- Email: [email protected]
172. Inhibition of Indoleamine 2,3-Dioxygenase in Dendritic Cells In Vivo Elicits Antitumor Responses
Cancer - Immunotherapy: Cytokine Gene Therapy, Dendritic Cells, and Modified Effector Cells challenged subcutaneously with N18 cells. Vaccination with G47∆infected N18 loaded FL-DCs caused a significant prolongation in median survival compared to mock treatment (RPMI injection) or FL-DCs pulsed with mock-infected N18 (p<0.05 log rank test). Mice vaccinated with G47∆-infected N18 loaded FL-DCs elicited a significant CTL response compared to the mock group. Furthermore, long-term immune memory was obtained as most mice treated with G47∆-infected N18 loaded FL-DCs remained tumor-free after a second challenge with a lethal dose of N18 cells 4 months after the first challenge. Taken together our results demonstrate that oHSVinfected tumor cells are a promising candidate to load/activate immature DCs.
172. Inhibition of Indoleamine 2,3-Dioxygenase in Dendritic Cells In Vivo Elicits Antitumor Responses Meng-Chi Yen,1,2 Chi-Chen Lin,1 Ming-Derg Lai.1,2 Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan; 2 Institute of Basic Medicincal Sciences of NCKU, Tainan City, Taiwan.
1
Indoleamine 2,3-dioxygenase (IDO), an enzyme that degrades tryptophan, is known as an immune regulatory molecule of dendritic cells. IDO-expressing dendritic cells from tumor-draining lymph nodes suppress T cell responses in vitro and may create an immune suppressive microenvironment in vivo. It was reported that intradermal administration of nucleic acids was efficiently delivered to skin resident dendritic cells which can migrate to lymph nodes in vivo. Thus, we hypothesized that silencing the IDO expressing in DCs in vivo via skin administration of IDO specific small interfering RNA (siRNA) by gene gun could alter the microenvironment in tumor draining lymph node and elicit the antitumor activity. In the present study, IDO siRNA was used to silence the IDO expression of dendritic cells in lymph nodes. IDO expression could be downregulated in CD11c+ cells from inguinal lymph node of mice treated with siRNA. Furthermore, IDO siRNA inhibited the tumor growth and prolonged the survival in a murine MBT-2 bladder tumor model. The number of tumor infiltrated T cells and then cytotoxic activity significantly increased in mice vaccinated with IDO siRNA. A similar antitumor effect was observed in a CT-26 colon tumor model. To further investigate whether the tumor antigen was more efficiently presented to dendritic cells on IDO siRNA treated mice, the combination effect of Her2/Neu DNA vaccine and IDO siRNA was examined in a neu- overexpression MBT-2 tumor model. The IDO siRNA could further enhance therapeutic efficacy of neu DNA vaccine. These results indicated that silencing the IDO expression in dendritic cells in vivo was sufficient to induce antitumor effect and serve as immune adjuvant for increasing therapeutic effect of DNA vaccine.
173. Dendritic Cells Transduced with Granulocyte Macrophage Colony Stimulating Factor Mediated by Non-Transmissible Sendai Virus Induce Therapeutic Antitumor Immunity in LLC Bearing Mice
Hiroyuki Inoue,1,2 Mutsunori Iga,1 Haruka Nabeta,1 Meng Xin,1 Ryo Kurita,1 Koichi Takayama,2 Makoto Inoue,3 Mamoru Hasegawa,3 Yoichi Nakanishi,2 Kenzaburo Tani.1 1 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Research Institute of Diseases of the Chest, Kyushu University, Fukuoka, Japan; 3DNAVEC Corporation, Tsukuba, Ibaraki, Japan.
Recently, several studies have shown that vaccine therapy using dendritic cells (DCs) genetically engineered to express inflammatory cytokines with or without a surrogate tumor antigen can effectively Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
induce antitumor immunity. In this study, murine bone marrow DCs were transduced with murine granulocyte macrophage colony stimulating factor (GM-CSF) by novel non-transmissible Sendai virus vector (SeV/dF) (DC/SeV/GM). SeV is a murine parainfluenza virus type I belonging to the family Paramyxoviridae and a cytoplasmic RNA vector considered to be another promising device in gene transduction for its capacity to infect and amplify in most mammalian cells including DCs. And it possesses an exclusively cytoplasmic replication cycle without any risk of integration into the genomic DNA. We examined whether antigen-specific CTL responses and therapeutic immunity could be induced in immunocompetent mice bearing LLC (murine Lewis lung carcinoma) vaccinated with GMCSF gene transduced and tumor lysate-pulsed mature DCs. SeV encoding green fluorescence protein (GFP), SeV/GFP, successfully transduced to bone marrow derived DCs (DC/SeV/GFP) with the optimum at MOI of 100. In vitro treatment with SeV/GFP and SeV/GM-CSF directed DCs to mature phenotype. The LLC tumor lysate pulse during DCs maturation did not largely influence on their phenotypic expression profiles. And the several inflammatory cytokines including GM-CSF in the supernatant of cultured mature DCs were quantified. Even though the expression level of DC activation surface markers of CD40 and B7-2 (CD86), did not attain the level induced by optimum level of LPS (1µg/ml), better antitumor effects to LLC were obtained with the use of DC/SeV/ GM compared with those seen with DC/LPS and DC/SeV/GFP in preestablished subcutaneous LLC model. During the therapeutic DCs vaccines, mice were well tolerated. These results demonstrated that SeV mediated GM-CSF gene transduction significantly enhanced the capacity of DCs to induce primary antitumor immune responses in vivo, although further investigation is required to achieve its optimum in vivo antitumor effects.
174. Effective Prevention of Lung Metastasis of Murine Model by Sendai Virus/Dendritic CellMediated Immunostimulatory Virotherapy
Atsushi Komaru,1,2 Yasuji Ueda,1 Hiroaki Kinoh,1 Tomonori Kato,1,2 Yui Harada,1,2 Hiroyoshi Suzuki,2 Aki Furuya,2 Kumi Ishida,2 Makoto Inoue,3 Mamoru Hasegawa,3 Tomohiko Ichikawa,2 Yoshikazu Yonemitsu.1 1 Gene Therapy at the 21st Century COE Program, Chiba University Graduate School of Medicine, Chiba, Japan; 2Urology, Chiba University Graduate School of Medicine, Chiba, Japan; 3 DNAVEC Corporation, Tsukuba, Japan. We recently demonstrated the highly efficient antitumor immunity against dermal tumor of B16F10 murine melanoma in use of dendritic cells (DCs) activated by non-transmissible recombinant Sendai virus (rSeV), proposing a new concept namely ‘immunostimulatory virotherapy’ for cancer immunotherapy. However, critical information regarding the efficacies 1) under more clinically relevant situations including metastatic diseases, 2) in use of clinically relevant vector types and transgene(s), 3) on other tumor type and other animal species, and 4) the related molecular/cellular mechanisms have been largely unknown. In this study, therefore, we investigated the efficacy of vaccination of DCs activated by fusion gene-deleted non-transmissible rSeV expressing GFP (rSeV/dF-GFP), on murine model of lung metastasis using mouse neuroblastoma, C1300 and mouse prostate cancer, RM9. rSeV/dF-GFP activated bone marrowderived DCs (rSeV/dF-GFP-DC), consistent results previously seen in murine DCs. Vaccination of rSeV/dF-GFP-DC was highly effective to prevent lung metastasis following intravenous load of tumor cells, compared with the effects seen with immature DCs. Importantly, rSeV/dF-DCs expressing dominant negative mutant of retinoic acid-inducible gene I (RIG-I) (rSeV/dF-RIGIC-DC), a RNA helicase that recognizes rSeV genome for inducing type I interferons, largely lost the expression of proinflammatory cytokines without any S65
172. Inhibition of Indoleamine 2,3-Dioxygenase in Dendritic Cells In Vivo Elicits Antitumor Responses Meng-Chi Yen,1,2 Chi-Chen Lin,1 Ming-Derg Lai.1,2 Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan; 2 Institute of Basic Medicincal Sciences of NCKU, Tainan City, Taiwan.
1
Indoleamine 2,3-dioxygenase (IDO), an enzyme that degrades tryptophan, is known as an immune regulatory molecule of dendritic cells. IDO-expressing dendritic cells from tumor-draining lymph nodes suppress T cell responses in vitro and may create an immune suppressive microenvironment in vivo. It was reported that intradermal administration of nucleic acids was efficiently delivered to skin resident dendritic cells which can migrate to lymph nodes in vivo. Thus, we hypothesized that silencing the IDO expressing in DCs in vivo via skin administration of IDO specific small interfering RNA (siRNA) by gene gun could alter the microenvironment in tumor draining lymph node and elicit the antitumor activity. In the present study, IDO siRNA was used to silence the IDO expression of dendritic cells in lymph nodes. IDO expression could be downregulated in CD11c+ cells from inguinal lymph node of mice treated with siRNA. Furthermore, IDO siRNA inhibited the tumor growth and prolonged the survival in a murine MBT-2 bladder tumor model. The number of tumor infiltrated T cells and then cytotoxic activity significantly increased in mice vaccinated with IDO siRNA. A similar antitumor effect was observed in a CT-26 colon tumor model. To further investigate whether the tumor antigen was more efficiently presented to dendritic cells on IDO siRNA treated mice, the combination effect of Her2/Neu DNA vaccine and IDO siRNA was examined in a neu- overexpression MBT-2 tumor model. The IDO siRNA could further enhance therapeutic efficacy of neu DNA vaccine. These results indicated that silencing the IDO expression in dendritic cells in vivo was sufficient to induce antitumor effect and serve as immune adjuvant for increasing therapeutic effect of DNA vaccine.
173. Dendritic Cells Transduced with Granulocyte Macrophage Colony Stimulating Factor Mediated by Non-Transmissible Sendai Virus Induce Therapeutic Antitumor Immunity in LLC Bearing Mice
Hiroyuki Inoue,1,2 Mutsunori Iga,1 Haruka Nabeta,1 Meng Xin,1 Ryo Kurita,1 Koichi Takayama,2 Makoto Inoue,3 Mamoru Hasegawa,3 Yoichi Nakanishi,2 Kenzaburo Tani.1 1 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Research Institute of Diseases of the Chest, Kyushu University, Fukuoka, Japan; 3DNAVEC Corporation, Tsukuba, Ibaraki, Japan.
Recently, several studies have shown that vaccine therapy using dendritic cells (DCs) genetically engineered to express inflammatory cytokines with or without a surrogate tumor antigen can effectively Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
induce antitumor immunity. In this study, murine bone marrow DCs were transduced with murine granulocyte macrophage colony stimulating factor (GM-CSF) by novel non-transmissible Sendai virus vector (SeV/dF) (DC/SeV/GM). SeV is a murine parainfluenza virus type I belonging to the family Paramyxoviridae and a cytoplasmic RNA vector considered to be another promising device in gene transduction for its capacity to infect and amplify in most mammalian cells including DCs. And it possesses an exclusively cytoplasmic replication cycle without any risk of integration into the genomic DNA. We examined whether antigen-specific CTL responses and therapeutic immunity could be induced in immunocompetent mice bearing LLC (murine Lewis lung carcinoma) vaccinated with GMCSF gene transduced and tumor lysate-pulsed mature DCs. SeV encoding green fluorescence protein (GFP), SeV/GFP, successfully transduced to bone marrow derived DCs (DC/SeV/GFP) with the optimum at MOI of 100. In vitro treatment with SeV/GFP and SeV/GM-CSF directed DCs to mature phenotype. The LLC tumor lysate pulse during DCs maturation did not largely influence on their phenotypic expression profiles. And the several inflammatory cytokines including GM-CSF in the supernatant of cultured mature DCs were quantified. Even though the expression level of DC activation surface markers of CD40 and B7-2 (CD86), did not attain the level induced by optimum level of LPS (1µg/ml), better antitumor effects to LLC were obtained with the use of DC/SeV/ GM compared with those seen with DC/LPS and DC/SeV/GFP in preestablished subcutaneous LLC model. During the therapeutic DCs vaccines, mice were well tolerated. These results demonstrated that SeV mediated GM-CSF gene transduction significantly enhanced the capacity of DCs to induce primary antitumor immune responses in vivo, although further investigation is required to achieve its optimum in vivo antitumor effects.
174. Effective Prevention of Lung Metastasis of Murine Model by Sendai Virus/Dendritic CellMediated Immunostimulatory Virotherapy
Atsushi Komaru,1,2 Yasuji Ueda,1 Hiroaki Kinoh,1 Tomonori Kato,1,2 Yui Harada,1,2 Hiroyoshi Suzuki,2 Aki Furuya,2 Kumi Ishida,2 Makoto Inoue,3 Mamoru Hasegawa,3 Tomohiko Ichikawa,2 Yoshikazu Yonemitsu.1 1 Gene Therapy at the 21st Century COE Program, Chiba University Graduate School of Medicine, Chiba, Japan; 2Urology, Chiba University Graduate School of Medicine, Chiba, Japan; 3 DNAVEC Corporation, Tsukuba, Japan. We recently demonstrated the highly efficient antitumor immunity against dermal tumor of B16F10 murine melanoma in use of dendritic cells (DCs) activated by non-transmissible recombinant Sendai virus (rSeV), proposing a new concept namely ‘immunostimulatory virotherapy’ for cancer immunotherapy. However, critical information regarding the efficacies 1) under more clinically relevant situations including metastatic diseases, 2) in use of clinically relevant vector types and transgene(s), 3) on other tumor type and other animal species, and 4) the related molecular/cellular mechanisms have been largely unknown. In this study, therefore, we investigated the efficacy of vaccination of DCs activated by fusion gene-deleted non-transmissible rSeV expressing GFP (rSeV/dF-GFP), on murine model of lung metastasis using mouse neuroblastoma, C1300 and mouse prostate cancer, RM9. rSeV/dF-GFP activated bone marrowderived DCs (rSeV/dF-GFP-DC), consistent results previously seen in murine DCs. Vaccination of rSeV/dF-GFP-DC was highly effective to prevent lung metastasis following intravenous load of tumor cells, compared with the effects seen with immature DCs. Importantly, rSeV/dF-DCs expressing dominant negative mutant of retinoic acid-inducible gene I (RIG-I) (rSeV/dF-RIGIC-DC), a RNA helicase that recognizes rSeV genome for inducing type I interferons, largely lost the expression of proinflammatory cytokines without any S65