- Email: [email protected]
553. Autologous Tumor Cell Vaccine Genetically Modified To Express GM-CSF and Block Expression of TGFβ2
CANCER - IMMUNOTHERAPY II 553. Autologous Tumor Cell Vaccine Genetically Modified To Express GM-CSF and Block Expression of TGFβ2
Phillip B. Maples,1 Padmasini Kumar,1 Yang Yu,1 Ila Oxendine,1 Joseph Kuhn,2 John Nemunaitis.1,3,4,5 1 Gradalis, Inc., Dallas, TX; 2General and Oncology Surgery Associates, Dallas, TX; 3Mary Crowley Cancer Research Centers, Dallas, TX; 4Baylor Sammons Cancer Center, Dallas, TX; 5Texas Oncology, P.A., Dallas, TX.
Gene modified cell-based cancer vaccines have demonstrated durable response in selected patients. We have developed a novel expression vector that we believe, when transfected into tumor cells, will evoke immune recognition /stimulation by two distinct routes. The nonviral vector system expresses both GM-CSF and a TGFβ2 antisense. GM-CSF transgene expression in tumor cells has been used with some success in generating immune responses to various cancers including NSCLC (Nemunaitis et al, JNCI 2004, 96: 326331). Similarly, TGFβ2 antisense-modified tumor cells have been used to generate positive clinical responses in NSCLC and glioma patients (Nemunaitis et al, JCO 2006, 24: 4721-4730). However, each of these approaches mediates activity through distinct mechanisms of action (i.e. dendritic activation and immune inhibitor inhibition). We hypothesize that combining GMCSF and TGFβ2AS would improve activity. Our Phase I clinical protocol (n=55 patients) was opened in May, 2008 (BB-IND 13650). Advanced cancer patients that have sufficient accessible tumor tissue are eligible for trial entry. Twentyeight patients have been consented for vaccine manufacturing. Tumor types collected include colorectal (7), NSCLC (5), breast (4) and melanoma (3). Of the 24 successful manufacturing processes, 8 patients’ vaccines are in the low dose cohort (1e7 cells per dose) and 16 patients’ vaccines are in the high dose cohort (2.5e7 cells per dose). There were 4 manufacturing failures, due to insufficient starting material (2) or contamination (2). The contaminations occurred with tumor tissue procured from colorectal cancer patients. We no longer harvest tumor for vaccine manufacturing from the visceral lumeral area. Product stability has been monitored over one year by sterility, GM-CSF mRNA and TGFβ2 antisense expression and other parameters. After each patient vaccine is manufactured, GMCSF and TGFβ2 protein expression are assessed by immunoassay comparing transfected and nontransfected tumor cell expression over 14 days. In all but two instances, GM-CSF secretion met our product release specification. TGFβ2 knockdown was evident in all instances although in 4 instances it did not reach the 30% knockdown goal. TGFβ1 expression was also assessed pre and post transfection (no effect) and TGFβ1 levels were typically ten-fold higher than TGFβ2 levels. TGFβ1 is thought to be the more dominant immunosuppressive agent in many cancers and these data underscore its presence in these tumors and vaccine products.
554. Nonionic Block Copolymer Pluronic P85 Is a Promising Adjuvant for MUC4 Plasmid DNA Immunotherapy
Zagit Z. Gaymalov,1 Caroline Roques,1 Surinder K. Batra,2 Alexander V. Kabanov.1 1 Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE; 2Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE.
Introduction: Gene therapy based vaccination is a promising approach to facilitate successful immunization against various cancers. Pancreatic cancer, a devastating and aggressive disease affecting over 37,000 people each year in US alone, is an attractive target for vaccination because of clearly marked MUC4 overexpression. S212
DNA vaccination approach through delivery and expression of the gene coding for MUC4 in aim to alert host immune system against MUC4 overexpressing cells, might be interesting prophylactic and therapeutic option. In this study we report, that Pluronic block copolymer P85 co-administered intramuscularly with plasmid DNA encoding MUC4 significantly enhances plasmid expression in the muscle, spleen and lymph nodes and stimulates expansion of T cells in mice. Methods: The expression of the transgene coding for a mini-MUC4 was assessed in muscle tissue as well as draining lymph node and spleen at day 4 after direct intramuscular injection of either DNA alone or DNA/Pluronic P85 formulation. Co-localization studies were performed on cryo-sections of each tissue to determine the cell-types expressing mini-MUC4. In addition, activation of the immune response was monitored on spleen and lymph nodes by direct immunostaining of lymphatic cells and further FACS analysis of various subsets of immune cells. Results: In the muscle tissue, MUC4 expression was observed in either myocytes or keratinocytes, with an increase in transfection efficiency when associated with P85. MUC4 expression also co-localized with antigen presenting cells (dendritic cells and macrophages) as well as T cells. The same pattern of expression was found in cells participating to the immune response in the draining lymph node (inguinal lymph node) and spleen. Moreover, FACS analysis showed a significant expansion of the CD8a+ and CD4+ T cells in the spleen, but no dendritic cell expansion for the P85/DNA formulation. Dendritic cells maturation was also monitored. Conclusion: MUC4/P85 formulations might be a promising strategy towards vaccination through gene delivery due to the improved expression of the transgene when associated to the Pluronic block copolymer P85, in addition to the expansion of both cytotoxic and helper T cells. This study was supported by the National Institute of Health grant R01 CA116591 (AVK).
555. PiggyBac, an Efficient Method for Genetic Modification of T Cells with Second Generation CD19-Specific Chimeric Antigen Receptor (CAR) for Treatment of B-Lineage Malignancies
Pallavi R. Manuri,1 Sourindra N. Maiti,1 Harjeet Singh,1 Tiejuan Mi,1 Simon Olivares,1 Margaret J. Dawson,1 Helen Huls,1 Dean A. Lee,1 Pulivarthi H. Rao,2 Joseph M. Kaminski,3 Partow Kebriaei,4 Matthew H. Wilson,5 Laurence J. N. Cooper.1 1 Pediatrics, U.T. M.D. Anderson Cancer Center, Houston, TX; 2Pediatrics Hematology/Oncology, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX; 3 Center for Molecular Chaperone, Radiobiology, and Cancer Virology, Medical College of Georgia, Augusta, GA; 4Stem Cell Transplantation and Cellular Therapy, U.T. M.D. Anderson Cancer Center, Houston, TX; 5Michael E. DeBakey VA Medical Center and Department of Medicine, Section of Nephrology, Baylor College of Medicine, Houston, TX. Non-viral integrating vectors can be used for expression of therapeutic genes. To improve gene transfer efficiency, we and others have used transposon/transposase systems from Sleeping Beauty to integrate desired transgenes, such as chimeric antigen receptors (CARs), into primary T cells. To determine whether transposition of CARs is adaptable to other systems, we now show that the piggyBac (PB) can be used to express a CD19-specific CAR in human T cells. We demonstrate that T cells electroporated to introduce PB transposon and transposase stably express CD19-specific CAR upon propagation on irradiated CD19+ artificial antigen presenting cells (aAPC; Figure) and maintain a diverse repertoire of endogenous T-cell receptors consistent with efficient gene transfer. The numerically expanded T cells display a phenotype associated with both memory and effector T-cell populations and exhibit CD19-dependent killing of tumor targets. The electro-transfer of PB transposon to express the CAR was not associated with genotoxicity based upon fluorescent in situ Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Phillip B. Maples,1 Padmasini Kumar,1 Yang Yu,1 Ila Oxendine,1 Joseph Kuhn,2 John Nemunaitis.1,3,4,5 1 Gradalis, Inc., Dallas, TX; 2General and Oncology Surgery Associates, Dallas, TX; 3Mary Crowley Cancer Research Centers, Dallas, TX; 4Baylor Sammons Cancer Center, Dallas, TX; 5Texas Oncology, P.A., Dallas, TX.
Gene modified cell-based cancer vaccines have demonstrated durable response in selected patients. We have developed a novel expression vector that we believe, when transfected into tumor cells, will evoke immune recognition /stimulation by two distinct routes. The nonviral vector system expresses both GM-CSF and a TGFβ2 antisense. GM-CSF transgene expression in tumor cells has been used with some success in generating immune responses to various cancers including NSCLC (Nemunaitis et al, JNCI 2004, 96: 326331). Similarly, TGFβ2 antisense-modified tumor cells have been used to generate positive clinical responses in NSCLC and glioma patients (Nemunaitis et al, JCO 2006, 24: 4721-4730). However, each of these approaches mediates activity through distinct mechanisms of action (i.e. dendritic activation and immune inhibitor inhibition). We hypothesize that combining GMCSF and TGFβ2AS would improve activity. Our Phase I clinical protocol (n=55 patients) was opened in May, 2008 (BB-IND 13650). Advanced cancer patients that have sufficient accessible tumor tissue are eligible for trial entry. Twentyeight patients have been consented for vaccine manufacturing. Tumor types collected include colorectal (7), NSCLC (5), breast (4) and melanoma (3). Of the 24 successful manufacturing processes, 8 patients’ vaccines are in the low dose cohort (1e7 cells per dose) and 16 patients’ vaccines are in the high dose cohort (2.5e7 cells per dose). There were 4 manufacturing failures, due to insufficient starting material (2) or contamination (2). The contaminations occurred with tumor tissue procured from colorectal cancer patients. We no longer harvest tumor for vaccine manufacturing from the visceral lumeral area. Product stability has been monitored over one year by sterility, GM-CSF mRNA and TGFβ2 antisense expression and other parameters. After each patient vaccine is manufactured, GMCSF and TGFβ2 protein expression are assessed by immunoassay comparing transfected and nontransfected tumor cell expression over 14 days. In all but two instances, GM-CSF secretion met our product release specification. TGFβ2 knockdown was evident in all instances although in 4 instances it did not reach the 30% knockdown goal. TGFβ1 expression was also assessed pre and post transfection (no effect) and TGFβ1 levels were typically ten-fold higher than TGFβ2 levels. TGFβ1 is thought to be the more dominant immunosuppressive agent in many cancers and these data underscore its presence in these tumors and vaccine products.
554. Nonionic Block Copolymer Pluronic P85 Is a Promising Adjuvant for MUC4 Plasmid DNA Immunotherapy
Zagit Z. Gaymalov,1 Caroline Roques,1 Surinder K. Batra,2 Alexander V. Kabanov.1 1 Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE; 2Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE.
Introduction: Gene therapy based vaccination is a promising approach to facilitate successful immunization against various cancers. Pancreatic cancer, a devastating and aggressive disease affecting over 37,000 people each year in US alone, is an attractive target for vaccination because of clearly marked MUC4 overexpression. S212
DNA vaccination approach through delivery and expression of the gene coding for MUC4 in aim to alert host immune system against MUC4 overexpressing cells, might be interesting prophylactic and therapeutic option. In this study we report, that Pluronic block copolymer P85 co-administered intramuscularly with plasmid DNA encoding MUC4 significantly enhances plasmid expression in the muscle, spleen and lymph nodes and stimulates expansion of T cells in mice. Methods: The expression of the transgene coding for a mini-MUC4 was assessed in muscle tissue as well as draining lymph node and spleen at day 4 after direct intramuscular injection of either DNA alone or DNA/Pluronic P85 formulation. Co-localization studies were performed on cryo-sections of each tissue to determine the cell-types expressing mini-MUC4. In addition, activation of the immune response was monitored on spleen and lymph nodes by direct immunostaining of lymphatic cells and further FACS analysis of various subsets of immune cells. Results: In the muscle tissue, MUC4 expression was observed in either myocytes or keratinocytes, with an increase in transfection efficiency when associated with P85. MUC4 expression also co-localized with antigen presenting cells (dendritic cells and macrophages) as well as T cells. The same pattern of expression was found in cells participating to the immune response in the draining lymph node (inguinal lymph node) and spleen. Moreover, FACS analysis showed a significant expansion of the CD8a+ and CD4+ T cells in the spleen, but no dendritic cell expansion for the P85/DNA formulation. Dendritic cells maturation was also monitored. Conclusion: MUC4/P85 formulations might be a promising strategy towards vaccination through gene delivery due to the improved expression of the transgene when associated to the Pluronic block copolymer P85, in addition to the expansion of both cytotoxic and helper T cells. This study was supported by the National Institute of Health grant R01 CA116591 (AVK).
555. PiggyBac, an Efficient Method for Genetic Modification of T Cells with Second Generation CD19-Specific Chimeric Antigen Receptor (CAR) for Treatment of B-Lineage Malignancies
Pallavi R. Manuri,1 Sourindra N. Maiti,1 Harjeet Singh,1 Tiejuan Mi,1 Simon Olivares,1 Margaret J. Dawson,1 Helen Huls,1 Dean A. Lee,1 Pulivarthi H. Rao,2 Joseph M. Kaminski,3 Partow Kebriaei,4 Matthew H. Wilson,5 Laurence J. N. Cooper.1 1 Pediatrics, U.T. M.D. Anderson Cancer Center, Houston, TX; 2Pediatrics Hematology/Oncology, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX; 3 Center for Molecular Chaperone, Radiobiology, and Cancer Virology, Medical College of Georgia, Augusta, GA; 4Stem Cell Transplantation and Cellular Therapy, U.T. M.D. Anderson Cancer Center, Houston, TX; 5Michael E. DeBakey VA Medical Center and Department of Medicine, Section of Nephrology, Baylor College of Medicine, Houston, TX. Non-viral integrating vectors can be used for expression of therapeutic genes. To improve gene transfer efficiency, we and others have used transposon/transposase systems from Sleeping Beauty to integrate desired transgenes, such as chimeric antigen receptors (CARs), into primary T cells. To determine whether transposition of CARs is adaptable to other systems, we now show that the piggyBac (PB) can be used to express a CD19-specific CAR in human T cells. We demonstrate that T cells electroporated to introduce PB transposon and transposase stably express CD19-specific CAR upon propagation on irradiated CD19+ artificial antigen presenting cells (aAPC; Figure) and maintain a diverse repertoire of endogenous T-cell receptors consistent with efficient gene transfer. The numerically expanded T cells display a phenotype associated with both memory and effector T-cell populations and exhibit CD19-dependent killing of tumor targets. The electro-transfer of PB transposon to express the CAR was not associated with genotoxicity based upon fluorescent in situ Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy