- Email: [email protected]
25. Combined Armed Oncolytic Virus and CAR-T Cells Counter the Hostile Environment of Solid Tumors
CANCER-ONCOLYTIC VIRUSES consistent with acute liver failure. Biochemical and histological analyses confirmed the diagnosis of severe liver injury, characterized by diffuse and severe hepatocellular damage, in addition to hepatic fibrosis. In conclusion, FAH-/- pigs provide the first genetically engineered large animal model of an inborn error of metabolism in the liver and should provide a significant advancement for translational research in metabolic liver disease using myriad gene and cell therapy approaches.
Cancer-Oncolytic Viruses 25. Combined Armed Oncolytic Virus and CAR-T Cells Counter the Hostile Environment of Solid Tumors
Nobuhiro Nishio,1 Iulia Diaconu,1 Vincenzo Cerullo,2 Hao Liu,3 Valentina Hoyos,1 Lisa Bouchier-Hayes,1 Barbara Savoldo,1 Gianpietro Dotti.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston; 2ImmunoViroTherapy, University of Helsinki, Helsinki, Finland; 3Biostatistics Core, Baylor College of Medicine, Houston. The clinical efficacy of chimeric antigen receptor (CAR)redirected T cells remains marginal in solid tumors compared to leukemia. Insufficient T-cell migration to the tumor and a highly immunosuppressive environment are major contributors to the failure of T-cell based immunotherapy in solid tumors. To overcome these obstacles, we have combined CAR-T cells with oncolytic virus (OV)-based therapy, with the rationale that the latter will have both a direct effect on infected malignant cells and can counter the unfavorable tumor environment if appropriately engineered. We first tested the combination of the OV Ad5Δ24 with T cells expressing a 3rd generation GD2-specific CAR (GD2.CAR-T cells) in a neuroblastoma (NB) model. Ex vivo, Ad5Δ24 exerted a potent, dose dependent, cytotoxic effect on 5/7 NB cell lines, while GD2. CAR-T cells were unaffected. When used in combination, Ad5Δ24 and GD2.CAR-T cells eliminated NB cells in co-culture experiments significantly faster than monotherapies (residual tumor cells by day 7; 30±7% in GD2.CAR-T cells alone, 33±7% in Ad5Δ24 and control T cells, and less than 5% when exposed to both Ad5Δ24 and GD2. CAR-T cells, p=0.01). Importantly, the superior antitumor effect of the combination therapy was mediated by a more rapid activation of the caspase cascade. In NSG mice engrafted with NB cells, combined Ad5Δ24 and GD2.CAR-T cells showed significantly better control of tumor growth/volume at the lowest dose (106 vp) of Ad5Δ24 tested compared to Ad5Δ24 alone (789 vs. 1452 mm3 at day 20, p=0.04) or GD2.CAR-T cells alone (789 vs. 2340 mm3 at day 20, p=0.02) treated mice. However, since T-cell migration and persistence at the tumor site remained suboptimal, we engineered the Ad5Δ24 to produce both the chemokine RANTES and the cytokine IL-15 (Ad5Δ24. RANTES.IL-15) upon infection of NB cells, to attract T cells at the tumor site and promote their local survival. NB cells infected with Ad5Δ24.RANTES.IL-15 produced both RANTES and IL-15 in vitro (2.2 and 2.1 ng/ml at day 3 post infection, respectively) and in vivo (5.8 and 1.7 ng/ml at the tumor site, 18-25 days post infection, respectively) without compromising the oncolytic properties of the OV. Both transgenes were functional, so that supernatants from Ad5Δ24.RANTES.IL-15-infected NB cells improved the migration and expansion of GD2.CAR-T cells in vitro. In the NB xenograft mouse model, bioluminescent imaging showed that both migration and proliferation of GD2.CAR-T cells at the Ad5Δ24.RANTES. IL-15-injected tumor sites were dramatically improved as compared to the combination of GD2.CAR-T cells and Ad5Δ24 (2.1x107 vs. 5.6x105 p/s/cm2/sr at day 12, respectively, p<0.001). This enhanced T-cell migration/expansion increased overall survival (73% vs. 44% at day 45, respectively, p=0.03). In conclusion our preclinical data S10
support an innovative platform of entirely biological components for therapy of solid tumors that overcomes the unfavorable cancer environment.
26. Measles Virus Rapid and Discreet Epithelial Spread Through Nectin-4: Implications for Oncolytic Virotherapy
Mathieu Mateo,1 Patrick L. Sinn,2 Chanakha K. Navaratnarajah,1 Crystal Mendoza,1 Ryan Donohue,1 Roberto Cattaneo.1 1 Molecular Medicine, Mayo Clinic, Rochester, MN; 2Pediatrics, University of Iowa, Iowa City, IA. Spread of several viruses used for cancer therapy is mainly cell-associated. Direct cell-cell spread without budding has three advantages: speed, immune evasion, and bypass of physical barriers. We discovered that measles virus (MeV) uses nectin-4, an adherens junction (AJ) protein with epithelium-organizing functions to spread in epithelia (1). We now present evidence that MeV takes advantage of the epithelium-organizing function of nectins in two ways. First, the MeV attachment protein hemagglutinin (H) binds nectin-4 through its adhesive surface, displacing interactions of nectins located on apposed cells. We showed this by generating mutants of the nectin-4 adhesive surface and assessing their ability to function as receptors. We focused on three loops of the variable domain: the BC, C’C’’ and FG loops. We identified three amino acids in the nectin-4 FG loop crucial for functional interactions with H; in addition, residues in other two loops influence these interactions. Using surface plasmon resonance, we demonstrated that the MeV attachment protein H requires integrity of the BC and FG loop to bind. Thus, H-binding to the nectin adhesive surface localizes the viral membrane fusion apparatus to the AJ. Second, we observed that MeV spreads rapidly in reconstituted human airways epithelial sheets without affecting function. In particular, we observed that cytoplasmic contents flow from infected to contiguous cells from the expected location of the AJ. This occurs without visible syncytia formation, and while transepithelial resistance is maintained. Thus fusion pores are formed but do not expand, possibly due to the tissue architecture around the AJ, which is stabilized by the actin and myosin cytoskeletal belt. We also observed that viral components are efficiently transported through secondarily infected cells, rapidly reaching the AJ connecting to the next cell. We are currently assessing which cytoskeletal filaments move the viral components. Our findings suggest that MeV infection induce the formation of canals: constrained membranes pores started by the viral fusion apparatus. These canals would connect the cytoskeletal belts of adjacent epithelial cells, thereby creating a rapid transit system for the viral components. These observations have implications for the spread of viruses used in other cancer clinical trials: nectins-1 and -2 are primary receptors for herpes simplex virus-1 and -2, and nectin-like protein 5 is the primary poliovirus receptor. Moreover, tight junction proteins like the junction adhesion molecule 1 and the coxackie-adenovirus receptor serve as receptors for reoviruses and adenoviruses, respectively. Spread of all these viruses could be facilitated by newly connected cytoskeletal networks of adjacent cells. Moreover, general accessibility of junction proteins in disordered cancer tissue would facilitate viral entry, contributing to efficient oncolysis. REFERENCE: (1) Muehlebach et al. (2011) Nature 480, 530-533.
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Cancer-Oncolytic Viruses 25. Combined Armed Oncolytic Virus and CAR-T Cells Counter the Hostile Environment of Solid Tumors
Nobuhiro Nishio,1 Iulia Diaconu,1 Vincenzo Cerullo,2 Hao Liu,3 Valentina Hoyos,1 Lisa Bouchier-Hayes,1 Barbara Savoldo,1 Gianpietro Dotti.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston; 2ImmunoViroTherapy, University of Helsinki, Helsinki, Finland; 3Biostatistics Core, Baylor College of Medicine, Houston. The clinical efficacy of chimeric antigen receptor (CAR)redirected T cells remains marginal in solid tumors compared to leukemia. Insufficient T-cell migration to the tumor and a highly immunosuppressive environment are major contributors to the failure of T-cell based immunotherapy in solid tumors. To overcome these obstacles, we have combined CAR-T cells with oncolytic virus (OV)-based therapy, with the rationale that the latter will have both a direct effect on infected malignant cells and can counter the unfavorable tumor environment if appropriately engineered. We first tested the combination of the OV Ad5Δ24 with T cells expressing a 3rd generation GD2-specific CAR (GD2.CAR-T cells) in a neuroblastoma (NB) model. Ex vivo, Ad5Δ24 exerted a potent, dose dependent, cytotoxic effect on 5/7 NB cell lines, while GD2. CAR-T cells were unaffected. When used in combination, Ad5Δ24 and GD2.CAR-T cells eliminated NB cells in co-culture experiments significantly faster than monotherapies (residual tumor cells by day 7; 30±7% in GD2.CAR-T cells alone, 33±7% in Ad5Δ24 and control T cells, and less than 5% when exposed to both Ad5Δ24 and GD2. CAR-T cells, p=0.01). Importantly, the superior antitumor effect of the combination therapy was mediated by a more rapid activation of the caspase cascade. In NSG mice engrafted with NB cells, combined Ad5Δ24 and GD2.CAR-T cells showed significantly better control of tumor growth/volume at the lowest dose (106 vp) of Ad5Δ24 tested compared to Ad5Δ24 alone (789 vs. 1452 mm3 at day 20, p=0.04) or GD2.CAR-T cells alone (789 vs. 2340 mm3 at day 20, p=0.02) treated mice. However, since T-cell migration and persistence at the tumor site remained suboptimal, we engineered the Ad5Δ24 to produce both the chemokine RANTES and the cytokine IL-15 (Ad5Δ24. RANTES.IL-15) upon infection of NB cells, to attract T cells at the tumor site and promote their local survival. NB cells infected with Ad5Δ24.RANTES.IL-15 produced both RANTES and IL-15 in vitro (2.2 and 2.1 ng/ml at day 3 post infection, respectively) and in vivo (5.8 and 1.7 ng/ml at the tumor site, 18-25 days post infection, respectively) without compromising the oncolytic properties of the OV. Both transgenes were functional, so that supernatants from Ad5Δ24.RANTES.IL-15-infected NB cells improved the migration and expansion of GD2.CAR-T cells in vitro. In the NB xenograft mouse model, bioluminescent imaging showed that both migration and proliferation of GD2.CAR-T cells at the Ad5Δ24.RANTES. IL-15-injected tumor sites were dramatically improved as compared to the combination of GD2.CAR-T cells and Ad5Δ24 (2.1x107 vs. 5.6x105 p/s/cm2/sr at day 12, respectively, p<0.001). This enhanced T-cell migration/expansion increased overall survival (73% vs. 44% at day 45, respectively, p=0.03). In conclusion our preclinical data S10
support an innovative platform of entirely biological components for therapy of solid tumors that overcomes the unfavorable cancer environment.
26. Measles Virus Rapid and Discreet Epithelial Spread Through Nectin-4: Implications for Oncolytic Virotherapy
Mathieu Mateo,1 Patrick L. Sinn,2 Chanakha K. Navaratnarajah,1 Crystal Mendoza,1 Ryan Donohue,1 Roberto Cattaneo.1 1 Molecular Medicine, Mayo Clinic, Rochester, MN; 2Pediatrics, University of Iowa, Iowa City, IA. Spread of several viruses used for cancer therapy is mainly cell-associated. Direct cell-cell spread without budding has three advantages: speed, immune evasion, and bypass of physical barriers. We discovered that measles virus (MeV) uses nectin-4, an adherens junction (AJ) protein with epithelium-organizing functions to spread in epithelia (1). We now present evidence that MeV takes advantage of the epithelium-organizing function of nectins in two ways. First, the MeV attachment protein hemagglutinin (H) binds nectin-4 through its adhesive surface, displacing interactions of nectins located on apposed cells. We showed this by generating mutants of the nectin-4 adhesive surface and assessing their ability to function as receptors. We focused on three loops of the variable domain: the BC, C’C’’ and FG loops. We identified three amino acids in the nectin-4 FG loop crucial for functional interactions with H; in addition, residues in other two loops influence these interactions. Using surface plasmon resonance, we demonstrated that the MeV attachment protein H requires integrity of the BC and FG loop to bind. Thus, H-binding to the nectin adhesive surface localizes the viral membrane fusion apparatus to the AJ. Second, we observed that MeV spreads rapidly in reconstituted human airways epithelial sheets without affecting function. In particular, we observed that cytoplasmic contents flow from infected to contiguous cells from the expected location of the AJ. This occurs without visible syncytia formation, and while transepithelial resistance is maintained. Thus fusion pores are formed but do not expand, possibly due to the tissue architecture around the AJ, which is stabilized by the actin and myosin cytoskeletal belt. We also observed that viral components are efficiently transported through secondarily infected cells, rapidly reaching the AJ connecting to the next cell. We are currently assessing which cytoskeletal filaments move the viral components. Our findings suggest that MeV infection induce the formation of canals: constrained membranes pores started by the viral fusion apparatus. These canals would connect the cytoskeletal belts of adjacent epithelial cells, thereby creating a rapid transit system for the viral components. These observations have implications for the spread of viruses used in other cancer clinical trials: nectins-1 and -2 are primary receptors for herpes simplex virus-1 and -2, and nectin-like protein 5 is the primary poliovirus receptor. Moreover, tight junction proteins like the junction adhesion molecule 1 and the coxackie-adenovirus receptor serve as receptors for reoviruses and adenoviruses, respectively. Spread of all these viruses could be facilitated by newly connected cytoskeletal networks of adjacent cells. Moreover, general accessibility of junction proteins in disordered cancer tissue would facilitate viral entry, contributing to efficient oncolysis. REFERENCE: (1) Muehlebach et al. (2011) Nature 480, 530-533.
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy