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317. Neuroblastoma Cell Vaccines Expressing Vesicular Stomatitis Virus G Protein Are Highly Immunogenic In Vivo
CANCER IMMUNOTHERAPY 316. Induction of Therapeutic Antitumor Antiangiogenesis by Intratumoral Injection of Genetically Engineered Endostatin-Producing Semliki Forest Virus Ryuya Yamanaka,1 Ryuichi Tanaka,1 Kleanthis Xanthopoulos.2 Neurosurgery, Brain Research Institute, Niigata University, Niigata, Japan; 2Clinical Gene Therapy, NHGRI/NIH, Bethesda, MD, United States. 1
Antiangiogenic therapy using Semliki Forest virus ( SFV) carrying Endostatin gene for malignant brain tumor was investigated to improve the therapeutic efficacy. The efficiency of SFV- mediated gene delivery was first evaluated for B 16 cells and compared with the efficiency in cells of endothelial origin ( HMVECs). HMVECs are more susceptible to SFV infection than B 16 cells. For the in vivo treatment model, phosphate- buffered saline, SFV- LacZ, retrovirus vector GCsap- Endostatin, and SFV- Endostatin were injected to mice bearing B 16 brain tumors. A very significant inhibition of tumor growth was observed in the group that had been treated with SFV- Endostatin. A marked reduction of intratumoral vascularization was seen in the tumor sections from the SFVEndostatin group compared with tumor sections from the SFVLacZ or GCsap- Endostatin groups. Moreover, at day 7 after intravenous administration of SFV- Endostatin, the serum level of endostatin was augmented more than 3- fold compared to that after intravenous administration of GCsap- Endostatin. The results indicated that treatment with SFV- Endostatin inhibited the angiogenesis with established tumors. Gene therapy with Endostatin delivered via SFV may be a candidate for the development of new therapy for brain tumors.
317. Neuroblastoma Cell Vaccines Expressing Vesicular Stomatitis Virus G Protein Are Highly Immunogenic In Vivo Vy Phan,1,2 Florence Neczyporenko,1 Bernard Bonnotte,1 Emmanouela Linardakis,1 Jill Thompson,1 Alan Melcher,4 Richard Vile.3 1 Molecular Medicine Program; 2Department of Tumor Biology; 3 Department of Immunology, Mayo Clinic, Rochester, MN; 4 Cancer Research UK Clinical Centre Oncology Unit, St. James’s Hospital, Leeds, United Kingdom. Neuroblastoma is a malignant cancer which, like many others, is poorly immunogenic due, in part, to the lack of costimulatory gene expression needed for T cell stimulation. We describe here a novel method to enhance the immunogenicity of neuroblastoma cells via gene transfer of cDNA encoding a viral fusogenic membrane glycoprotein, Vesicular Stomatitis Virus G protein (VSV-G), which is normally involved in the binding of the rhabdoviral particle to the target cell receptor as well as in fusion of the viral and cellular membranes. In this report, mouse neuroblastoma Neuro-2a (N-2a) cells were transfected with wild-type VSV-G cDNA (potent fusogenicity) and 60% cell-cell fusion was observed, at pH 7.0 and 5.7 conditions. Inoculation of live VSV-G modified cells in immunocompetent animals (but not in immunodeficient animals) yielded significantly smaller tumors than unmodified cells did (mean tumor volume of 0.2 cm³ versus 0.4 cm³ by Day 28), suggesting that the foreign-derived G protein can act as a potent immunogen in vivo to retard tumor growth. N2a cell vaccines prepared with VSV-G pH 7.0 and 5.7 conditions and administered prior to tumor challenge with parental cells led to significant increases in survival of mice (80-90%, compared to 10% with unmodified cells and 0% with PBS). Interestingly, vaccines transfected with dead VSV-G cDNA (impaired fusogenicity) also led to enhanced survival (70-80% survival), further corroborating that the introduction of a foreign viral protein (even in the absence of cell-cell fusion and the resulting Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
immunostimulatory effects) is sufficient to enhance the immunogenicity of the cell in which it is expressed. The mechanism of immune stimulation by VSV-G expressing vaccines is dependent on T-cells and might also, more specifically, be dependent on nonapoptotic death pathways, since VSV-G transfection in N-2a cells produced no significant DNA laddering and at most only 6% apoptosis by nuclear assessment. We therefore speculate that ex vivo modification of human neuroblastoma cells with foreign viral genes, such as VSV-G, can serve to generate potent and effective vaccines against residual and possibly recurring disease.
