- Email: [email protected]
415. The Potency of a Histone Deacetylase Inhibitor and Reolysin in Head and Neck Squamous Cell Carcinoma
Cancer-Oncolytic Viruses I 414. A Novel System to Systematically Analyze Diverse Types of Conditionally Replicating Adenoviruses Expressing Immunostimulatory Genes in Syngenic Hamster Models Nobuhiro Ijichi, Hirotaka Kawakami, Yuki Obama, Ken-ichiro Kosai Department of Gene Therapy and Regenerative Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
We developed a method to efficiently construct diverse “conditionally replicating adenoviruses regulated with multiple cancer-specific factors” (m-CRAs) for an ideal gene-viro-therapy (Gene Ther 2005), after we had developed the immunological gene therapy, including granulocyte macrophage colony-stimulating factor (GM-CSF) (PNAS 1995, 1996, Cancer Res 1996). We then identified by systematic m-CRA analyses that survivin-responsive m-CRA (Surv.m-CRA), in which E1A is regulated by survivin promoter, could achieve stronger and more cancer-selective potentials than competing m-CRAs (Cancer Res 2005). The new Surv.m-CRA, of which an altered E1B promoter is regulated with another cancerspecific promoter (CS-Pr), further increased cancer specificity without reduced anticancer effects (Cancer Gene Ther 2011). Moreover, the unique feature of the increased effectiveness against cancer stem cells suggests that Surv.m-CRA is promising anticancer agent (J Trans Med 2014). In addition, we have developed a novel m-CRA strategy that specifically eliminates undifferentiated cells in pluripotent stem cell-based regenerative medicine (Mol Ther Methods Clin Dev 2015). Taken altogether, we are about to start its clinical trial in Japan. As the first-in-class oncolytic virus expressing GM-CSF was recently approved, m-CRAs expressing immunostimulatory genes should be promising. In general, the development of oncolytic adenoviruses with cytokines has been hampered by two technical limitations in the vector construction and the viral replication-permissive animals. Here we demonstrate a novel system to efficiently generate diverse types of m-CRAs expressing candidate immunostimulatory genes under candidate promoters, and systematically analyze their potentials in syngenic hamster models. We tested the feasibility of this methodology, and succeeded in the rapid generation of the new Surv.m-CRAs comprising 18 different therapeutic units, including a mouse or human cytokine gene downstream to a ubiquitously active promoter (UA-Pr) or CS-Pr. To test the utility of our experimental system, we first analyzed in vivo antitumor efficiencies and toxicities of Surv.m-CRA expressing GM-CSF under a strong UAPr in xenograft syngeneic tumor of syrian hamster. An intratumoral injection of the control Surv.m-CRA without GM-CSF inhibited a tumor growth and extended a survival to some degrees. Whereas Surv.m-CRAs expressing mouse GM-CSF more drastically reduced tumor growth, some of these animals died of excessive GM-CSF expressions (showing the remarkable splenomegaly) within 3 days. The results importantly suggest the necessity of systematical analyses to identify the best combination of cytokine genes and promoters to achieve the optimal cytokine levels (Int J Oncol 2004), and we are now analyzing 18 different Surv.m-CRAs, including those expressing GM-CSF at a moderate level and/or in the cancer-specific manner. In conclusion, we have developed a novel system to efficiently generate the candidate m-CRAs expressing diverse immunostimulatory genes under several promoters and to systematically analyze them in syngenic hamster models.
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415. The Potency of a Histone Deacetylase Inhibitor and Reolysin in Head and Neck Squamous Cell Carcinoma
Matthew Old1, Cristina Jamie-Ramirez2, Jun-Ge Yu1, Bhavna Kumar1, Pawan Kumar1, Quintin Pan1, John Lee3, Theodoros Teknos1, Flavia Pichiorri4, Balveen Kaur2 1 Otolaryngology-Head and Neck Surgery, The Ohio State University, Columbus, OH, 2Department of Neurological Surgery, The Ohio State University, Columbus, OH, 3OtolaryngologyHead and Neck Surgery, Sanford Health, Sioux Falls, SD, 4 Comprehensive Cancer Center, The Ohio State University, Columbus, OH Introduction: There is a clear and pressing need for novel therapies with activity against locally advanced head and neck cancers which still carry a dismal prognosis. Oncolytic viruses are powerful, targeted anti-cancer agents. Reovirus is a naturally occurring non-pathogenic virus that was isolated from the human respiratory and gastrointestinal tracts. Moreover, Reovirus type 3 Dearing (Reolysin; Oncolytics Biotech Inc., Calgary, AB, Canada) is currently being tested in phase I-III clinical trials in a variety of tumor types. Histone deacetylase inhibitors (HDACi) comprise a structurally diverse class of compounds that are targeted anticancer agents. The first FDA approved HDACi, vorinostat (suberoylanilide hydroxamic acid-SAHA), is highly effective in the treatment of cutaneous Tcell lymphoma. SAHA is currently being testing in head and neck cancer clinical trials. We previously found a synergistic combination of SAHA and Reolysin in a nude mouse model. Preclinical models of oncolytics are often in immunocompromised mice, negating the significant impact of the immune system. Mounting evidence demonstrates that the immune system is critically important in oncolytic viral response. In this study, we sought to investigate the impact of this combination in an immunocompetent model. Methods: Cell survival experiments were performed with reovirus and SAHA in shPTP-BL-Ras SCC cells. IC50 values were interpolated from a sigmoidal dose-response curve fit of the log-transformed survival data. JAM-1 surface levels were assessed via flow cytometric analysis. Cells were collected after 48 hours and cell death was assessed via Annexin V and PI staining. JAM-1 levels were assessed using anti-JAM-1-PE staining as compared to a control isotype-PE antibody. Whole splenocytes were isolated at the time of death for C57BL/6 mice bearing MTE tumors treated in 4 groups (control, SAHA, reovirus, and combination). Three independent mice were stained per group for the following: B cells (CD19+), NK cells (CD49b+), activated NK cells (CD49b+NKp46+), dendritic cells (CD11c+) and activated DCs (CD11c+MHCII+ or Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Cancer-Oncolytic Viruses I CD11c+CD86+), CD4 T helper (CD3+CD4+), and CD8 cytotoxic cells (CD3+CD8+). Results: Experiments demonstrated significant efficacy of SAHA and Reolysin treatment in vitro and in the immunocompetent mouse model. Combination therapy exhibited a synergistic anti-tumor effect with a significant increased survival of mice compared to any of the agents alone. The Jam1 receptor was upregulated on tumor cells allowing enhanced reovirus uptake. There was marked and significant reduction in circulating B cells in combination treated mice versus all other groups. Activated NK cells were decreased in the combination group. T cells and dendritic cells (CD11c+MHCII+) were reduced in the SAHA groups. SAHA model withdrawal experiments show a significant synergistic response and immune system rebound after SAHA cessation. Conclusion: This data demonstrates that combination of reovirus plus SAHA therapy has significant activity in the treatment of SCCHN, even in an immunocompetent model. Immune inhibition due to SAHA as well as increased Jam1 receptor expression on tumor cells results in a synergistic effect of the combination therapy. Immune system rebound likely plays a significant role in the long-term anti-tumor response. This strong preclinical evidence supports the translation of this combination to phase-I clinical trials.
416. Enhanced Oncolytic Effect of Tumor Necrosis Factor Alpha Armed Engineered Oncolytic Measles Virus Therapy for Ovarian Carcinoma Cell Line
Ahmed M. Al-Shammari1, Patrycja J. Lech2, Stephen J. Russell2 1 Experimental Therapy Department, Iraqi Center for Cancer and Medical Genetic Research, Baghdad, Iraq, 2Molecular Medicine, Mayo Clinic, Rochester, MN Introduction. Tumor necrosis factor alpha (TNFa) has been recognized having anticancer properties, and proposed as a cancer treatment early in the 1980s, But its toxicity lead to limited use. Replication competent oncolytic viruses such as live attenuated measles virus (MV), targeting cancer cells selectively, have shown very promising results in both pre and clinical stages. one of its most useful aspects is their ability to produce high amounts of transgene products locally, resulting in high local tumor microenvironment versus systemic concentrations. Therefore, we developed Gene therapy approach to increase anti-tumor activity results from the combination of oncolytic activity, transgene-mediated direct cytotoxic effect such as TNFa-mediated apoptosis. Methods, we created novel oncolytic MV expressing murine TNFa by cloning TNFa gene into MV genome. the gene was amplified by PCR using primers that introduce restriction sites at either end of the coding sequence. The gene cloned into a plasmid encoding the entire measles genome. The insertion site for the TNFa gene was between H and L in the first version. In the second version, transgene was inserted upstream of N. The resulting full-length plasmids [MV-TNFa1 and MV-TNFa2] used to rescue the corresponding recombinant measles virus on 2933-46 helper cells that was transfected with the MV plasmids. We evaluated the 2 recombinant versions of the MV expressing TNFa with 2 other viruses of MV, one having the sodium iodide symporter (NIS) between H and L, and the second has GFP upstream of N for oncolytic potential against ovarian carcinoma cell line SKOV-3. Infectivity, syncytium formation and cytotoxicity of recombinant MV-TNFs in SKOV-3 cells were evaluated by inverted microscopy and the MTT assay. Transgene expression in SKOV-3 cells after infection was assessed for TNFa functionality and concentration using cells that are genetically engineered for NF-κB Signaling Pathway and TNFa quantitative ELISA assay. Results, we were successful to generate the two versions of the virus with TNFa in different genome locations for each version. MV-TNFa2 shown to produce more TNFa than MV-TNFa1 (Figure-1a), but both viruses were producing Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
functional TNFa by activating NF-κB Signaling Pathway (Figure-1b). Recombinant MV-TNFs viruses were efficiently infected SKOV-3 cell line, resulting in extensive syncytium formation followed by cell death. There was enhanced killing for the MV-TNFa1 infected cells comparing to all other viruses (Figure-1c). In conclusion, engineered MV-TNFs may be a potent and novel cancer gene therapy system. MV expressing TNFa elicited oncolytic effects in ovarian cancer cells, enhancing the killing effect which depend on the insert location.
Figure-1, Experiments on MV-TNFs, A- shown MV-TNFa2 to produce more TNFa than MV-TNFa1. B- Secreted TNFa is activating NF-κB Signaling Pathway. C- enhanced killing for the MV-TNFa1 infected cells comparing to all other viruses.
417. Intratumoral Delivery of Oncolytic Adenovirus Expressing Decorin Inhibits Growth and Metastases of 4T1 Breast Tumors in Syngeneic Immune Competent BALB/c Mice Model
Weidong Xu1, Yuefeng Yang2, Thomas Neill3, Hua Wang2, Chi-Hsiung Wang4, Chae-Ok Yun5, Theresa Guise6, Charles B. Brendler4, Renato V. Iozzo3, Lisheng Wang2, Prem Seth1 1 Gene Therapy Program, Department of Medicine, NorthShore Research Institute, an affiliate of the University of Chicago, Evanston, IL, 2Beijing Institute of Radidation Medicine, Beijing, China, 3Department of Pathology, Anatomy and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, 4 Department of Surgery, NorthShore Research Institute, Evanston, IL, 5Department of Bioengineering, Hanyang University, Seoul, Korea, Republic of, 6Department of Medicine, Indiana University, Indianapolis, IN
During the advanced stage of breast cancer, majority of the patients develop distal metastases that eventually lead to mortality. Unfortunately, conventional strategies generally invoke limited therapeutic responses in patients with tumor metastases. Therefore, the development of novel and highly effective therapies for the treatment of distal metastases of breast cancer is an unmet need in medicine. Decorin (DCN) is a small leucine-rich proteoglycan and is often down-regulated in tumor stroma of breast cancer patients. Our previous work demonstrated that systemic delivery of Ad.dcn, an oncolytic adenovirus expressing DCN, significantly inhibited skeletal metastases and the tumor induced bone destruction in MDAMB-231 bone metastasis model. However, the therapeutic responses of Ad.dcn in immune-competent models have never been examined. In this study, we showed that non-replicated adenovirus Ad(E1-). dcn mediated high level expression of human DCN in 4T1 cells, and inhibited the expression of tyrosine kinase receptor MET, β-catenin S165
We developed a method to efficiently construct diverse “conditionally replicating adenoviruses regulated with multiple cancer-specific factors” (m-CRAs) for an ideal gene-viro-therapy (Gene Ther 2005), after we had developed the immunological gene therapy, including granulocyte macrophage colony-stimulating factor (GM-CSF) (PNAS 1995, 1996, Cancer Res 1996). We then identified by systematic m-CRA analyses that survivin-responsive m-CRA (Surv.m-CRA), in which E1A is regulated by survivin promoter, could achieve stronger and more cancer-selective potentials than competing m-CRAs (Cancer Res 2005). The new Surv.m-CRA, of which an altered E1B promoter is regulated with another cancerspecific promoter (CS-Pr), further increased cancer specificity without reduced anticancer effects (Cancer Gene Ther 2011). Moreover, the unique feature of the increased effectiveness against cancer stem cells suggests that Surv.m-CRA is promising anticancer agent (J Trans Med 2014). In addition, we have developed a novel m-CRA strategy that specifically eliminates undifferentiated cells in pluripotent stem cell-based regenerative medicine (Mol Ther Methods Clin Dev 2015). Taken altogether, we are about to start its clinical trial in Japan. As the first-in-class oncolytic virus expressing GM-CSF was recently approved, m-CRAs expressing immunostimulatory genes should be promising. In general, the development of oncolytic adenoviruses with cytokines has been hampered by two technical limitations in the vector construction and the viral replication-permissive animals. Here we demonstrate a novel system to efficiently generate diverse types of m-CRAs expressing candidate immunostimulatory genes under candidate promoters, and systematically analyze their potentials in syngenic hamster models. We tested the feasibility of this methodology, and succeeded in the rapid generation of the new Surv.m-CRAs comprising 18 different therapeutic units, including a mouse or human cytokine gene downstream to a ubiquitously active promoter (UA-Pr) or CS-Pr. To test the utility of our experimental system, we first analyzed in vivo antitumor efficiencies and toxicities of Surv.m-CRA expressing GM-CSF under a strong UAPr in xenograft syngeneic tumor of syrian hamster. An intratumoral injection of the control Surv.m-CRA without GM-CSF inhibited a tumor growth and extended a survival to some degrees. Whereas Surv.m-CRAs expressing mouse GM-CSF more drastically reduced tumor growth, some of these animals died of excessive GM-CSF expressions (showing the remarkable splenomegaly) within 3 days. The results importantly suggest the necessity of systematical analyses to identify the best combination of cytokine genes and promoters to achieve the optimal cytokine levels (Int J Oncol 2004), and we are now analyzing 18 different Surv.m-CRAs, including those expressing GM-CSF at a moderate level and/or in the cancer-specific manner. In conclusion, we have developed a novel system to efficiently generate the candidate m-CRAs expressing diverse immunostimulatory genes under several promoters and to systematically analyze them in syngenic hamster models.
S164
415. The Potency of a Histone Deacetylase Inhibitor and Reolysin in Head and Neck Squamous Cell Carcinoma
Matthew Old1, Cristina Jamie-Ramirez2, Jun-Ge Yu1, Bhavna Kumar1, Pawan Kumar1, Quintin Pan1, John Lee3, Theodoros Teknos1, Flavia Pichiorri4, Balveen Kaur2 1 Otolaryngology-Head and Neck Surgery, The Ohio State University, Columbus, OH, 2Department of Neurological Surgery, The Ohio State University, Columbus, OH, 3OtolaryngologyHead and Neck Surgery, Sanford Health, Sioux Falls, SD, 4 Comprehensive Cancer Center, The Ohio State University, Columbus, OH Introduction: There is a clear and pressing need for novel therapies with activity against locally advanced head and neck cancers which still carry a dismal prognosis. Oncolytic viruses are powerful, targeted anti-cancer agents. Reovirus is a naturally occurring non-pathogenic virus that was isolated from the human respiratory and gastrointestinal tracts. Moreover, Reovirus type 3 Dearing (Reolysin; Oncolytics Biotech Inc., Calgary, AB, Canada) is currently being tested in phase I-III clinical trials in a variety of tumor types. Histone deacetylase inhibitors (HDACi) comprise a structurally diverse class of compounds that are targeted anticancer agents. The first FDA approved HDACi, vorinostat (suberoylanilide hydroxamic acid-SAHA), is highly effective in the treatment of cutaneous Tcell lymphoma. SAHA is currently being testing in head and neck cancer clinical trials. We previously found a synergistic combination of SAHA and Reolysin in a nude mouse model. Preclinical models of oncolytics are often in immunocompromised mice, negating the significant impact of the immune system. Mounting evidence demonstrates that the immune system is critically important in oncolytic viral response. In this study, we sought to investigate the impact of this combination in an immunocompetent model. Methods: Cell survival experiments were performed with reovirus and SAHA in shPTP-BL-Ras SCC cells. IC50 values were interpolated from a sigmoidal dose-response curve fit of the log-transformed survival data. JAM-1 surface levels were assessed via flow cytometric analysis. Cells were collected after 48 hours and cell death was assessed via Annexin V and PI staining. JAM-1 levels were assessed using anti-JAM-1-PE staining as compared to a control isotype-PE antibody. Whole splenocytes were isolated at the time of death for C57BL/6 mice bearing MTE tumors treated in 4 groups (control, SAHA, reovirus, and combination). Three independent mice were stained per group for the following: B cells (CD19+), NK cells (CD49b+), activated NK cells (CD49b+NKp46+), dendritic cells (CD11c+) and activated DCs (CD11c+MHCII+ or Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Cancer-Oncolytic Viruses I CD11c+CD86+), CD4 T helper (CD3+CD4+), and CD8 cytotoxic cells (CD3+CD8+). Results: Experiments demonstrated significant efficacy of SAHA and Reolysin treatment in vitro and in the immunocompetent mouse model. Combination therapy exhibited a synergistic anti-tumor effect with a significant increased survival of mice compared to any of the agents alone. The Jam1 receptor was upregulated on tumor cells allowing enhanced reovirus uptake. There was marked and significant reduction in circulating B cells in combination treated mice versus all other groups. Activated NK cells were decreased in the combination group. T cells and dendritic cells (CD11c+MHCII+) were reduced in the SAHA groups. SAHA model withdrawal experiments show a significant synergistic response and immune system rebound after SAHA cessation. Conclusion: This data demonstrates that combination of reovirus plus SAHA therapy has significant activity in the treatment of SCCHN, even in an immunocompetent model. Immune inhibition due to SAHA as well as increased Jam1 receptor expression on tumor cells results in a synergistic effect of the combination therapy. Immune system rebound likely plays a significant role in the long-term anti-tumor response. This strong preclinical evidence supports the translation of this combination to phase-I clinical trials.
416. Enhanced Oncolytic Effect of Tumor Necrosis Factor Alpha Armed Engineered Oncolytic Measles Virus Therapy for Ovarian Carcinoma Cell Line
Ahmed M. Al-Shammari1, Patrycja J. Lech2, Stephen J. Russell2 1 Experimental Therapy Department, Iraqi Center for Cancer and Medical Genetic Research, Baghdad, Iraq, 2Molecular Medicine, Mayo Clinic, Rochester, MN Introduction. Tumor necrosis factor alpha (TNFa) has been recognized having anticancer properties, and proposed as a cancer treatment early in the 1980s, But its toxicity lead to limited use. Replication competent oncolytic viruses such as live attenuated measles virus (MV), targeting cancer cells selectively, have shown very promising results in both pre and clinical stages. one of its most useful aspects is their ability to produce high amounts of transgene products locally, resulting in high local tumor microenvironment versus systemic concentrations. Therefore, we developed Gene therapy approach to increase anti-tumor activity results from the combination of oncolytic activity, transgene-mediated direct cytotoxic effect such as TNFa-mediated apoptosis. Methods, we created novel oncolytic MV expressing murine TNFa by cloning TNFa gene into MV genome. the gene was amplified by PCR using primers that introduce restriction sites at either end of the coding sequence. The gene cloned into a plasmid encoding the entire measles genome. The insertion site for the TNFa gene was between H and L in the first version. In the second version, transgene was inserted upstream of N. The resulting full-length plasmids [MV-TNFa1 and MV-TNFa2] used to rescue the corresponding recombinant measles virus on 2933-46 helper cells that was transfected with the MV plasmids. We evaluated the 2 recombinant versions of the MV expressing TNFa with 2 other viruses of MV, one having the sodium iodide symporter (NIS) between H and L, and the second has GFP upstream of N for oncolytic potential against ovarian carcinoma cell line SKOV-3. Infectivity, syncytium formation and cytotoxicity of recombinant MV-TNFs in SKOV-3 cells were evaluated by inverted microscopy and the MTT assay. Transgene expression in SKOV-3 cells after infection was assessed for TNFa functionality and concentration using cells that are genetically engineered for NF-κB Signaling Pathway and TNFa quantitative ELISA assay. Results, we were successful to generate the two versions of the virus with TNFa in different genome locations for each version. MV-TNFa2 shown to produce more TNFa than MV-TNFa1 (Figure-1a), but both viruses were producing Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
functional TNFa by activating NF-κB Signaling Pathway (Figure-1b). Recombinant MV-TNFs viruses were efficiently infected SKOV-3 cell line, resulting in extensive syncytium formation followed by cell death. There was enhanced killing for the MV-TNFa1 infected cells comparing to all other viruses (Figure-1c). In conclusion, engineered MV-TNFs may be a potent and novel cancer gene therapy system. MV expressing TNFa elicited oncolytic effects in ovarian cancer cells, enhancing the killing effect which depend on the insert location.
Figure-1, Experiments on MV-TNFs, A- shown MV-TNFa2 to produce more TNFa than MV-TNFa1. B- Secreted TNFa is activating NF-κB Signaling Pathway. C- enhanced killing for the MV-TNFa1 infected cells comparing to all other viruses.
417. Intratumoral Delivery of Oncolytic Adenovirus Expressing Decorin Inhibits Growth and Metastases of 4T1 Breast Tumors in Syngeneic Immune Competent BALB/c Mice Model
Weidong Xu1, Yuefeng Yang2, Thomas Neill3, Hua Wang2, Chi-Hsiung Wang4, Chae-Ok Yun5, Theresa Guise6, Charles B. Brendler4, Renato V. Iozzo3, Lisheng Wang2, Prem Seth1 1 Gene Therapy Program, Department of Medicine, NorthShore Research Institute, an affiliate of the University of Chicago, Evanston, IL, 2Beijing Institute of Radidation Medicine, Beijing, China, 3Department of Pathology, Anatomy and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, 4 Department of Surgery, NorthShore Research Institute, Evanston, IL, 5Department of Bioengineering, Hanyang University, Seoul, Korea, Republic of, 6Department of Medicine, Indiana University, Indianapolis, IN
During the advanced stage of breast cancer, majority of the patients develop distal metastases that eventually lead to mortality. Unfortunately, conventional strategies generally invoke limited therapeutic responses in patients with tumor metastases. Therefore, the development of novel and highly effective therapies for the treatment of distal metastases of breast cancer is an unmet need in medicine. Decorin (DCN) is a small leucine-rich proteoglycan and is often down-regulated in tumor stroma of breast cancer patients. Our previous work demonstrated that systemic delivery of Ad.dcn, an oncolytic adenovirus expressing DCN, significantly inhibited skeletal metastases and the tumor induced bone destruction in MDAMB-231 bone metastasis model. However, the therapeutic responses of Ad.dcn in immune-competent models have never been examined. In this study, we showed that non-replicated adenovirus Ad(E1-). dcn mediated high level expression of human DCN in 4T1 cells, and inhibited the expression of tyrosine kinase receptor MET, β-catenin S165