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63. Immunogenicity of Self Tumor Associated Antigens Is Enhanced Through Protein Truncation
Cancer-Oncolytic RNA Viruses 63. Immunogenicity of Self Tumor Associated Antigens Is Enhanced Through Protein Truncation
Tim Kottke1, Kevin G. Shim1, Laura Evgin1, Vanesa AlonsoCamino1, Shane Zaidi1, Rosa Diaz1, Jose Pulido1, Jill Thompson1, Karishma Rajani1, Amanda Huff1, Elizabeth Ilett2, Hardev Pandha3, Kevin Harrington4, Peter Selby2, Alan Melcher2, Richard Vile1 1 Mayo Clinic, Rochester, MN, 2University of Leeds, Leeds, United Kingdom, 3University of Surrey, Guildford, United Kingdom, 4The Institute of Cancer Research, London, United Kingdom We showed previously that expressing tumor-associated antigens (TAA) from Vesicular Stomatitis Virus (VSV) eradicates established tumors. We show here that truncation of TAA expressed from VSV can occur to preserve the ability of the virus to replicate efficiently. We observed that truncation of VSV-expressed TAA affects the processing of the antigen, causing a bias towards an IL-17 anti-tumor response which was raised by cumulative signaling from different types of APC, each presenting specific, truncated antigens. Whereas processing of full length, self-TAA invoked an IFN-γ based, CD8+ dependent response, truncated versions of the same self-TAA (likely to be poorly and incompletely folded) were processed through a class II-dependent pathway, and invoked an IL-17 based response. Significantly, the IL-17-mediated anti tumor response was both more therapeutic, and durable, than the response against full length selfTAA. These data show that the type/potency of anti-tumor immune responses against self-TAA can be manipulated in vivo through the nature of the self protein (full length or truncated), inclusion of multiple TAA to recruit the optimal combination of APC, and the resultant skewing of the T cell response to either an IFN-γ or IL-17 producing phenotype. Therefore, in addition to generation of neoantigens through sequence mutation, immunological tolerance against self-TAA can be broken through manipulation of protein integrity, allowing for rational design of better self immunogens for cancer immunotherapy.
64. Generation of Tumor Cells Resistant to Oncolysis Is Mediated Through Virus Induced APOBEC Expression
Karishma Rajani1, Kevin G. Shim1, Nazanin Yeganeh Kazemi1, William Gendron1, Tim Kottke1, Amy Molan1, Christopher Driscoll1, Reuben Harris2, Richard Vile1 1 Mayo Clinic, Rochester, MN, 2University of Alberta, Edmonton, ON, Canada In both pre-clinical, and clinical models, we have observed that treatment of primary tumors with oncolytic viruses can lead to very significant tumor regressions, often with apparent disappearance of the tumor. However, in many cases, tumor subsequently recurs, often extremely aggressively. We have been able to mimic this effect in vitro by growing several different types of tumor cell lines in the presence of different oncolytic viruses at low m.o.i. over several weeks. Under such conditions, we consistently observe the emergence of cells which survive over long periods of time in culture, despite the demonstrable presence of ongoing viral replication. We identified APOBEC3 from a screen of genes which are induced in tumor cells undergoing continual exposure to either reovirus or Vesicular Stomatitis Virus (VSV). In this respect, overexpression of APOBEC3B, a cytidine deaminase, has been identified in human tumors associated with mutations that may drive tumorigenesis. Therefore, we tested the hypothesis that, upon infection with oncolytic viruses, APOBEC3 may help drive tumor cell mutation leading to protection from viral cytotoxicity. Consistent with this, both mRNA and protein levels of APOBEC3 were rapidly induced within 24-72 hours of low M.O.I infection by reovirus, or VSV, of B16 melanoma, GL261 glioma and TC2 prostate tumor murine cell lines. Similar low level infection of human tumor cell lines was associated with rapid induction of the human APOBEC3B S28
mRNA. Interestingly, engineered over-expression of APOBEC3B in tumor lines significantly enhanced the ability of these cells to resist killing by either VSV or reovirus. This effect was inhibited by blockade of PKC signaling upon viral infection but was enhanced by the presence of type I interferons. Correspondingly, inhibition of APOBEC3 using shRNA decreased the frequency of emergence of VSV-resistant tumor populations. These data suggest that infection of tumor cells by oncolytic viruses at low M.O.I (as is likely to be the case during clinical treatments) leads to the induction of cellular proteins, which enhance the ability of resistance to oncolysis to develop. Deep sequencing studies are currently underway to determine the genetic changes in both virus, and target tumor cells, which are associated with APOBEC3 over-expression during chronic exposure to oncolytic virus infection. Finally, data will be presented on how these findings allow the construction of improved viruses for cancer therapy by targeting APOBEC3 induction to improve primary killing and prevent emergence of treatment resistant populations.
65. Oncolytic Measles Virus Differentially Affects Mitochondrial Biogenesis in Transformed versus Non-Transformed BM-Derived MSCs
Sarah Aref1, Aditi Dey1, Katharine Bailey1, Benjamin Rosen2, Anna Castleton1, Gyorgy Szabadkai3, Adele K. Fielding1 1 UCL Cancer Institute, Paul O’Gorman Building, University College London, London, United Kingdom, 2University College London, London, United Kingdom, 3Division of Biosciences, University College of London, London, United Kingdom Vaccine strain measles virus (MV) is oncolytic in numerous models of malignancy. The mechanism behind the selectivity of MV for transformed cells is poorly understood. To investigate further, an established step-wise model of cellular transformation was used; in which progressive oncogenic hits were stably and additively expressed in human bone marrow derived mesenchymal stromal cells following retroviral transfer of human telomerase reverse transcriptase (hTERT), human papilloma virus16 E6 and E7 (3H), SV40 small T antigen (4+V), finally, H-RAS (5H) (Funes at al, 2007). The most highly transformed cells (5H) were more permissive to oncolytic MV infection than any of the less transformed counterparts, with significantly greater viral titres. MV-induced cell-death increased progressively with progressive transformation. This was not explained by any differences in MV receptors CD46, SLAM or Nectin-4 expression. Investigation of anti-viral type 1 IFN response in this model 24 and 48 hours post MV infection (hpi) by ELISA demonstrated a robust induction of IFNβ (to a lesser extent IFNα) in hTERT cells, which was significantly and progressively reduced in 3H, 4+V and 5H according to level of transformation, suggesting that defective IFN pathway is a potential mechanism for the enhanced MV permissiveness observed in transformed cells. Examination of integrity of the RLR signalling pathway, which triggers IFNα/β production, revealed that expression levels of RIG-I, MDA5 and MAVS, determined by RQ-PCR at 6, 12, 24 and 48 hpi, were lowest in 5H and highest in hTERT cells, proposing a role for the RLR pathway in MV-mediated oncolysis. To gain insight into the differential effects of MV infection in transformed versus non-transformed cells, metabolic effects post MV infection were investigated. A significant increase in oxygen consumption rate (OCR) was observed 24 hpi, proportional to the stage of transformation, followed by a steep decline at 48 hpi. This increase was abrogated in the presence of fusion inhibitory peptide (FIP), implicating the role of syncytia formation, the cytopathic effect of MV infection, in MV-mediated oncolysis in vitro. A similar pattern was observed in ATP levels post MV infection; highest at 24hpi in 4+V and 5H and dropping off dramatically at 48hpi when cell viability is compromised. To further characterize the consistent increase in OCR and ATP levels Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Tim Kottke1, Kevin G. Shim1, Laura Evgin1, Vanesa AlonsoCamino1, Shane Zaidi1, Rosa Diaz1, Jose Pulido1, Jill Thompson1, Karishma Rajani1, Amanda Huff1, Elizabeth Ilett2, Hardev Pandha3, Kevin Harrington4, Peter Selby2, Alan Melcher2, Richard Vile1 1 Mayo Clinic, Rochester, MN, 2University of Leeds, Leeds, United Kingdom, 3University of Surrey, Guildford, United Kingdom, 4The Institute of Cancer Research, London, United Kingdom We showed previously that expressing tumor-associated antigens (TAA) from Vesicular Stomatitis Virus (VSV) eradicates established tumors. We show here that truncation of TAA expressed from VSV can occur to preserve the ability of the virus to replicate efficiently. We observed that truncation of VSV-expressed TAA affects the processing of the antigen, causing a bias towards an IL-17 anti-tumor response which was raised by cumulative signaling from different types of APC, each presenting specific, truncated antigens. Whereas processing of full length, self-TAA invoked an IFN-γ based, CD8+ dependent response, truncated versions of the same self-TAA (likely to be poorly and incompletely folded) were processed through a class II-dependent pathway, and invoked an IL-17 based response. Significantly, the IL-17-mediated anti tumor response was both more therapeutic, and durable, than the response against full length selfTAA. These data show that the type/potency of anti-tumor immune responses against self-TAA can be manipulated in vivo through the nature of the self protein (full length or truncated), inclusion of multiple TAA to recruit the optimal combination of APC, and the resultant skewing of the T cell response to either an IFN-γ or IL-17 producing phenotype. Therefore, in addition to generation of neoantigens through sequence mutation, immunological tolerance against self-TAA can be broken through manipulation of protein integrity, allowing for rational design of better self immunogens for cancer immunotherapy.
64. Generation of Tumor Cells Resistant to Oncolysis Is Mediated Through Virus Induced APOBEC Expression
Karishma Rajani1, Kevin G. Shim1, Nazanin Yeganeh Kazemi1, William Gendron1, Tim Kottke1, Amy Molan1, Christopher Driscoll1, Reuben Harris2, Richard Vile1 1 Mayo Clinic, Rochester, MN, 2University of Alberta, Edmonton, ON, Canada In both pre-clinical, and clinical models, we have observed that treatment of primary tumors with oncolytic viruses can lead to very significant tumor regressions, often with apparent disappearance of the tumor. However, in many cases, tumor subsequently recurs, often extremely aggressively. We have been able to mimic this effect in vitro by growing several different types of tumor cell lines in the presence of different oncolytic viruses at low m.o.i. over several weeks. Under such conditions, we consistently observe the emergence of cells which survive over long periods of time in culture, despite the demonstrable presence of ongoing viral replication. We identified APOBEC3 from a screen of genes which are induced in tumor cells undergoing continual exposure to either reovirus or Vesicular Stomatitis Virus (VSV). In this respect, overexpression of APOBEC3B, a cytidine deaminase, has been identified in human tumors associated with mutations that may drive tumorigenesis. Therefore, we tested the hypothesis that, upon infection with oncolytic viruses, APOBEC3 may help drive tumor cell mutation leading to protection from viral cytotoxicity. Consistent with this, both mRNA and protein levels of APOBEC3 were rapidly induced within 24-72 hours of low M.O.I infection by reovirus, or VSV, of B16 melanoma, GL261 glioma and TC2 prostate tumor murine cell lines. Similar low level infection of human tumor cell lines was associated with rapid induction of the human APOBEC3B S28
mRNA. Interestingly, engineered over-expression of APOBEC3B in tumor lines significantly enhanced the ability of these cells to resist killing by either VSV or reovirus. This effect was inhibited by blockade of PKC signaling upon viral infection but was enhanced by the presence of type I interferons. Correspondingly, inhibition of APOBEC3 using shRNA decreased the frequency of emergence of VSV-resistant tumor populations. These data suggest that infection of tumor cells by oncolytic viruses at low M.O.I (as is likely to be the case during clinical treatments) leads to the induction of cellular proteins, which enhance the ability of resistance to oncolysis to develop. Deep sequencing studies are currently underway to determine the genetic changes in both virus, and target tumor cells, which are associated with APOBEC3 over-expression during chronic exposure to oncolytic virus infection. Finally, data will be presented on how these findings allow the construction of improved viruses for cancer therapy by targeting APOBEC3 induction to improve primary killing and prevent emergence of treatment resistant populations.
65. Oncolytic Measles Virus Differentially Affects Mitochondrial Biogenesis in Transformed versus Non-Transformed BM-Derived MSCs
Sarah Aref1, Aditi Dey1, Katharine Bailey1, Benjamin Rosen2, Anna Castleton1, Gyorgy Szabadkai3, Adele K. Fielding1 1 UCL Cancer Institute, Paul O’Gorman Building, University College London, London, United Kingdom, 2University College London, London, United Kingdom, 3Division of Biosciences, University College of London, London, United Kingdom Vaccine strain measles virus (MV) is oncolytic in numerous models of malignancy. The mechanism behind the selectivity of MV for transformed cells is poorly understood. To investigate further, an established step-wise model of cellular transformation was used; in which progressive oncogenic hits were stably and additively expressed in human bone marrow derived mesenchymal stromal cells following retroviral transfer of human telomerase reverse transcriptase (hTERT), human papilloma virus16 E6 and E7 (3H), SV40 small T antigen (4+V), finally, H-RAS (5H) (Funes at al, 2007). The most highly transformed cells (5H) were more permissive to oncolytic MV infection than any of the less transformed counterparts, with significantly greater viral titres. MV-induced cell-death increased progressively with progressive transformation. This was not explained by any differences in MV receptors CD46, SLAM or Nectin-4 expression. Investigation of anti-viral type 1 IFN response in this model 24 and 48 hours post MV infection (hpi) by ELISA demonstrated a robust induction of IFNβ (to a lesser extent IFNα) in hTERT cells, which was significantly and progressively reduced in 3H, 4+V and 5H according to level of transformation, suggesting that defective IFN pathway is a potential mechanism for the enhanced MV permissiveness observed in transformed cells. Examination of integrity of the RLR signalling pathway, which triggers IFNα/β production, revealed that expression levels of RIG-I, MDA5 and MAVS, determined by RQ-PCR at 6, 12, 24 and 48 hpi, were lowest in 5H and highest in hTERT cells, proposing a role for the RLR pathway in MV-mediated oncolysis. To gain insight into the differential effects of MV infection in transformed versus non-transformed cells, metabolic effects post MV infection were investigated. A significant increase in oxygen consumption rate (OCR) was observed 24 hpi, proportional to the stage of transformation, followed by a steep decline at 48 hpi. This increase was abrogated in the presence of fusion inhibitory peptide (FIP), implicating the role of syncytia formation, the cytopathic effect of MV infection, in MV-mediated oncolysis in vitro. A similar pattern was observed in ATP levels post MV infection; highest at 24hpi in 4+V and 5H and dropping off dramatically at 48hpi when cell viability is compromised. To further characterize the consistent increase in OCR and ATP levels Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy