatic agent and that it may act as a useful therapeutic transgene within a breast cancer gene therapy strategy. To test this hypothesis, we have developed a K5-expressing retroviral vector, gene-modified murine DA3 mammary cells to produce soluble human K5 protein and characterized the anti-tumor potency of the de novo produced K5 peptide in vivo. METHODS & RESULTS: The 381bp K5 domain cDNA Pro447–Asp546 of human plasminogen (InvivoGen, San Diego, CA) was His-tagged at the C terminus and cloned into a bicistronic retroviral vector comprising the enhanced green fluorescent protein reporter (GFP) gene. Upon transfection of the K5 retrovector plasmid into 293GPG retroviral packaging cells, single clones were drug selected and characterized. Stable K5-expressing 293GPG cells were utilized to transduce murine DA3 mammary cancer cells. Genemodified polyclonal DA3-K5-GFP cells were GFP positive as assessed by flow cytometry analysis and capable of secreting soluble K5 protein as detected by anti-His immunoblot analysis. Upon subcutaneous implantation of one million DA3-K5-GFP cells in immunocompetent BALB/c mice, tumor growth was strongly suppressed as early as 7 days post-implantation (P = 0.01 by t test) as compared to DA3 control implanted mice and the anti-tumor effect remained sustained for over 3 months. Three months postimplantation 100% of DA3-K5-GFP implanted mice are tumorfree (possessing tumors ≤15mm3) as compared to 20% in the control group (P = 0.0002 by log-rank). As a result, the DA3-K5-GFP implanted mice possessed a clear survival advantage with 100% of mice surviving over 3 months as compared to 20% in the control cohort (P = 0.0003 by log-rank). CONCLUSION: We provide compelling evidence that soluble K5 protein expressed by retrovirally-engineered mammary cancer cells possesses potent anti-tumor properties which induces longterm tumor regression in immunocompetent mice. These findings demonstrate for the first time that soluble K5 protein holds promise in breast cancer gene therapy and further supports its potency as an anti-angiogenic agent. RELEVANCE: This study focuses on the development of an innovative gene therapy for breast cancer and exploits the use of a potent endogenous soluble protein to inhibit blood vessel formation in vivo in a clinically relevant animal model. Findings will ultimately provide further understanding of the biology of breast cancer angiogenesis.
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