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646. Viral Spread of Adenovirus Serotype 5 Versus Serotype 3 in Epithelial Tumors
CANCER-ONCOLYTIC VIRUSES II the stage for efficient oncolysis by HSV-1 derived oncolytic viral vectors. Since bortezomib is FDA approved, the significance of this study is that it can offer a novel therapeutic treatment strategy for cancer therapy that can be rapidly translated in patients with multiple solid tumors.
646. Viral Spread of Adenovirus Serotype 5 Versus Serotype 3 in Epithelial Tumors
Maximilian Richter,1 Roma Yumul,1 Zuozhuang Lu,1 Hongjie Wang,1 Andre Lieber.1 1 Medicine, University of Washington, Seattle, WA. Most oncolytic adenoviruses are based on Ad5, a serotype that uses CAR as a receptor. More recently oncolytic vectors based on the desmoglein 2 (DSG2-interacting Ad3 and Ad11 serotypes have shown promise for cancer therapy. In normal polarized epithelial cells and epithelial tumors, DSG2 as well as CAR are sequestered in cell-cell junctions, and consequently relatively inaccessible for Ad infection. Therefore, both DSG2 and CAR are considered receptors primarily supporting the lateral spread of de novo produced virus. CAR- and DSG2-interacting Ads have evolved different strategies to support viral dissemination in epithelial tissues. During replication of Ad5, excess production of fiber or fiber/penton base complexes results in the disruption of epithelial junctions either by interfering with CAR dimerization or by triggering intracellular signaling that leads to reorganization of intercellular junctions. A hallmark of DSG2interacting Ads (such as Ad3) is the formation of large amounts of penton-dodecahedra (PtDd) during viral replication. PtDds are selfassembling subviral particles, 30nm in diameter, consisting of twelve fiber/penton-base hetero-oligomers called pentons. Dodecamerization of pentons is required for high affinity binding to DSG2. We showed that PtDds are released from Ad3 infected cells before virus-triggered cytolysis and can be found in the paracellular space in epithelial tumor cell cultures. Binding of PtDds to DSG2 on non-infected neighboring epithelial cells triggers intracellular signaling resulting in the transient opening of junctions between epithelial cells, thus supporting lateral spread of de novo produced Ad3. A penton-base mutant Ad3 vector disabled for PtDd production (Ad3D) was identical to wtAd3 in the efficiency of viral entry, replication, and release of progeny virus, but was impeded in spread in epithelial colon cancer T84 cell cultures and in T84 xenograft tumors (PLoS Pathog. 2013:e1003718). To compare Ad5 and Ad3, we infected polarized T84 cells grown in transwell chambers on membranes with 0.4 mm pore size. T84 cells formed multiple layers of cells linked by tight junctions. GFP-expressing wtAd3 and wtAd5 vectors were added into the inner chamber at an MOI of 10 vp/cell. Initial transduction (measured based on GFPexpressing units at day 1 p.i. ) were comparable for both vectors. Upon viral spread through the layers of T84 cells, de novo produced virus penetrates the membrane and is released into the outer chamber. Titers of de novo produced virus measured at day 9 p.i. in the outer chambers were orders of magnitude higher for Ad3 compared to Ad5, indicating more efficient spread of Ad3. The PtDd-mutant Ad3D vector had no advantage over Ad5. We are currently consolidating these finding in tumor cell spheroids and xenograft tumors. Considering that most solid tumors in humans are of epithelial origin and retain epithelial junctions, finding an adenovirus platform that can best overcome these physical obstacles is relevant for virotherapy of cancer.
647. Neutrophils (PMNs) in Measles Virus (MV) Mediated Leukaemia Therapy: Good or Bad? A. Dey, A. Castleton, B. Patel, A. Fielding. 1 UCL, London, United Kingdom. 1
1
1
1
We have previously shown that PMNs are involved in MVmediated tumor regression in a Raji tumor SCID mouse model, where responses correlated closely with the level of PMN infiltration. S250
When MV expressing murine GMCSF was used, higher levels of PMN infiltration were observed, and correlated with superior tumor regression. We also showed that unmodified MV (MV-NSe) is oncolytic in a Nalm-6 acute lymphoblastic leukaemia (ALL) SCID xenograft model. To investigate the role of PMNs in this model, we depleted PMN using the Ly6G antibody. Nalm-6 tumors responded to MV therapy as compared to UV-inactivated controls (MV-UV), but depletion of PMNs in the Nalm-6 model did not abrogate the therapeutic effect in-vivo. However, when we examined PMNmediated cytotoxicity in-vitro using Cr+ assays, where MV-infected Nalm-6 cells were incubated with PMNs from 10 healthy donors, we observed that MV significantly enhanced PMN-mediated cytotoxicity, suggesting that PMNs are playing a key role in MV-infected tumor cell cytotoxicity in-vitro but not in-vivo in this model. To investigate the intriguing and different roles of PMNs in MVmediated oncolysis in the Nalm-6 model, we cloned and rescued MV expressing the human cytokine, granulocyte colony stimulating factor (MVhGCSF), which also has significant activity at the murine GCSF receptor. Counter to expectations, MVhGCSF generated less PMNmediated cytotoxicity in-vitro, using Cr+ assay, than the unmodified MV. Next, we evaluated the effect of MVhGCSF on a disseminated Nalm-6 SCID xenograft model. The survival of MV-treated mice was superior to that of the MVhGCSF treated mice. Nine of 10 mice in the MVhGCSF-treated group had to be culled on the basis of preassigned humane end points (more than 20% weight loss and/or hind limb paralysis {HLP}) by day 71 post tumor inoculation, compared to only 1 in 4 in the MV-treated group. At humane end point, all the mice were evaluated for leukaemia burden in bone marrow (BM) and spleen (SP), size of SP, and the level of hGCSF in their serum. All SPs were subjected to flow cytometric analysis for assessment of CD11b, Ly6G6C, NK and Mac3. For all treatment groups, human CD10/ CD19 expressing cells were present, demonstrating the presence of ALL in all mice at the time of death. SPs were significantly larger in the MVhGCSF treated group compared to the MV or MV-UV group, but were heavily infiltrated by PMNs, rather than ALL, with 35-40% positivity for Cd11b and Ly6G6C as compared to the MV (2%) and MV-UV (10%) treated groups. Significant levels of hGCSF were detected in the serum of MVhGCSF-treated mice (0.3-1.13ng/ml) by ELISA. In terms of survival, mice treated with MVhGCSF did worse as compared to the unmodified MV-treated mice, and this was unexpected. The large SP size, heavy splenic infiltration by PMNs, and high levels of hGCSF in the serum of MVhGCSF-treated mice was suggestive of significant GCSF-mediated toxicity in this group, which may have contributed to their inferior survival. However, levels of leukaemic infiltration in the BM were comparable to the MV-treated group, suggesting no therapeutic benefit of MVhGCSF over MV-NSe. Work is ongoing to dissect whether PMNs might be unexpectedly operating to promote tumor growth in the MVhGSCFtreated Nalm-6 ALL disseminated model.
648. Combinatorial Treatment of Oncolytic Ad With Helper-Dependent Ad Augments the AntiTumor Effect of Adenoviral Cancer Gene Therapy
Lisa Farzad,1 Vincenzo Cerullo,2 Akseli Hemminki,2 Cliona Rooney,1 Brendan Lee,1 Malcolm Brenner,1 Masataka Suzuki.1 1 Center for Cell and Gene Therapy, Houston Methodist Hospital, Texas Children’s Hospital and Baylor College of Medicine, Houston, TX; 2University of Helsinki, Helsinki, Finland. Adenoviral vectors (Ads) can effectively enter malignant cells and express high levels of lytic or immunogenic transgenes, making them attractive agents for cancer gene therapy. However, the antitumor activity of oncolytic Ads rarely eliminates patient tumors. Investigators have therefore incorporated immunomodulatory molecules such as pro-inflammatory cytokines that can enhance anti-tumor immunity in host animals as well as in patients with Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
646. Viral Spread of Adenovirus Serotype 5 Versus Serotype 3 in Epithelial Tumors
Maximilian Richter,1 Roma Yumul,1 Zuozhuang Lu,1 Hongjie Wang,1 Andre Lieber.1 1 Medicine, University of Washington, Seattle, WA. Most oncolytic adenoviruses are based on Ad5, a serotype that uses CAR as a receptor. More recently oncolytic vectors based on the desmoglein 2 (DSG2-interacting Ad3 and Ad11 serotypes have shown promise for cancer therapy. In normal polarized epithelial cells and epithelial tumors, DSG2 as well as CAR are sequestered in cell-cell junctions, and consequently relatively inaccessible for Ad infection. Therefore, both DSG2 and CAR are considered receptors primarily supporting the lateral spread of de novo produced virus. CAR- and DSG2-interacting Ads have evolved different strategies to support viral dissemination in epithelial tissues. During replication of Ad5, excess production of fiber or fiber/penton base complexes results in the disruption of epithelial junctions either by interfering with CAR dimerization or by triggering intracellular signaling that leads to reorganization of intercellular junctions. A hallmark of DSG2interacting Ads (such as Ad3) is the formation of large amounts of penton-dodecahedra (PtDd) during viral replication. PtDds are selfassembling subviral particles, 30nm in diameter, consisting of twelve fiber/penton-base hetero-oligomers called pentons. Dodecamerization of pentons is required for high affinity binding to DSG2. We showed that PtDds are released from Ad3 infected cells before virus-triggered cytolysis and can be found in the paracellular space in epithelial tumor cell cultures. Binding of PtDds to DSG2 on non-infected neighboring epithelial cells triggers intracellular signaling resulting in the transient opening of junctions between epithelial cells, thus supporting lateral spread of de novo produced Ad3. A penton-base mutant Ad3 vector disabled for PtDd production (Ad3D) was identical to wtAd3 in the efficiency of viral entry, replication, and release of progeny virus, but was impeded in spread in epithelial colon cancer T84 cell cultures and in T84 xenograft tumors (PLoS Pathog. 2013:e1003718). To compare Ad5 and Ad3, we infected polarized T84 cells grown in transwell chambers on membranes with 0.4 mm pore size. T84 cells formed multiple layers of cells linked by tight junctions. GFP-expressing wtAd3 and wtAd5 vectors were added into the inner chamber at an MOI of 10 vp/cell. Initial transduction (measured based on GFPexpressing units at day 1 p.i. ) were comparable for both vectors. Upon viral spread through the layers of T84 cells, de novo produced virus penetrates the membrane and is released into the outer chamber. Titers of de novo produced virus measured at day 9 p.i. in the outer chambers were orders of magnitude higher for Ad3 compared to Ad5, indicating more efficient spread of Ad3. The PtDd-mutant Ad3D vector had no advantage over Ad5. We are currently consolidating these finding in tumor cell spheroids and xenograft tumors. Considering that most solid tumors in humans are of epithelial origin and retain epithelial junctions, finding an adenovirus platform that can best overcome these physical obstacles is relevant for virotherapy of cancer.
647. Neutrophils (PMNs) in Measles Virus (MV) Mediated Leukaemia Therapy: Good or Bad? A. Dey, A. Castleton, B. Patel, A. Fielding. 1 UCL, London, United Kingdom. 1
1
1
1
We have previously shown that PMNs are involved in MVmediated tumor regression in a Raji tumor SCID mouse model, where responses correlated closely with the level of PMN infiltration. S250
When MV expressing murine GMCSF was used, higher levels of PMN infiltration were observed, and correlated with superior tumor regression. We also showed that unmodified MV (MV-NSe) is oncolytic in a Nalm-6 acute lymphoblastic leukaemia (ALL) SCID xenograft model. To investigate the role of PMNs in this model, we depleted PMN using the Ly6G antibody. Nalm-6 tumors responded to MV therapy as compared to UV-inactivated controls (MV-UV), but depletion of PMNs in the Nalm-6 model did not abrogate the therapeutic effect in-vivo. However, when we examined PMNmediated cytotoxicity in-vitro using Cr+ assays, where MV-infected Nalm-6 cells were incubated with PMNs from 10 healthy donors, we observed that MV significantly enhanced PMN-mediated cytotoxicity, suggesting that PMNs are playing a key role in MV-infected tumor cell cytotoxicity in-vitro but not in-vivo in this model. To investigate the intriguing and different roles of PMNs in MVmediated oncolysis in the Nalm-6 model, we cloned and rescued MV expressing the human cytokine, granulocyte colony stimulating factor (MVhGCSF), which also has significant activity at the murine GCSF receptor. Counter to expectations, MVhGCSF generated less PMNmediated cytotoxicity in-vitro, using Cr+ assay, than the unmodified MV. Next, we evaluated the effect of MVhGCSF on a disseminated Nalm-6 SCID xenograft model. The survival of MV-treated mice was superior to that of the MVhGCSF treated mice. Nine of 10 mice in the MVhGCSF-treated group had to be culled on the basis of preassigned humane end points (more than 20% weight loss and/or hind limb paralysis {HLP}) by day 71 post tumor inoculation, compared to only 1 in 4 in the MV-treated group. At humane end point, all the mice were evaluated for leukaemia burden in bone marrow (BM) and spleen (SP), size of SP, and the level of hGCSF in their serum. All SPs were subjected to flow cytometric analysis for assessment of CD11b, Ly6G6C, NK and Mac3. For all treatment groups, human CD10/ CD19 expressing cells were present, demonstrating the presence of ALL in all mice at the time of death. SPs were significantly larger in the MVhGCSF treated group compared to the MV or MV-UV group, but were heavily infiltrated by PMNs, rather than ALL, with 35-40% positivity for Cd11b and Ly6G6C as compared to the MV (2%) and MV-UV (10%) treated groups. Significant levels of hGCSF were detected in the serum of MVhGCSF-treated mice (0.3-1.13ng/ml) by ELISA. In terms of survival, mice treated with MVhGCSF did worse as compared to the unmodified MV-treated mice, and this was unexpected. The large SP size, heavy splenic infiltration by PMNs, and high levels of hGCSF in the serum of MVhGCSF-treated mice was suggestive of significant GCSF-mediated toxicity in this group, which may have contributed to their inferior survival. However, levels of leukaemic infiltration in the BM were comparable to the MV-treated group, suggesting no therapeutic benefit of MVhGCSF over MV-NSe. Work is ongoing to dissect whether PMNs might be unexpectedly operating to promote tumor growth in the MVhGSCFtreated Nalm-6 ALL disseminated model.
648. Combinatorial Treatment of Oncolytic Ad With Helper-Dependent Ad Augments the AntiTumor Effect of Adenoviral Cancer Gene Therapy
Lisa Farzad,1 Vincenzo Cerullo,2 Akseli Hemminki,2 Cliona Rooney,1 Brendan Lee,1 Malcolm Brenner,1 Masataka Suzuki.1 1 Center for Cell and Gene Therapy, Houston Methodist Hospital, Texas Children’s Hospital and Baylor College of Medicine, Houston, TX; 2University of Helsinki, Helsinki, Finland. Adenoviral vectors (Ads) can effectively enter malignant cells and express high levels of lytic or immunogenic transgenes, making them attractive agents for cancer gene therapy. However, the antitumor activity of oncolytic Ads rarely eliminates patient tumors. Investigators have therefore incorporated immunomodulatory molecules such as pro-inflammatory cytokines that can enhance anti-tumor immunity in host animals as well as in patients with Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy