- Email: [email protected]
522. Tumor-Selective Suicide Gene Therapy by Transcriptionally Targeted Replicating Retrovirus Vectors
CANCER-ONCOLYTIC VIRUSES I 521. Armed and Targeted Measles Virus for Virotherapy of Chemoresistant Pancreatic Cancer
Christian Grossardt,1 Sascha Bossow,1 Achim Temme,2 Mathias F. Leber,1 Stefanie Sawall,1 Roberto Cattaneo,3 Christof von Kalle,1 Guy Ungerechts.1 1 Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany; 2Department of Neurosurgery, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany; 3Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, MN. Pancreatic cancer is one of the most drug-resistant types of malignancy, with extremely poor prognosis and survival rate. Over the past 10 years, gemcitabine has been the front line chemotherapy to treat non-resectable and metastasized pancreatic cancer, ultimately not preventing a progressive disease. Measles viruses (MV) derived from vaccine strain have shown considerable oncolytic activity against lymphomas and a wide variety of solid adenocarcinomas from different entities and are a promising tool to treat pancreatic cancer. Since prostate stem cell antigen (PSCA), originally identified as a marker for prostate cancer, is also expressed on pancreatic adenocarcinoma but not on non-neoplastic tissue, we generated a fully retargeted MV for the treatment of pancreatic adenocarcinoma. Therefore, we fused a single chain antibody (scFv) specific for the extracellular domain of PSCA as a C-terminal extension to the MV attachment protein H which was blinded for the natural MV receptors CD46/SLAM. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cancer cell lines. We were able to show that cell lines with high- and low-level PSCA expression were susceptible to PSCA-retargeted MV. Furthermore, we engineered a PSCA-targeted MV armed with the prodrug convertase purine nucleoside phosphorylase (PNP) – MV-PNPantiPSCA. PNP converts the prodrug fludarabine to the highly toxic and diffusible 2-fluoroadenine substantially enhancing the oncolytic efficacy of the virus on infected and bystander cells. Synergistic therapeutic effects were demonstrated in a subcutaneous BxPC-3 tumor xenograft model of pancreatic adenocarcinoma. The combined chemovirotherapy of intratumoral MV-PNP-antiPSCA application together with intraperitoneal administration of fludarabine yielded a potent synergistic oncolytic effect and resulted in a significant tumor regression compared to mice treated with virus or prodrug only. Importantly, with regard to future clinical trials no cross resistance of various gemcitabine-resistant pancreatic adenocarcinoma cell lines to MV mediated oncolysis and activated prodrug was detected.
522. Tumor-Selective Suicide Gene Therapy by Transcriptionally Targeted Replicating Retrovirus Vectors Yi-Hui Lai,1 Chien-Kuo Tai.1 Department of Life Science, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan.
1
Replicating virus vectors are attractive tools for anticancer gene therapy, but the potential for adverse events due to uncontrolled spread of the vectors has been a foremost concern of investigators in the field of retroviral gene therapy. To design a tumor-selective replicating retrovirus vector (RRV), we replaced the 3’ U3 region of the RRV ACE-GFP with a regulatory sequence consisting of the HBV enhancer II and the human alpha-fetoprotein (AFP) core promoter to produce a hepatocelluar carcinoma (HCC)-targeting RRV ACE-GFP-EIIAFP. Similar to the vector ACE-GFP, ACE-GFP-EIIAFP exhibited robust GFP expression in various human HCC cells. Most importantly, ACEGFP-EIIAFP showed tumor-specific replication in HCC cells but not in non-HCC tumor cells or primary normal liver cells. To examine the Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
structural stability of the ACE-GFP-EIIAFP vector genome during its replicative spread, we sequenced the promoter region of the vector collected from each replication cycle, and we found that the vector could retain the hybrid regulatory sequence in the genome over a long period. In vitro studies showed highly efficient cell killing to HCC cells by ACE-CD-EIIAFP which carries a suicide gene yeast cytosine deaminase after treatment with the prodrug 5-fluorocytosine (5-FC). For in vivo studies, ACE-CD-EIIAFP was injected intratumorally in a pre-established murine subcutaneous tumor model, and two weeks later 5-FC was administered intraperitoneally. Significant tumor suppression was observed in the therapeutic treatment and notably, no systemic spread of the vector to extratumoral tissues was detected by sensitive quantitative PCR. These results suggest that tumorselective RRV encoding suicide gene may show great promise for cancer gene therapy.
523. BioKnife, a Urokinase-Targeted Oncolytic Sendai Virus, Eliminates Pleural Spread of Malignant Mesothelioma
Yosuke Morodomi,1 Tokujiro Yano,1 Hiroaki Kinoh,3 Yui Harada,2 Satoru Saito,2 Tsukihisa Yoshida,1 Kensaku Ito,1 Yasunori Shikada,1 Riichiroh Maruyama,1 Kumi Yoshida,2 Yasuji Ueda,3 Mamoru Hasegawa,3 Yoshihiko Maehara,1 Yoshikazu Yonemitsu.2 1 Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2R&D Laboratory for Innovative Biotherapeutics, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan; 3 DNAVEC Corp, Tsukuba, Ibaraki, Japan. Background: Malignant pleural mesothelioma (MPM) is highly intractable: therefore, novel therapeutics are much desired. It has been largely known that malignant tumors including MPM showing invasive and metastatic activity frequently express urokinase-type plasminogen activator (uPA) or its receptor (uPAR). We have recently developed a novel type of oncolytic virus based on Sendai virus, that shows fusogenic oncolysis via specific targeting to uPA/uPAR system (named as ‘BioKnife: Kinoh H, et al. Gene Ther 2008, and Hasegawa Y, et al, Mol Ther 2010); therefore, we here investigated the therapeutic potential of the BioKnife to treat mesothelioma. Methods: Orthotropic mouse models of MPM were established by inoculation human MPM cell lines (MSTO-211H and NCI-H226) into the pleural cavity of nu/nu mice. BioKnife expressing green fluororescent protein (BioKnife-GFP) were administered to the pleural cavity of the tumor bearing mice. Results: The both human MPM cells exhibited multiple tumor nodules in the pleural cavity on day 7, and the all tumor bearing mice were dead within 50 days (MSTO-211H: uPAR high and uPA high) or 140 days (NCI-H226: uPAR high but uPA low). Dissecting fluorescent microscope demonstrated that GFP signal was detected specifically in the tumors but rarely in normal tissues, and immunofluorescent study revealed that extensive tunnel positive cells were detected in the tumors treated with BioKnife-GFP. Unexpectedly, repeated administration of BioKnife-GFP significantly prolonged survival of mice in both models (p<0.001), irrespective of the marked difference of uPA expression in these cells. Importantly, we here found that infection of BioKnife itself dramatically stimulated uPA expression from NCI-H226 via RIG-I/NK-kB dependent system, resulting the efficient oncolysis of this tumor. Conclusions: We here show that BioKnife was highly effective to eliminate uPARexpressing MPM in vivo, irrespective of its base-line expression level of uPA. These results illustrate the potential of BioKnife as a novel therapeutic agent to treat MPM in clinical setting.
S201
Christian Grossardt,1 Sascha Bossow,1 Achim Temme,2 Mathias F. Leber,1 Stefanie Sawall,1 Roberto Cattaneo,3 Christof von Kalle,1 Guy Ungerechts.1 1 Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany; 2Department of Neurosurgery, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany; 3Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, MN. Pancreatic cancer is one of the most drug-resistant types of malignancy, with extremely poor prognosis and survival rate. Over the past 10 years, gemcitabine has been the front line chemotherapy to treat non-resectable and metastasized pancreatic cancer, ultimately not preventing a progressive disease. Measles viruses (MV) derived from vaccine strain have shown considerable oncolytic activity against lymphomas and a wide variety of solid adenocarcinomas from different entities and are a promising tool to treat pancreatic cancer. Since prostate stem cell antigen (PSCA), originally identified as a marker for prostate cancer, is also expressed on pancreatic adenocarcinoma but not on non-neoplastic tissue, we generated a fully retargeted MV for the treatment of pancreatic adenocarcinoma. Therefore, we fused a single chain antibody (scFv) specific for the extracellular domain of PSCA as a C-terminal extension to the MV attachment protein H which was blinded for the natural MV receptors CD46/SLAM. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cancer cell lines. We were able to show that cell lines with high- and low-level PSCA expression were susceptible to PSCA-retargeted MV. Furthermore, we engineered a PSCA-targeted MV armed with the prodrug convertase purine nucleoside phosphorylase (PNP) – MV-PNPantiPSCA. PNP converts the prodrug fludarabine to the highly toxic and diffusible 2-fluoroadenine substantially enhancing the oncolytic efficacy of the virus on infected and bystander cells. Synergistic therapeutic effects were demonstrated in a subcutaneous BxPC-3 tumor xenograft model of pancreatic adenocarcinoma. The combined chemovirotherapy of intratumoral MV-PNP-antiPSCA application together with intraperitoneal administration of fludarabine yielded a potent synergistic oncolytic effect and resulted in a significant tumor regression compared to mice treated with virus or prodrug only. Importantly, with regard to future clinical trials no cross resistance of various gemcitabine-resistant pancreatic adenocarcinoma cell lines to MV mediated oncolysis and activated prodrug was detected.
522. Tumor-Selective Suicide Gene Therapy by Transcriptionally Targeted Replicating Retrovirus Vectors Yi-Hui Lai,1 Chien-Kuo Tai.1 Department of Life Science, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan.
1
Replicating virus vectors are attractive tools for anticancer gene therapy, but the potential for adverse events due to uncontrolled spread of the vectors has been a foremost concern of investigators in the field of retroviral gene therapy. To design a tumor-selective replicating retrovirus vector (RRV), we replaced the 3’ U3 region of the RRV ACE-GFP with a regulatory sequence consisting of the HBV enhancer II and the human alpha-fetoprotein (AFP) core promoter to produce a hepatocelluar carcinoma (HCC)-targeting RRV ACE-GFP-EIIAFP. Similar to the vector ACE-GFP, ACE-GFP-EIIAFP exhibited robust GFP expression in various human HCC cells. Most importantly, ACEGFP-EIIAFP showed tumor-specific replication in HCC cells but not in non-HCC tumor cells or primary normal liver cells. To examine the Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
structural stability of the ACE-GFP-EIIAFP vector genome during its replicative spread, we sequenced the promoter region of the vector collected from each replication cycle, and we found that the vector could retain the hybrid regulatory sequence in the genome over a long period. In vitro studies showed highly efficient cell killing to HCC cells by ACE-CD-EIIAFP which carries a suicide gene yeast cytosine deaminase after treatment with the prodrug 5-fluorocytosine (5-FC). For in vivo studies, ACE-CD-EIIAFP was injected intratumorally in a pre-established murine subcutaneous tumor model, and two weeks later 5-FC was administered intraperitoneally. Significant tumor suppression was observed in the therapeutic treatment and notably, no systemic spread of the vector to extratumoral tissues was detected by sensitive quantitative PCR. These results suggest that tumorselective RRV encoding suicide gene may show great promise for cancer gene therapy.
523. BioKnife, a Urokinase-Targeted Oncolytic Sendai Virus, Eliminates Pleural Spread of Malignant Mesothelioma
Yosuke Morodomi,1 Tokujiro Yano,1 Hiroaki Kinoh,3 Yui Harada,2 Satoru Saito,2 Tsukihisa Yoshida,1 Kensaku Ito,1 Yasunori Shikada,1 Riichiroh Maruyama,1 Kumi Yoshida,2 Yasuji Ueda,3 Mamoru Hasegawa,3 Yoshihiko Maehara,1 Yoshikazu Yonemitsu.2 1 Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2R&D Laboratory for Innovative Biotherapeutics, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan; 3 DNAVEC Corp, Tsukuba, Ibaraki, Japan. Background: Malignant pleural mesothelioma (MPM) is highly intractable: therefore, novel therapeutics are much desired. It has been largely known that malignant tumors including MPM showing invasive and metastatic activity frequently express urokinase-type plasminogen activator (uPA) or its receptor (uPAR). We have recently developed a novel type of oncolytic virus based on Sendai virus, that shows fusogenic oncolysis via specific targeting to uPA/uPAR system (named as ‘BioKnife: Kinoh H, et al. Gene Ther 2008, and Hasegawa Y, et al, Mol Ther 2010); therefore, we here investigated the therapeutic potential of the BioKnife to treat mesothelioma. Methods: Orthotropic mouse models of MPM were established by inoculation human MPM cell lines (MSTO-211H and NCI-H226) into the pleural cavity of nu/nu mice. BioKnife expressing green fluororescent protein (BioKnife-GFP) were administered to the pleural cavity of the tumor bearing mice. Results: The both human MPM cells exhibited multiple tumor nodules in the pleural cavity on day 7, and the all tumor bearing mice were dead within 50 days (MSTO-211H: uPAR high and uPA high) or 140 days (NCI-H226: uPAR high but uPA low). Dissecting fluorescent microscope demonstrated that GFP signal was detected specifically in the tumors but rarely in normal tissues, and immunofluorescent study revealed that extensive tunnel positive cells were detected in the tumors treated with BioKnife-GFP. Unexpectedly, repeated administration of BioKnife-GFP significantly prolonged survival of mice in both models (p<0.001), irrespective of the marked difference of uPA expression in these cells. Importantly, we here found that infection of BioKnife itself dramatically stimulated uPA expression from NCI-H226 via RIG-I/NK-kB dependent system, resulting the efficient oncolysis of this tumor. Conclusions: We here show that BioKnife was highly effective to eliminate uPARexpressing MPM in vivo, irrespective of its base-line expression level of uPA. These results illustrate the potential of BioKnife as a novel therapeutic agent to treat MPM in clinical setting.
S201