- Email: [email protected]
207. Effect of Interleukin-15 and Dendritic Cells on the Phenotype and Cytotoxicity of Cytokine-Induced Killer Cells
CANCER - IMMUNOTHERAPY I that defined CMV sequences were preferentially selected. Overall, our results highlight the robustness of TCR-LAM direct sequencing in dissecting the TCR repertoire in different immunological conditions and will be highly valuable in preclinical and clinical gene therapy affecting the immune system.
205.
Aptamer Targeted Antigen Delivery
Matthew Levy,1 Brian C. Wengerter,1 Debborah Palliser,2 Joseph Katakowski.3 1 Biochemistry, Albert Einstein College of Medicine, Bronx, NY; 2 Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY. Nucleic acid aptamers present a promising alternative to protein-based targeting approaches. We have selected aptamers that specifically bind the murine receptor, DEC205, a C-type lectin expressed predominantly on the surface of CD8+ dendritic cells (DCs) that has been shown to be particularly efficient at facilitating antigen cross-presentation and subsequent CD8+ T cell activation. A minimized DEC205 aptamer was conjugated to the model antigen ovalbumin (OVA), and the DEC205 aptamer-OVA conjugate was delivered in vitro to murine CD11c+ splenocytes. Antigen crosspresentation was verified in vitro and in vivo by proliferation and cytokine production by primary murine CD8+ T cells expressing a T cell receptor (TCR) specific for the MHC I-restricted OVA257264 peptide SIINFEKL. Compared with a nonspecific modified RNA of similar length, DEC205 aptamer-OVA-mediated antigen delivery stimulated strong proliferation and interferon gamma (IFN) secretion. Our results demonstrate a new application of aptamer technology and potentially an improved approach for the development of effective T cell-mediated vaccines.
206. FANG™ Cytokine Expression Analyses Update
Padmasini Kumar,1 Connor Phalon,1 Beena O. Pappen,1 Gladice Wallraven,1 Yang Yu,1 Fabienne Norvell,1 Christopher M. Jay,1 Zhaohui Wang,1 Donald D. Rao,1 John Nemunaitis,1,2,3,4 Neil Senzer,1,2 Phillip B. Maples.1 1 Gradalis, Inc, Dallas; 2Mary Crowley Cancer Research Centers, Dallas; 3Medical City HCA, Dallas; 4Texas Oncology, P.A., Dallas. We have developed an autologous whole cell tumor vaccine, FANG™, incorporating a plasmid encoding GMCSF and a novel bifunctional short hairpin RNA (bi-shRNA) targeting FURIN. FURIN, also known as furin/PACE/SPC1, is an enzyme, which belongs to the subtilisin-like proprotein convertase family. The proprotein convertases processes latent precursor proteins into their biologically active products. One of the furin substrates is the family of transforming growth factor beta (TGF) precursors which have been shown to play a significant role in tumor progression, metastasis, and tumor induced immunosuppression. For Phase I study of FANG™, two preclinical and 54 clinical vaccines were manufactured. An aliquot of cells from before and after transfection with FANG™ plasmid were placed in culture and incubated at 37ºC. Cells and supernatants were collected on Days 0, 1, 2, 3, 4, 7, 10 and 14. GMCSF, TGF1, TGF2 (RD Systems) and Furin (USCN Life Sciences) proteins were quantified by enzyme-linked immunosorbent assay (ELISA) kits. We demonstrated (shown below) an average increase (n=85) in the expression of GMCSF from 7.3 to 1,108 pg/106cells/ml at Day 4. Mean TGF1 and 2 effective target knockdown was 93.5 and 92.5% from baseline, respectively (Senzer et.al. Mol. Ther 2011). Furin knockdown was 85.09%.
S80
The percentage knockdown of TGF1 and TGF2 were significant with p <0.001 and consistent with the Furin knockdown also with p<0.001 results. Evaluation of these cytokines from FANG Phase II studies with Vaccines manufactured in Phase II Ovarian, Phase II Melanoma and Phase II Colorectal Carcinoma are analyzed and will be presented at this meeting. The increase in the expression of GMCSF and knockdown of TGF1, TGF2 and FURIN are consistently achieved in all tumor cells transfected with FANG plasmid.
207. Effect of Interleukin-15 and Dendritic Cells on the Phenotype and Cytotoxicity of CytokineInduced Killer Cells Xianghui He,1 Zhaohui Lai,1 Na Zhao,1 Liwei Zhu.1 Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China.
1
Objective: To investigate the effect of interleukin 15 (IL-15) and dendritic cells (DC) on the phenotype and cytotoxicity of cytokineinduced killer (CIK) cells. Methods:Spleen mononuclear cells were harvested from C57BL/6 mice and cultured in anti-CD3 antibodycoated plate with IFN-and IL-2 containing medium to induce CIK cells. Bone marrow derived- DC was prepared. IL-15, DC or both were added to the CIK culture medium. CIK, CIK, IL-15-CIK, DC-CIK, DC/IL-15-CIK cells were produced, respectively. The percentage of CD3+CD8+,CD3+NK1.1+ cells in these CIK cells were analyzed with FCM. Cytotoxicity of different CIK cells on mouse liver carcinoma hepal-6 cells were determined through detection of lactate dehydrogenase (LDH) release. Result: The ratio of CD3+CD8+and CD3+NK1.1+ cells in the spleen mononuclear cells of C57BL/6 mice before stimulation was (12.3±2.1)% and (1.1±0.5)%, respectively. After culturing for 14 days in vitro, the ratio of CD3+CD8+ cells in CIK, IL-15-CIK, DC-CIK and DC/IL-15-CIK cells was (18.8±2.4)%, (22.1±2.0)%, (28.2±2.6)% and (34.7±2.3)5, respectively. The ratio of CD3+NK1.1+ cells was (17.1±2.6)%, (18.9±3.4)%, (22.5±1.5)% and (25.9±1.8)%. There was significant difference between IL-15 treatment group and control. The cytotoxicity of DC-CIK and DC/ IL-15-CIK cells on liver carcinoma Hepal-6 cells were significantly higher than CIK cells as effector-target ration range from 2.5:1 to 20:1 (p<0.05). Cytotoxicity of DC-CIK and DC/IL-15-CIK was (54.9±3.1)% and (58.75±4.34)% as effector-target ratio was 20:1. Conclusion: Both IL-15 and DC increase the ratio of CD3+CD8+and CD3+NK1.1+ cells in CIK cells, and synergetic effects also present. CIK cells stimulated with DC showed increased anti-tumor effect, and IL-15 can further enhance the antitumor effect of DC-CIK cells.
208. Investigating Macrophage Induction of Metastasis: A Precursor To Identify Therapeutic Targets
Trevor Memmott,1 Evita Weagel,1 Justin Livingston,1 Pingguo Liu,1 Atif Elnaggar,1 Richard Robison,1 Kim O’Neill.1 1 Microbiology and Molecular Biology, Brigham Young University, Provo, UT. Metastasis is the leading cause of cancer-related deaths. In patients, tumors that don’t metastasize have a better prognosis. Many of the molecular mechanisms behind metastases have yet to be elucidated. Recently, it has been suggested that macrophages in the tumor microenvironment could play a major role in regulating the genes involved in metastatic tumor development. There are two different macrophage phenotypes: the tumor associated macrophage (generally M2) and the naturally occurring macrophage (generally Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
205.
Aptamer Targeted Antigen Delivery
Matthew Levy,1 Brian C. Wengerter,1 Debborah Palliser,2 Joseph Katakowski.3 1 Biochemistry, Albert Einstein College of Medicine, Bronx, NY; 2 Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY. Nucleic acid aptamers present a promising alternative to protein-based targeting approaches. We have selected aptamers that specifically bind the murine receptor, DEC205, a C-type lectin expressed predominantly on the surface of CD8+ dendritic cells (DCs) that has been shown to be particularly efficient at facilitating antigen cross-presentation and subsequent CD8+ T cell activation. A minimized DEC205 aptamer was conjugated to the model antigen ovalbumin (OVA), and the DEC205 aptamer-OVA conjugate was delivered in vitro to murine CD11c+ splenocytes. Antigen crosspresentation was verified in vitro and in vivo by proliferation and cytokine production by primary murine CD8+ T cells expressing a T cell receptor (TCR) specific for the MHC I-restricted OVA257264 peptide SIINFEKL. Compared with a nonspecific modified RNA of similar length, DEC205 aptamer-OVA-mediated antigen delivery stimulated strong proliferation and interferon gamma (IFN) secretion. Our results demonstrate a new application of aptamer technology and potentially an improved approach for the development of effective T cell-mediated vaccines.
206. FANG™ Cytokine Expression Analyses Update
Padmasini Kumar,1 Connor Phalon,1 Beena O. Pappen,1 Gladice Wallraven,1 Yang Yu,1 Fabienne Norvell,1 Christopher M. Jay,1 Zhaohui Wang,1 Donald D. Rao,1 John Nemunaitis,1,2,3,4 Neil Senzer,1,2 Phillip B. Maples.1 1 Gradalis, Inc, Dallas; 2Mary Crowley Cancer Research Centers, Dallas; 3Medical City HCA, Dallas; 4Texas Oncology, P.A., Dallas. We have developed an autologous whole cell tumor vaccine, FANG™, incorporating a plasmid encoding GMCSF and a novel bifunctional short hairpin RNA (bi-shRNA) targeting FURIN. FURIN, also known as furin/PACE/SPC1, is an enzyme, which belongs to the subtilisin-like proprotein convertase family. The proprotein convertases processes latent precursor proteins into their biologically active products. One of the furin substrates is the family of transforming growth factor beta (TGF) precursors which have been shown to play a significant role in tumor progression, metastasis, and tumor induced immunosuppression. For Phase I study of FANG™, two preclinical and 54 clinical vaccines were manufactured. An aliquot of cells from before and after transfection with FANG™ plasmid were placed in culture and incubated at 37ºC. Cells and supernatants were collected on Days 0, 1, 2, 3, 4, 7, 10 and 14. GMCSF, TGF1, TGF2 (RD Systems) and Furin (USCN Life Sciences) proteins were quantified by enzyme-linked immunosorbent assay (ELISA) kits. We demonstrated (shown below) an average increase (n=85) in the expression of GMCSF from 7.3 to 1,108 pg/106cells/ml at Day 4. Mean TGF1 and 2 effective target knockdown was 93.5 and 92.5% from baseline, respectively (Senzer et.al. Mol. Ther 2011). Furin knockdown was 85.09%.
S80
The percentage knockdown of TGF1 and TGF2 were significant with p <0.001 and consistent with the Furin knockdown also with p<0.001 results. Evaluation of these cytokines from FANG Phase II studies with Vaccines manufactured in Phase II Ovarian, Phase II Melanoma and Phase II Colorectal Carcinoma are analyzed and will be presented at this meeting. The increase in the expression of GMCSF and knockdown of TGF1, TGF2 and FURIN are consistently achieved in all tumor cells transfected with FANG plasmid.
207. Effect of Interleukin-15 and Dendritic Cells on the Phenotype and Cytotoxicity of CytokineInduced Killer Cells Xianghui He,1 Zhaohui Lai,1 Na Zhao,1 Liwei Zhu.1 Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China.
1
Objective: To investigate the effect of interleukin 15 (IL-15) and dendritic cells (DC) on the phenotype and cytotoxicity of cytokineinduced killer (CIK) cells. Methods:Spleen mononuclear cells were harvested from C57BL/6 mice and cultured in anti-CD3 antibodycoated plate with IFN-and IL-2 containing medium to induce CIK cells. Bone marrow derived- DC was prepared. IL-15, DC or both were added to the CIK culture medium. CIK, CIK, IL-15-CIK, DC-CIK, DC/IL-15-CIK cells were produced, respectively. The percentage of CD3+CD8+,CD3+NK1.1+ cells in these CIK cells were analyzed with FCM. Cytotoxicity of different CIK cells on mouse liver carcinoma hepal-6 cells were determined through detection of lactate dehydrogenase (LDH) release. Result: The ratio of CD3+CD8+and CD3+NK1.1+ cells in the spleen mononuclear cells of C57BL/6 mice before stimulation was (12.3±2.1)% and (1.1±0.5)%, respectively. After culturing for 14 days in vitro, the ratio of CD3+CD8+ cells in CIK, IL-15-CIK, DC-CIK and DC/IL-15-CIK cells was (18.8±2.4)%, (22.1±2.0)%, (28.2±2.6)% and (34.7±2.3)5, respectively. The ratio of CD3+NK1.1+ cells was (17.1±2.6)%, (18.9±3.4)%, (22.5±1.5)% and (25.9±1.8)%. There was significant difference between IL-15 treatment group and control. The cytotoxicity of DC-CIK and DC/ IL-15-CIK cells on liver carcinoma Hepal-6 cells were significantly higher than CIK cells as effector-target ration range from 2.5:1 to 20:1 (p<0.05). Cytotoxicity of DC-CIK and DC/IL-15-CIK was (54.9±3.1)% and (58.75±4.34)% as effector-target ratio was 20:1. Conclusion: Both IL-15 and DC increase the ratio of CD3+CD8+and CD3+NK1.1+ cells in CIK cells, and synergetic effects also present. CIK cells stimulated with DC showed increased anti-tumor effect, and IL-15 can further enhance the antitumor effect of DC-CIK cells.
208. Investigating Macrophage Induction of Metastasis: A Precursor To Identify Therapeutic Targets
Trevor Memmott,1 Evita Weagel,1 Justin Livingston,1 Pingguo Liu,1 Atif Elnaggar,1 Richard Robison,1 Kim O’Neill.1 1 Microbiology and Molecular Biology, Brigham Young University, Provo, UT. Metastasis is the leading cause of cancer-related deaths. In patients, tumors that don’t metastasize have a better prognosis. Many of the molecular mechanisms behind metastases have yet to be elucidated. Recently, it has been suggested that macrophages in the tumor microenvironment could play a major role in regulating the genes involved in metastatic tumor development. There are two different macrophage phenotypes: the tumor associated macrophage (generally M2) and the naturally occurring macrophage (generally Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy