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1017. Selective Induction of Tumor-Associated Antigens in Murine Pulmonary Vasculature Using Double-Targeted Adenoviral Vectors
CANCER-TARGETED GENE THERAPY: TARGETING OF ADENOVIRAL VECTORS administration of recombinant human endostatin protein (rhEndo) for 9 days, the ratio of T/C (rhEndo group versus PBS group) was less than 47%. However, two days after rhEndo treatment ceased, the ratio of T/C was more than 50%. The peak of expression of endostatin mRNA in tumor tissue was at 2 or 3 days after administration intratumorally with Ad/hEndo and gradually dropped undetectable by day 7. Dynamic analysis of endostatin concentration in tumor tissue showed that the highest level of mRNA is up at third day after injection, and dropped to basal level three weeks later. Conclusion Endostatin gene therapy mediated by a recombinant adenoviral vector had significantly inhibited the growth of hepatocellular carcinoma BEL-7402 xenografted tumors at a high dose of 1 × 109 pfu compared with other groups. The analysis of dynamic expression of endostatin in vivo indicated that Ad/hEndo had acquired a high-level, relatively long-term expression in vivo and bioactivity capability.
1016. Retargeted Selectively Replicating Adenoviruses in Combination with Antiangiogenic Therapy for the Therapy of Colon Cancer Krisztian Homicsko,1 Alexander Lukashev,1 Richard Iggo.1 Department of Oncogenes, ISREC, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland.
1
In virtually all colon cancer cell lines β-catenin is activated. The target genes of β-catenin are activated through the interaction of βcatenin with the transcription factor TCF. Previously in our laboratory conditionally replicating adenoviruses with TCF sites in the early viral promoters were constructed. We inserted TCF sites into the E1A, E1B and E4 promoters. This virus, vKH1, shows 1000 fold selectivity for colon cancer cell lines compared to wild type Ad5. In vivo after i.v. injection of vKH1, the virus can reach the s.c SW620 colon cancer xenograft. It replicates and gradually spreads within the tumour as shown by in situ hybridization and Q-PCR. However the virus alone can only delay the growth of the SW620 xenograft with 2 weeks but due to the uninfected regions that overgrow the infected regions the tumour can not be treated. Combination with conventional chemotherapeutics are of potential interest though almost all of them interfere with the viral replication. Growing evidence supports that antiangiogenic drugs are effective and promising antitumour agents. These drugs interfere less with the viral life cycle. RAD001 is a rapamycin derivative and it inhibits mTOR a protein kinase in the PI3K pathway. RAD001 inhibits cell growth, has strong antiangiogenic and immunosuppressive effect. RAD001 in vitro does not effect CPE. Viral gene expression and viral burst size were also not changed by the drug. In vivo vKH1 and RAD001 has an additive effect in delaying the tumour growth however the tumour growth is still not completely inhibited. One of the major reasons of the poor viral effect is the initial infection rate of the tumour cells and the viral spread within the tumour. The native adenoviral receptor CAR is poorly expressed or downregulated in tumours and in tumour associated vessels. By inserting an RGD motif in the adenoviral fibre protein we targeted adenovirus to αvβ3 and αvβ5 integring receptors that are highly expressed both on tumour cells and on the neoangiogenic vasculature. This virus ,vKH6, shows 10 to 100 fold more CPE than the parental vKH1 virus while the selectivity is mentained. In vivo vKH6 produces more foci and spread faster than vKH1. VKH6 alone and also in combination with RAD001 has more potent antitumour effect than vKH1 alone or in combination.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
1017. Selective Induction of Tumor-Associated Antigens in Murine Pulmonary Vasculature Using Double-Targeted Adenoviral Vectors Maaike Everts,1 SangAe Kim-Park,1 Meredith A. Preuss,1 Michael J. Passineau,1 Joel N. Glasgow,1 Alexander V. Pereboev,1 Parameshwar J. Mahasreshti,1,2 William E. Grizzle,3 Paul N. Reynolds,4 David T. Curiel.1 1 Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL; 2Department of Obstetrics and Gyneacology, University of Alabama at Birmingham, Birmingham, AL; 3Department of Pathology, University of Alabama at Birmingham, Birmingham, AL; 4Department of Thoracic Medicine, Royal Adelaide Hospital, Adelaide, Australia. Targeted therapies directed to tumor-associated antigens are being investigated for the treatment of cancer. However, there are few suitable animal models for testing the ability to target these tumor markers. Therefore, we have exploited mice transgenic for the human coxsackie and adenovirus receptor (hCAR) to establish a new model for transient expression of human tumor-associated antigens in the pulmonary vasculature. Systemic administration of Ad in hCAR mice resulted in an increase in transgene expression in the lungs compared to wild type mice, as determined using a luciferase reporter gene. To reduce transgene expression in the liver, the predominant organ of ectopic Ad localization and transgene expression following systemic administration, we utilized the endothelial-specific flt-1 promoter, which resulted in a further increased lung-to-liver ratio of luciferase expression. Administration of an adenoviral vector encoding the tumor-associated antigen carcinoembryonic antigen (CEA) under transcriptional control of the flt-1 promoter resulted in selective expression of this antigen in the pulmonary vasculature of hCAR mice. Feasibility of targeting to expressed CEA was subsequently demonstrated using adenoviral vectors pre-incubated with a bifunctional adapter molecule recognizing this tumor-associated antigen, thus demonstrating utility of this transient transgenic animal model.
1018. Infectivity Enhancement for Ovarian Cancer and Glioma Via a Novel Human Adenoviral Vector with a Chimeric Fiber Derived from Canine Adenovirus Type 1 Mariam A. Stoff-Khalili,1,2 Angel A. Rivera,1 Joel N. Glasgow,1 Long P. Le,1 Alexander Stoff,1,3 Maaike Everts,1 Yuko Tsuruta,1 Yusoke Kawakami,1 J. Michael Mathis,4 Larisa Pereboeva,1 Peter Dall,2 David T. Curiel.1 1 Division of Human Gene Therapy, UAB, Birmingham, AL; 2 Department of Obstetrics and Gynecology, University of Duesseldorf, Duesseldorf, NRW, Germany; 3Department of Plastic and Reconstructive Surgery, Dreifaltigkeitskrankenhaus, Koeln, NRW, Germany; 4Department of Cellular Biology and Anatomy, Lousiana State University Health Sciences Center, Shreveport, LA. The exploitation of novel therapy strategies merits a high priority in the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on “xenotype” knob switching with nonhuman adenovirus have been exploited. Herein, we present the S393
1016. Retargeted Selectively Replicating Adenoviruses in Combination with Antiangiogenic Therapy for the Therapy of Colon Cancer Krisztian Homicsko,1 Alexander Lukashev,1 Richard Iggo.1 Department of Oncogenes, ISREC, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland.
1
In virtually all colon cancer cell lines β-catenin is activated. The target genes of β-catenin are activated through the interaction of βcatenin with the transcription factor TCF. Previously in our laboratory conditionally replicating adenoviruses with TCF sites in the early viral promoters were constructed. We inserted TCF sites into the E1A, E1B and E4 promoters. This virus, vKH1, shows 1000 fold selectivity for colon cancer cell lines compared to wild type Ad5. In vivo after i.v. injection of vKH1, the virus can reach the s.c SW620 colon cancer xenograft. It replicates and gradually spreads within the tumour as shown by in situ hybridization and Q-PCR. However the virus alone can only delay the growth of the SW620 xenograft with 2 weeks but due to the uninfected regions that overgrow the infected regions the tumour can not be treated. Combination with conventional chemotherapeutics are of potential interest though almost all of them interfere with the viral replication. Growing evidence supports that antiangiogenic drugs are effective and promising antitumour agents. These drugs interfere less with the viral life cycle. RAD001 is a rapamycin derivative and it inhibits mTOR a protein kinase in the PI3K pathway. RAD001 inhibits cell growth, has strong antiangiogenic and immunosuppressive effect. RAD001 in vitro does not effect CPE. Viral gene expression and viral burst size were also not changed by the drug. In vivo vKH1 and RAD001 has an additive effect in delaying the tumour growth however the tumour growth is still not completely inhibited. One of the major reasons of the poor viral effect is the initial infection rate of the tumour cells and the viral spread within the tumour. The native adenoviral receptor CAR is poorly expressed or downregulated in tumours and in tumour associated vessels. By inserting an RGD motif in the adenoviral fibre protein we targeted adenovirus to αvβ3 and αvβ5 integring receptors that are highly expressed both on tumour cells and on the neoangiogenic vasculature. This virus ,vKH6, shows 10 to 100 fold more CPE than the parental vKH1 virus while the selectivity is mentained. In vivo vKH6 produces more foci and spread faster than vKH1. VKH6 alone and also in combination with RAD001 has more potent antitumour effect than vKH1 alone or in combination.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
1017. Selective Induction of Tumor-Associated Antigens in Murine Pulmonary Vasculature Using Double-Targeted Adenoviral Vectors Maaike Everts,1 SangAe Kim-Park,1 Meredith A. Preuss,1 Michael J. Passineau,1 Joel N. Glasgow,1 Alexander V. Pereboev,1 Parameshwar J. Mahasreshti,1,2 William E. Grizzle,3 Paul N. Reynolds,4 David T. Curiel.1 1 Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL; 2Department of Obstetrics and Gyneacology, University of Alabama at Birmingham, Birmingham, AL; 3Department of Pathology, University of Alabama at Birmingham, Birmingham, AL; 4Department of Thoracic Medicine, Royal Adelaide Hospital, Adelaide, Australia. Targeted therapies directed to tumor-associated antigens are being investigated for the treatment of cancer. However, there are few suitable animal models for testing the ability to target these tumor markers. Therefore, we have exploited mice transgenic for the human coxsackie and adenovirus receptor (hCAR) to establish a new model for transient expression of human tumor-associated antigens in the pulmonary vasculature. Systemic administration of Ad in hCAR mice resulted in an increase in transgene expression in the lungs compared to wild type mice, as determined using a luciferase reporter gene. To reduce transgene expression in the liver, the predominant organ of ectopic Ad localization and transgene expression following systemic administration, we utilized the endothelial-specific flt-1 promoter, which resulted in a further increased lung-to-liver ratio of luciferase expression. Administration of an adenoviral vector encoding the tumor-associated antigen carcinoembryonic antigen (CEA) under transcriptional control of the flt-1 promoter resulted in selective expression of this antigen in the pulmonary vasculature of hCAR mice. Feasibility of targeting to expressed CEA was subsequently demonstrated using adenoviral vectors pre-incubated with a bifunctional adapter molecule recognizing this tumor-associated antigen, thus demonstrating utility of this transient transgenic animal model.
1018. Infectivity Enhancement for Ovarian Cancer and Glioma Via a Novel Human Adenoviral Vector with a Chimeric Fiber Derived from Canine Adenovirus Type 1 Mariam A. Stoff-Khalili,1,2 Angel A. Rivera,1 Joel N. Glasgow,1 Long P. Le,1 Alexander Stoff,1,3 Maaike Everts,1 Yuko Tsuruta,1 Yusoke Kawakami,1 J. Michael Mathis,4 Larisa Pereboeva,1 Peter Dall,2 David T. Curiel.1 1 Division of Human Gene Therapy, UAB, Birmingham, AL; 2 Department of Obstetrics and Gynecology, University of Duesseldorf, Duesseldorf, NRW, Germany; 3Department of Plastic and Reconstructive Surgery, Dreifaltigkeitskrankenhaus, Koeln, NRW, Germany; 4Department of Cellular Biology and Anatomy, Lousiana State University Health Sciences Center, Shreveport, LA. The exploitation of novel therapy strategies merits a high priority in the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on “xenotype” knob switching with nonhuman adenovirus have been exploited. Herein, we present the S393