- Email: [email protected]
Production of endothelial no-synthase gene-enhanced autologous progenitor cell therapies for cardio-pulmonary diseases
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Oral Abstracts
patients before or immediately after mCTL infusion. In the absence of conventional therapy, 8 of the 11 patients with CMV infection became negative for CMV in the blood within 7d of mCTL infusion, with a coinciding rise in CMV-specific CTL in PB. Each of 8 patients with high EBV loads cleared their virus, as did 7 of 7 patients with adenoviral infections/disease. Overall the response rate in both groups was 88%. This study demonstrates that mCTL derived from the PB of seropositive donors as well as the CB of virus naive donors expand in vivo and are active against multiple viruses. Furthermore, by restoring immunity to multiple viruses simultaneously, the need for continued prophylaxis with pharmacotherapy is eliminated, thus, improving the efficiency and cost effectiveness of protecting SCT and CBT recipients from these potentially lethal viruses. 5 ANTI-CD123 CHIMERIC ANTIGEN RECEPTOR REDIRECTED T CELLS FOR RELAPSED B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA M Ruella1, O Shestova1, S Kenderian1, D Barrett2, S Grupp2, J Scholler1, S Lacey1, M Kalos1, CH June1, S Gill1 1 Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States, 2Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States
Figure 2A. Comparison of in vitro anti-tumor activity of CART123 and CART19.
Relapsing/refractory (R/R) B-cell Acute Lymphoblastic Leukemia (ALL) is associated with a poor prognosis. We have previously shown that anti CD19 Chimeric Antigen Receptor T cells (CART19) induce significant responses in this population. However, occasional CD19-ve relapses have occurred, likely due to selective pressure from CART19 cells. Hence, CAR-based therapies against additional antigens may be useful in the treatment of B-ALL. Twenty R/R ALL samples, including two CD19-ve relapses, were screened for 30 potential secondary targets using a custom Quantigene RNA panel (Affymetrix) and results were validated by flow cytometry. CD123 was amongst the most highly and homogeneously expressed, present in >60% of blasts in 16/20 R/RALL including 2/2 CD19-ve relapses (Figure 1). We utilized the anti-CD123 CAR T cells co-stimulated via 4-1BB (CART123) that we had previously
Figure 2B. In vitro anti-tumor activity of CART19 and CART123 against ALL. generated for the treatment of acute myeloid leukemia (AML) (Gill et al, ISCT 2013) and performed in vitro comparisons between CART123 and CART19. These investigations revealed similar proliferation, degranulation, cytokine production and cytotoxicity (Figure 2 A) and suggested that CART123 could be used to treat B-ALL. We therefore injected NSG mice with 0.5-2x106 cells of the CD19+ve CD123dim +ve B-ALL cell line Nalm6, and treated them with CART19, CART123 or control T cells (1x106 each). As expected, mice treated with control T cells succumbed quickly to disease. Mice treated with CART19 experienced enhanced survival (*).Mice treated with CART123 had improved survival (*) compared with control T cell treatment, and intermediate between control and CART19 (Figure 2 B). Hence, CART123 may represent an additional approach to treating CD19+ve malignancy. 6 PRODUCTION OF ENDOTHELIAL NO-SYNTHASE GENEENHANCED AUTOLOGOUS PROGENITOR CELL THERAPIES FOR CARDIO-PULMONARY DISEASES DW Courtman, L Comanita, DJ Stewart Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
Figure 1. CD123 expression on ALL blasts at baseline and at CD19-ve relapse.
Endothelial progenitor cells (EPCs) derived from the circulation hold promise for the treatment of cardiovascular diseases. Yet preclinical studies suggest that patient derived cells are less effective stimulators of vascular repair. We have therefore pioneered the development of gene enhanced autologous cell based treatments for pulmonary hypertension and myocardial ischemia. Our first-inhuman clinical trial (PHACeT, Pulmonary Hypertension: Assessment of Cell Therapy, NCT00469027) was a dose escalation safety trial using peripheral blood derived early outgrowth EPCs transiently transfected via electroporation with a human eNOS plasmid and delivered directly to the pulmonary
20th Annual ISCT Meeting
circulation. We demonstrated that administration of eNOS-transfected EPCs to patients with stable, severe PAH was well tolerated, and resulted in a trend towards short-term hemodynamic improvement. Although, there was no long term sustained hemodynamic improvement in this small (7 patient), uncontrolled trial; there were significant increases in 6 minute walk time seen at both 1 month, and persisting to 3and 6 months post cell based gene therapy. To apply our therapy to cardiac patients we developed a non-mobilized automated apheresis procedure combined with GMP manufacturing in environmentally controlled isolator units and in addition adopted a cationic polymer (manose modified linear polyethylenimine) for eNOS transfection to limit cellular manipulations. Enhanced eNOS expression was verified by Western Blotting and cell identity by flow cytometry. With these manufacturing modifications in place a placebo controlled randomized trial (Enhanced Angiogenic Cell Therapy-Acute Myocardial Infraction trial, NCT00936819) testing the role of eNOS transfection in autologous EPCs was initiated with 4 patients treated to date. Our early results suggest that eNOS transfected autologous EPCs are safe and potentially effective treatments for cardiovascular disorders.
7 GENERATION OF DISEASE-SPECIFIC INDUCED PLURIPOTENT STEM CELLS FROM HUMAN FETAL EXTRA-EMBRYONIC TISSUES P Spitalieri, V Talarico, A Luchetti, F Brancati, A Botta, G Novelli, F Sangiuolo Biomedicine and Prevention, University of Rome “Tor Vergata,” Rome, Italy The generation of induced pluripotent stem cells (iPSCs) is a innovative personalized-regenerative technology, which can transform own-self cells into embryonic stem elike cells, which have regarded as a promising candidate for cell-based therapy, as well as an ideal target for disease modeling and drug testing and drug discovery, thus enabling researchers to undertake studies for treating diseases. The objective of the present study was to reprogramming patient-specific fetal cells deriving from prenatal diagnosis for several genetic disorder as Cystic Fibrosis (FC), Myotonic Dystrophy (DM1), b-Thalassemia (b-Thal), Spinal Muscolar Atrophy (SMA1), Lymphema-Distichiasis Syndrome (FOXC2) and healthy cells. The cell type used for create iPSCs can significantly influence the reprogramming efficiency and kinetics. Here, we show that amniotic fluid (LA) and chorionic villus sampling (CVS) represent an ideal cell resource for rapid and efficient generation of human iPSCs. The reprogramming were done using a polycistronic lentiviral vector (hSTEMCCA-loxP) encoding Oct4, Sox2, Klf4 and c-Myc genes necessary to cell reprogramming. Moreover loxP sites can be excised with Cre recombinase. Stem cells specific morphological, molecular and immunocytochemical markers (ALP; OCT4; SSEA4; TRA1-60;TRA1-81) confirmed the successful reprogramming. Additionally, we evaluated their ability to differentiate into the three embryonic germ layers (ecto, endo and mesoderm) by immunocytochemical characterization and their ability to in vivo form teratomas. To date, this represents the first example of iPS cells derived from a very early extra-embryonic fetal tissues like chorionic villi (hIPS-CVS). These data suggest that hIPS-CVS/LA can be considered a valid cell model to accomplish pathogenesis studies and possibly represent a valid tool for future therapeutic applications.
treat HBV-associated liver cancer. We designed a chimeric antigen receptor (CAR) that is composed of a single chain antibody fragment binding to HBsAg and CD28 / CD3z signaling domains. This study aimed to proof feasibility of this approach in vivo addressing the following challenges: (i) T cell-depletion to generate space for cell engraftment in chronic virus carriers is too perilous, (ii) viral antigens circulating in high amounts may inactivate transferred T cells or (iii) trigger uncontrolled immune damage. Methods: Primary murine CD8+ T cells were isolated, stimulated using an optimized protocol and grafted with CARs by retroviral transduction. A CAR that binds HBV envelope proteins and transfers activation signals to the T cell was compared to a control CAR without a proper signaling domain and a CAR not binding HBV proteins. Results: CD8+ T cells engineered to express an HBV-specific CAR, which recognizes HBV envelope proteins of various subtypes on infected hepatocytes, were able to engraft and expand in immune competent HBV transgenic mice. Following adoptive transfer CAR-grafted T cells targeted the liver, remained functional in vivo, rapidly and efficiently controlled HBV replication while causing only transient liver damage. The large amount of circulating viral antigens neither impaired nor over-activated the transferred T cells. Conclusion: HBV-specific cell therapy with CAR-engineered T cells bears the potential to treat chronic hepatitis B and HBV-associated hepatocellular carcinoma irrespective of the patient’s individual HLA-type. 9 BONE MARROW MONONUCLEAR CELLS FOR ISCHEMIC CARDIAC FAILURE - A PROSPECTIVE, CONTROLLED, RANDOMIZED, DOUBLE-BLINDED STUDY OF CELL TRANSPLANTATION COMBINED WITH CORONARY BYPASS SURGERY T Pätilä1, M Lehtinen1, A Vento1, J Schildt2, J Sinisalo3, M Laine3, P Hämmäinen1, A Nihtinen4, R Alitalo5, P Nikkinen2, A Ahonen2, M Holmström6, K Lauerma6, R Pöyhiä7, M Kupari3, E Kankuri8, A Harjula1,8 1 Department of Cardiothoracic Surgery, Helsinki University Central Hospital, Helsinki, Finland, 2Department of Clinical Physiology, Helsinki University Central Hospital, Helsinki, Finland, 3Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland, 4Department of Hematology, Helsinki University Central Hospital, Helsinki, Finland, 5Stem Cell Laboratory, HUSLAB, Helsinki, Finland, 6Department of Radiology, Helsinki University Central Hospital, Helsinki, Finland, 7Department of Anesthesiology and Intensive Care, Helsinki University Central Hospital, Helsinki, Finland, 8Institute of Biomedicine, University of Helsinki, Helsinki, Finland Objectives: Worldwide, millions of people are killed every year by ischemic heart failure. Bone marrow mononuclear cell (BMMC) transplantation is a
8 T CELLS REDIRECTED BY A CHIMERIC ANTIGEN RECEPTOR RECOGNIZING HBSAG EFFICIENTLY CONTROL HBV IN VIVO IN TRANSGENIC MICE K Krebs1, N Böttinger1, L Huang2, M Chmielewski3, W Uckert4, H Abken3, M Heikenwälder1, P Knolle2, U Protzer1 1 Institute of Virology, Technische Universität München / Helmholtz Center München, München, Germany, 2Institute of Molecular Medicine and Experimental Immunology, University of Bonn, Bonn, Germany, 3Department of Internal Medicine I, University Hospital Cologne, Cologne, Germany, 4Molecular Cell Biology and Gene Therapy, Max Delbrück Center for Molecular Medicine, Berlin, Germany Background & aims: Current antivirals suppress HBV but do not clear the infection. For virus clearance strong effector T cell responses are needed, which are sparse in chronically infected individuals. Cell therapy using T cells redirected by HBV-specific receptors may clear HBV and help to prevent and
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Scar size at injection sites preoperatively and after follow-up.
Oral Abstracts
patients before or immediately after mCTL infusion. In the absence of conventional therapy, 8 of the 11 patients with CMV infection became negative for CMV in the blood within 7d of mCTL infusion, with a coinciding rise in CMV-specific CTL in PB. Each of 8 patients with high EBV loads cleared their virus, as did 7 of 7 patients with adenoviral infections/disease. Overall the response rate in both groups was 88%. This study demonstrates that mCTL derived from the PB of seropositive donors as well as the CB of virus naive donors expand in vivo and are active against multiple viruses. Furthermore, by restoring immunity to multiple viruses simultaneously, the need for continued prophylaxis with pharmacotherapy is eliminated, thus, improving the efficiency and cost effectiveness of protecting SCT and CBT recipients from these potentially lethal viruses. 5 ANTI-CD123 CHIMERIC ANTIGEN RECEPTOR REDIRECTED T CELLS FOR RELAPSED B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA M Ruella1, O Shestova1, S Kenderian1, D Barrett2, S Grupp2, J Scholler1, S Lacey1, M Kalos1, CH June1, S Gill1 1 Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States, 2Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States
Figure 2A. Comparison of in vitro anti-tumor activity of CART123 and CART19.
Relapsing/refractory (R/R) B-cell Acute Lymphoblastic Leukemia (ALL) is associated with a poor prognosis. We have previously shown that anti CD19 Chimeric Antigen Receptor T cells (CART19) induce significant responses in this population. However, occasional CD19-ve relapses have occurred, likely due to selective pressure from CART19 cells. Hence, CAR-based therapies against additional antigens may be useful in the treatment of B-ALL. Twenty R/R ALL samples, including two CD19-ve relapses, were screened for 30 potential secondary targets using a custom Quantigene RNA panel (Affymetrix) and results were validated by flow cytometry. CD123 was amongst the most highly and homogeneously expressed, present in >60% of blasts in 16/20 R/RALL including 2/2 CD19-ve relapses (Figure 1). We utilized the anti-CD123 CAR T cells co-stimulated via 4-1BB (CART123) that we had previously
Figure 2B. In vitro anti-tumor activity of CART19 and CART123 against ALL. generated for the treatment of acute myeloid leukemia (AML) (Gill et al, ISCT 2013) and performed in vitro comparisons between CART123 and CART19. These investigations revealed similar proliferation, degranulation, cytokine production and cytotoxicity (Figure 2 A) and suggested that CART123 could be used to treat B-ALL. We therefore injected NSG mice with 0.5-2x106 cells of the CD19+ve CD123dim +ve B-ALL cell line Nalm6, and treated them with CART19, CART123 or control T cells (1x106 each). As expected, mice treated with control T cells succumbed quickly to disease. Mice treated with CART19 experienced enhanced survival (*).Mice treated with CART123 had improved survival (*) compared with control T cell treatment, and intermediate between control and CART19 (Figure 2 B). Hence, CART123 may represent an additional approach to treating CD19+ve malignancy. 6 PRODUCTION OF ENDOTHELIAL NO-SYNTHASE GENEENHANCED AUTOLOGOUS PROGENITOR CELL THERAPIES FOR CARDIO-PULMONARY DISEASES DW Courtman, L Comanita, DJ Stewart Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
Figure 1. CD123 expression on ALL blasts at baseline and at CD19-ve relapse.
Endothelial progenitor cells (EPCs) derived from the circulation hold promise for the treatment of cardiovascular diseases. Yet preclinical studies suggest that patient derived cells are less effective stimulators of vascular repair. We have therefore pioneered the development of gene enhanced autologous cell based treatments for pulmonary hypertension and myocardial ischemia. Our first-inhuman clinical trial (PHACeT, Pulmonary Hypertension: Assessment of Cell Therapy, NCT00469027) was a dose escalation safety trial using peripheral blood derived early outgrowth EPCs transiently transfected via electroporation with a human eNOS plasmid and delivered directly to the pulmonary
20th Annual ISCT Meeting
circulation. We demonstrated that administration of eNOS-transfected EPCs to patients with stable, severe PAH was well tolerated, and resulted in a trend towards short-term hemodynamic improvement. Although, there was no long term sustained hemodynamic improvement in this small (7 patient), uncontrolled trial; there were significant increases in 6 minute walk time seen at both 1 month, and persisting to 3and 6 months post cell based gene therapy. To apply our therapy to cardiac patients we developed a non-mobilized automated apheresis procedure combined with GMP manufacturing in environmentally controlled isolator units and in addition adopted a cationic polymer (manose modified linear polyethylenimine) for eNOS transfection to limit cellular manipulations. Enhanced eNOS expression was verified by Western Blotting and cell identity by flow cytometry. With these manufacturing modifications in place a placebo controlled randomized trial (Enhanced Angiogenic Cell Therapy-Acute Myocardial Infraction trial, NCT00936819) testing the role of eNOS transfection in autologous EPCs was initiated with 4 patients treated to date. Our early results suggest that eNOS transfected autologous EPCs are safe and potentially effective treatments for cardiovascular disorders.
7 GENERATION OF DISEASE-SPECIFIC INDUCED PLURIPOTENT STEM CELLS FROM HUMAN FETAL EXTRA-EMBRYONIC TISSUES P Spitalieri, V Talarico, A Luchetti, F Brancati, A Botta, G Novelli, F Sangiuolo Biomedicine and Prevention, University of Rome “Tor Vergata,” Rome, Italy The generation of induced pluripotent stem cells (iPSCs) is a innovative personalized-regenerative technology, which can transform own-self cells into embryonic stem elike cells, which have regarded as a promising candidate for cell-based therapy, as well as an ideal target for disease modeling and drug testing and drug discovery, thus enabling researchers to undertake studies for treating diseases. The objective of the present study was to reprogramming patient-specific fetal cells deriving from prenatal diagnosis for several genetic disorder as Cystic Fibrosis (FC), Myotonic Dystrophy (DM1), b-Thalassemia (b-Thal), Spinal Muscolar Atrophy (SMA1), Lymphema-Distichiasis Syndrome (FOXC2) and healthy cells. The cell type used for create iPSCs can significantly influence the reprogramming efficiency and kinetics. Here, we show that amniotic fluid (LA) and chorionic villus sampling (CVS) represent an ideal cell resource for rapid and efficient generation of human iPSCs. The reprogramming were done using a polycistronic lentiviral vector (hSTEMCCA-loxP) encoding Oct4, Sox2, Klf4 and c-Myc genes necessary to cell reprogramming. Moreover loxP sites can be excised with Cre recombinase. Stem cells specific morphological, molecular and immunocytochemical markers (ALP; OCT4; SSEA4; TRA1-60;TRA1-81) confirmed the successful reprogramming. Additionally, we evaluated their ability to differentiate into the three embryonic germ layers (ecto, endo and mesoderm) by immunocytochemical characterization and their ability to in vivo form teratomas. To date, this represents the first example of iPS cells derived from a very early extra-embryonic fetal tissues like chorionic villi (hIPS-CVS). These data suggest that hIPS-CVS/LA can be considered a valid cell model to accomplish pathogenesis studies and possibly represent a valid tool for future therapeutic applications.
treat HBV-associated liver cancer. We designed a chimeric antigen receptor (CAR) that is composed of a single chain antibody fragment binding to HBsAg and CD28 / CD3z signaling domains. This study aimed to proof feasibility of this approach in vivo addressing the following challenges: (i) T cell-depletion to generate space for cell engraftment in chronic virus carriers is too perilous, (ii) viral antigens circulating in high amounts may inactivate transferred T cells or (iii) trigger uncontrolled immune damage. Methods: Primary murine CD8+ T cells were isolated, stimulated using an optimized protocol and grafted with CARs by retroviral transduction. A CAR that binds HBV envelope proteins and transfers activation signals to the T cell was compared to a control CAR without a proper signaling domain and a CAR not binding HBV proteins. Results: CD8+ T cells engineered to express an HBV-specific CAR, which recognizes HBV envelope proteins of various subtypes on infected hepatocytes, were able to engraft and expand in immune competent HBV transgenic mice. Following adoptive transfer CAR-grafted T cells targeted the liver, remained functional in vivo, rapidly and efficiently controlled HBV replication while causing only transient liver damage. The large amount of circulating viral antigens neither impaired nor over-activated the transferred T cells. Conclusion: HBV-specific cell therapy with CAR-engineered T cells bears the potential to treat chronic hepatitis B and HBV-associated hepatocellular carcinoma irrespective of the patient’s individual HLA-type. 9 BONE MARROW MONONUCLEAR CELLS FOR ISCHEMIC CARDIAC FAILURE - A PROSPECTIVE, CONTROLLED, RANDOMIZED, DOUBLE-BLINDED STUDY OF CELL TRANSPLANTATION COMBINED WITH CORONARY BYPASS SURGERY T Pätilä1, M Lehtinen1, A Vento1, J Schildt2, J Sinisalo3, M Laine3, P Hämmäinen1, A Nihtinen4, R Alitalo5, P Nikkinen2, A Ahonen2, M Holmström6, K Lauerma6, R Pöyhiä7, M Kupari3, E Kankuri8, A Harjula1,8 1 Department of Cardiothoracic Surgery, Helsinki University Central Hospital, Helsinki, Finland, 2Department of Clinical Physiology, Helsinki University Central Hospital, Helsinki, Finland, 3Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland, 4Department of Hematology, Helsinki University Central Hospital, Helsinki, Finland, 5Stem Cell Laboratory, HUSLAB, Helsinki, Finland, 6Department of Radiology, Helsinki University Central Hospital, Helsinki, Finland, 7Department of Anesthesiology and Intensive Care, Helsinki University Central Hospital, Helsinki, Finland, 8Institute of Biomedicine, University of Helsinki, Helsinki, Finland Objectives: Worldwide, millions of people are killed every year by ischemic heart failure. Bone marrow mononuclear cell (BMMC) transplantation is a
8 T CELLS REDIRECTED BY A CHIMERIC ANTIGEN RECEPTOR RECOGNIZING HBSAG EFFICIENTLY CONTROL HBV IN VIVO IN TRANSGENIC MICE K Krebs1, N Böttinger1, L Huang2, M Chmielewski3, W Uckert4, H Abken3, M Heikenwälder1, P Knolle2, U Protzer1 1 Institute of Virology, Technische Universität München / Helmholtz Center München, München, Germany, 2Institute of Molecular Medicine and Experimental Immunology, University of Bonn, Bonn, Germany, 3Department of Internal Medicine I, University Hospital Cologne, Cologne, Germany, 4Molecular Cell Biology and Gene Therapy, Max Delbrück Center for Molecular Medicine, Berlin, Germany Background & aims: Current antivirals suppress HBV but do not clear the infection. For virus clearance strong effector T cell responses are needed, which are sparse in chronically infected individuals. Cell therapy using T cells redirected by HBV-specific receptors may clear HBV and help to prevent and
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Scar size at injection sites preoperatively and after follow-up.