318. Head to Head Comparison of a Genetic with a Conventional Protein Vaccine Against Malignant Melanoma: Induction of AntigenSpecific Protective Anti-Tumor Immunity by a pDNA-Based Vaccine but Not by Recombinant Protein Achim Schneeberger,1 Tatjana K. Weimann,2 Christine Wagner,1 Anja Zemann,2 Petra Luehrs,1 Manfred Goos,2 Georg Stingl,1 Stephan N. Wagner.1 1 DIAID, Dept. of Dermatology, University School of Medicine, Vienna, Austria; 2Dept. of Dermatology, University School of Medicine, Essen, Germany. MAA Pmel17/gp100 represents an attractive target antigen for the development of antigen-specific immunotherapeutic strategies against malignant melanoma. The recent development of genetic vaccines now raises the question whether these genetic vaccines are superior to conventional immunization strategies employing the respective protein for the induction of protective T cell responses. We therefore cloned the 1.9 kb full length cDNA murine homologue of human Pmel17, generated a recombinant baculovirus containing the respective cDNA sequence by ligation independent cloning, expressed the recombinant protein in Sf9 insect cells, and confirmed its identity by SDS-PAGE, Western blot analysis, and ELISA. After large-scale purification by preparative SDS-PAGE, electroelution, dialysis, and sterile filtration this recombinant full-length protein was compared with a pDNA vaccine for the induction of protective antigen-specific T cell responses in the experimental Cloudman M3/ DBA/2 mouse melanoma model. Mice genetically immunized with mPmel17-encoding pDNA showed significant protection against subsequent tumor challenge with Pmel17+-M3 melanoma cells whereas mice immunized with the recombinant protein in different adjuvants did not (p<0.001 at day 15). Induction of an antigenspecific T cell response by mPmel17-encoding pDNA could be demonstrated by (i) induction of protection against Pmel17+- but not Pmel17-- M3 melanoma sublines, (ii) complete loss of protection by in vivo CD4+/CD8+ T cell depletion, (iii) the detection of a Pmel17/gp100-specific T cell response in CTL assays. Antigenspecific T cell responses could not be detected in mice immunized with recombinant protein despite the induction of antigen-specific IgG responses of both the IgG1 and IgG2a subtypes. These results demonstrate that genetic immunization with antigen-encoding pDNA is superior to immunization with the respective recombinant protein for the induction of antigen-specific, protective anti-tumor T cell responses in vivo.
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Antiangiogenic therapy using Semliki Forest virus ( SFV) carrying Endostatin gene for malignant brain tumor was investigated to improve the therapeutic efficacy. The efficiency of SFV- mediated gene delivery was first evaluated for B 16 cells and compared with the efficiency in cells of endothelial origin ( HMVECs). HMVECs are more susceptible to SFV infection than B 16 cells. For the in vivo treatment model, phosphate- buffered saline, SFV- LacZ, retrovirus vector GCsap- Endostatin, and SFV- Endostatin were injected to mice bearing B 16 brain tumors. A very significant inhibition of tumor growth was observed in the group that had been treated with SFV- Endostatin. A marked reduction of intratumoral vascularization was seen in the tumor sections from the SFVEndostatin group compared with tumor sections from the SFVLacZ or GCsap- Endostatin groups. Moreover, at day 7 after intravenous administration of SFV- Endostatin, the serum level of endostatin was augmented more than 3- fold compared to that after intravenous administration of GCsap- Endostatin. The results indicated that treatment with SFV- Endostatin inhibited the angiogenesis with established tumors. Gene therapy with Endostatin delivered via SFV may be a candidate for the development of new therapy for brain tumors.
317. Neuroblastoma Cell Vaccines Expressing Vesicular Stomatitis Virus G Protein Are Highly Immunogenic In Vivo Vy Phan,1,2 Florence Neczyporenko,1 Bernard Bonnotte,1 Emmanouela Linardakis,1 Jill Thompson,1 Alan Melcher,4 Richard Vile.3 1 Molecular Medicine Program; 2Department of Tumor Biology; 3 Department of Immunology, Mayo Clinic, Rochester, MN; 4 Cancer Research UK Clinical Centre Oncology Unit, St. James’s Hospital, Leeds, United Kingdom. Neuroblastoma is a malignant cancer which, like many others, is poorly immunogenic due, in part, to the lack of costimulatory gene expression needed for T cell stimulation. We describe here a novel method to enhance the immunogenicity of neuroblastoma cells via gene transfer of cDNA encoding a viral fusogenic membrane glycoprotein, Vesicular Stomatitis Virus G protein (VSV-G), which is normally involved in the binding of the rhabdoviral particle to the target cell receptor as well as in fusion of the viral and cellular membranes. In this report, mouse neuroblastoma Neuro-2a (N-2a) cells were transfected with wild-type VSV-G cDNA (potent fusogenicity) and 60% cell-cell fusion was observed, at pH 7.0 and 5.7 conditions. Inoculation of live VSV-G modified cells in immunocompetent animals (but not in immunodeficient animals) yielded significantly smaller tumors than unmodified cells did (mean tumor volume of 0.2 cm³ versus 0.4 cm³ by Day 28), suggesting that the foreign-derived G protein can act as a potent immunogen in vivo to retard tumor growth. N2a cell vaccines prepared with VSV-G pH 7.0 and 5.7 conditions and administered prior to tumor challenge with parental cells led to significant increases in survival of mice (80-90%, compared to 10% with unmodified cells and 0% with PBS). Interestingly, vaccines transfected with dead VSV-G cDNA (impaired fusogenicity) also led to enhanced survival (70-80% survival), further corroborating that the introduction of a foreign viral protein (even in the absence of cell-cell fusion and the resulting Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
immunostimulatory effects) is sufficient to enhance the immunogenicity of the cell in which it is expressed. The mechanism of immune stimulation by VSV-G expressing vaccines is dependent on T-cells and might also, more specifically, be dependent on nonapoptotic death pathways, since VSV-G transfection in N-2a cells produced no significant DNA laddering and at most only 6% apoptosis by nuclear assessment. We therefore speculate that ex vivo modification of human neuroblastoma cells with foreign viral genes, such as VSV-G, can serve to generate potent and effective vaccines against residual and possibly recurring disease.
318. Head to Head Comparison of a Genetic with a Conventional Protein Vaccine Against Malignant Melanoma: Induction of AntigenSpecific Protective Anti-Tumor Immunity by a pDNA-Based Vaccine but Not by Recombinant Protein Achim Schneeberger,1 Tatjana K. Weimann,2 Christine Wagner,1 Anja Zemann,2 Petra Luehrs,1 Manfred Goos,2 Georg Stingl,1 Stephan N. Wagner.1 1 DIAID, Dept. of Dermatology, University School of Medicine, Vienna, Austria; 2Dept. of Dermatology, University School of Medicine, Essen, Germany. MAA Pmel17/gp100 represents an attractive target antigen for the development of antigen-specific immunotherapeutic strategies against malignant melanoma. The recent development of genetic vaccines now raises the question whether these genetic vaccines are superior to conventional immunization strategies employing the respective protein for the induction of protective T cell responses. We therefore cloned the 1.9 kb full length cDNA murine homologue of human Pmel17, generated a recombinant baculovirus containing the respective cDNA sequence by ligation independent cloning, expressed the recombinant protein in Sf9 insect cells, and confirmed its identity by SDS-PAGE, Western blot analysis, and ELISA. After large-scale purification by preparative SDS-PAGE, electroelution, dialysis, and sterile filtration this recombinant full-length protein was compared with a pDNA vaccine for the induction of protective antigen-specific T cell responses in the experimental Cloudman M3/ DBA/2 mouse melanoma model. Mice genetically immunized with mPmel17-encoding pDNA showed significant protection against subsequent tumor challenge with Pmel17+-M3 melanoma cells whereas mice immunized with the recombinant protein in different adjuvants did not (p<0.001 at day 15). Induction of an antigenspecific T cell response by mPmel17-encoding pDNA could be demonstrated by (i) induction of protection against Pmel17+- but not Pmel17-- M3 melanoma sublines, (ii) complete loss of protection by in vivo CD4+/CD8+ T cell depletion, (iii) the detection of a Pmel17/gp100-specific T cell response in CTL assays. Antigenspecific T cell responses could not be detected in mice immunized with recombinant protein despite the induction of antigen-specific IgG responses of both the IgG1 and IgG2a subtypes. These results demonstrate that genetic immunization with antigen-encoding pDNA is superior to immunization with the respective recombinant protein for the induction of antigen-specific, protective anti-tumor T cell responses in vivo.
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