- Email: [email protected]
Grb10 protein in delaying progression of duchenne muscular dystrophy (DMD) with combined therapy of glucocorticoid corticosteroids
S52
Poster abstracts
Union (EU), to facilitate their access to market and to foster European competitiveness while guaranteeing the highest level of health protection for patients. Academic GMP facilities are major contributors to the development of ATMPs. This study was designed to assess the impact of Regulation (EC) No 1394/2007 and related Directives on them. Respondents were gathered by accessibility sampling. 747 European contact points in academic and non-industrial facilities were identified. Of these, 85 responded to a first survey and 50 to a longer questionnaire. Experienced centers were selected in every member state and approached with semistructured interviews. Successful Academic and Hospital GMP facilities can be identified, independent of the country of origin. However, the implementation of EU Regulation is highly heterogeneous between member states, with evidence of substantial differences in the criteria used to define ATMPs and in the approved manufacturing environment. In ATMP Development and Manufacture, academic institutions fill an illdefined gap where commercialisation is improbable, such as rare diseases or highly patient-specific therapies. Investigator-initiated trials of ATMPs critically depend on academic GMP facilities, as does the biopharmaceutical industry which mostly uses these trials as its pipeline. However, EU legislators do not engage with Academia when considering new legislation. We have found evidence of poor harmonisation at the level of delivery across the Member States and uncertainty about the regulatory process; stifling development and commercialisation of these therapies. Most disturbing is the detrimental effect on translation to early phase trials which remain largely academic investigator-led. Academic GMP practitioners should strengthen their political visibility and contribute to the development of functional and effective EU legislation in this field.
use. The contribution of the manufacture of cell-based medicinal products (CBMPs) and gene transfer medicinal products (GTMPs) to clinical research is consistently underestimated. In addition, the establishment of an Academic GMP Facility is constrained by factors from within, such as limited GMP expertise, a lack of awareness of costs associated with drug development and manufacture, and insufficient personnel. The role of academia in the provision of potentially curative cell-based therapies is recognized but implementation of the infrastructure remains ill-defined. In Munich, an academic GMP facility for the manufacture of ATMPs has been established as a joint effort by the Technische Universität München, the Klinikum rechts der Isar and the Helmholtz Zentrum München. In doing so, the problems for GMP facilities are taken into consideration, as is the specific situation in Academia. This includes, among other factors: providing a central core facility for Academia in the region of Munich, open access for projects from various research groups, a contract-based provision of manufacturing capacity (time, equipment, personnel) according to the specific project needs, the recognition of local expertise and technical clusters, the enforcement of a ‘common trunk’ of technical platforms that lead to the GMP upscaling of selected technologies and processes, a lean, cost-effective management structure, interaction with GMP facilities in other Academic Centers in an attempt to exchange expertise and to avoid replication and the establishment of common structures in Quality Assurance for GMP and GCP in the institution. The academic GMP facility for production of ATMPs has been completed and is scheduled to be operative in the beginning of 2013. Details of the scheduled projects, management and operating structure will be presented.
184 ENABLING CELL BASED THERAPY MANUFACTURING SCALING UP CELL CULTURE FROM MULTITRAY STACK TO CLOSED AND CONTROLLED MULTIPLATE BIOREACTOR J Castillo, M Egloff, F Moncaubeig ATMI LifeSciences, Brussels, Belgium
186 A NEW CELL THERAPY SECTOR ARISING FROM THE CONVERGENCE OF CELL AND GENE THERAPY EJ Culme-Seymour1, S Edwards-Parton2, J Carmen3, W Folkerts3, D Smith3, C Mason2 1 London Regenerative Medicine Network, London, United Kingdom, 2Advanced Centre for Biochemical Engineering, University College London, London, United Kingdom, 3Lonza, Walkersville, MD
A successful transition from laboratory scale, which is suitable for supplying early clinical trials, to an efficient and robust good manufacturing practice (GMP) process is key and represents a major challenge for cell based products manufacturing. Implementing innovative technologies is essential to support such industrialization. Single-use bioreactors - now well established in biopharmaceutical productionrepresent a solution by minimizing manual handling, realizing economies of scale and delivering the benefits of a closed system. However process development and scale-up is challenging as stem cells are highly sensitive to the microenvironment and its physical parameters with factors such as surface material, pH, dissolved oxygen and shear stress. The XpansionÔ multiplate bioreactors (ATMI LifeSciences) have been specifically designed to enable an easy transfer from existing multitray stack process by offering the same cell growth environment -2D hydrophylized polystyrene plates- in a compact, closed system. Critical cell culture parameters as pH and dissolved oxygen are controlled in Xpansion and cell density could be automaticaly monitored via specific holographic microscopy. This presentation illustrates with different cases studies the efficient transition from traditional multitray stack systems to Xpansion bioreactor. Cell density and viability of human Mesenchymal Stem Cells were maintained after two consecutive passages for 3 different donors in Xpansion-10 plates compared to multitray stack based process. Second case demonstrates consistency of culture through scale-up. Cell density and viability were successfully maintained to the final scale Xpansion-200 plates (growth surface of 122,400 cm2 - equivalent to 20 multitray stack). Cell quality was assayed and showed equivalence between all Xpansion scales and standard multitray stack process. Trends analysis of pH and DO for several cell cultures, as well as cell confluence monitoring, highlight efficiency of controls and monitoring to develop a robust scalable process. Value and benefits are also examined through COGs analysis.
The cell therapy industry (CTI) is a rapidly growing new global healthcare sector. Encompassed within this sector is the unique offering of a combined cell and gene therapy application, which is starting to emerge as a clear focus for future product development. However, such a unique product offering presents numerous challenges that in many cases face the CTI as a whole: solving issues on mechanisms of action, manufacturing, regulation and clinical trial design. Whilst the cell therapy clinical trials pipeline has been widely published following a number of categorization strategies, the converged cell and gene therapy clinical pipeline is yet to be understood. A search was performed on the website clinicaltrials.gov using the embedded search-engine and key terms relating to ‘gene therapy’. 2,978 files were extracted (7/09/2012) encompassing all trials registered over the past decade, and individually screened for relevance to ensure the clinical procedure involved the in vivo delivery of a gene-modified cell. After screening, the resulting small number of cell and gene therapy combination trials were then categorized and core information collated including: phase, cell type, vector, recruitment status and start date. Key results from the chronology of active trials at either recruiting or non-recruiting stages highlight the progression of cell and gene therapy combinations reaching clinical development, and the data highlights current favored cell types and choice of vector, as well as the main indications targeted. This data has implications for discovery scientists, clinicians, and manufacturers looking at addressing market needs, and highlights the need for careful consideration on regulatory and product development strategy for these advanced therapies.
185 TUMCELLS: MANUFACTURE OF ADVANCED THERAPIES IN ACADEMIA A Slobodianski, M Hildebrandt, M Neuenhahn TUMCells, Munich, Germany
CELLULAR
The availability of dedicated GMP (Good Manufacturing Practice) facilities is crucial for the development and provision of cell-based therapies for clinical
187 GRB10 PROTEIN IN DELAYING PROGRESSION OF DUCHENNE MUSCULAR DYSTROPHY (DMD) WITH COMBINED THERAPY OF GLUCOCORTICOID CORTICOSTEROIDS TC Chang University of California, San Diego, La Jolla, CA Duchenne Muscular Dystrophy (DMD) is the most severe form of muscular dystrophy. Patients experience rapid muscle deterioration/wasting, motor
19th Annual ISCT Meeting
function loss, and eventually myocardium abnormality and death. The pathological cause of DMD is the production of non-functional dystrophin from mutated gene. Dystrophin, a vital component that connects cytoskeleton of muscle fiber to surrounding extracellular matrix (ECM), shield muscle fibers from injury, and promoting stable cell signaling. With dysfunctional dystrophin, muscle fibers are damaged under routine contraction and relaxation; intracellular signaling is disrupted (ICS), and eventually leads to myofiber necrosis. Recent study of glucocorticoid corticosteroids therapy (GCT) may strengthen and retains functionality of muscle to delay progression of condition. However, strengthening muscle alone might not be sufficient to prevent premature death caused by myocardium deterioration or diaphragm collapse. Recent study on protein Grb10 implicates possible treatment for DMD. Growth factor receptor-bound protein 10 (Grb10), originally responsible for signaling of insulin/insulin-like growth factor 1 (IGF1) is found to actively influences the growth of muscle fibers. In recent study, global inhibition or deletion of Grb10 in rodents shows substantial and permanent growth in muscle . Although the exact pathway of affect in human is not yet understood, with enhanced growth of muscle fibers, patient with DMD may benefit from increased amount of functional muscles and allows more physical-related muscle strengthening therapy to be performed. This research is aimed at combining GCT and Grb10 inhibition to determine the efficacy on patient with DMD and also any possible contraindication. 188 CHIMERIC ANTIGEN RECEPTOR FOR SPECIFIC TARGETING OF ACUTE MYELOID LEUKEMIA I Pizzitola1,2, F Anjos-Afonso1, K Rouault-Pierre1, F Lassailly1, A Biondi2, E Biagi2, D Bonnet1 1 Cancer Research UK, London Research Institute, London, United Kingdom, 2M.Tettamanti Research Center, Clinica Pediatrica, Università Milano-Bicocca, Monza, Italy Chimeric antigen receptors (CARs) have recently emerged as a powerful tool to redirect T-cell activity against tumors. Acute myeloid leukemia (AML) is a good target for a CAR strategy due the over-expression of CD33 and CD123 molecules. Since CD33 is also expressed on hematopoietic stem/progenitors cells (HSPCs), CD123 has emerged as new attractive molecules being still overexpressed by AML population, and at the same time less expressed on HSPCs. We describe the in-vivo efficacy and the safety of this approach based on Cytokine-Induced-Killers (CIK)-cells genetically modified to express CAR specific for the CD33 or CD123 antigen. Once injected into low-level AML engrafted mice, genetically modify T-cells had a potent antitumor effect. Bone-marrow of untreated animals or mice treated with un-manipulated CIK-cells, was infiltrated by leukemic cells (86% and 81% leukemic engraftment), while in 7/8 anti-CD33-CD28-OX40-z and 8/ 10 anti-CD123-CD28-OX40-z treated mice we couldn’t detect any AML-cells. Similar results have been obtained with high AML burden. One week after the last CIK’s injection the level of AML engraftment was 96%, 87%, 0.35% and 0.34% for untreated mice, mice treated with un-manipulated CIK-cells and with anti-CD33-CD28-OX40-z and anti-CD123-CD28-OX40-z transduced CIK-cells respectively. With residual AML-cells we performed secondary transplantation and mice were treated again with transduced CIKcells. AML-cells were still sensitive to CARs approach, leading to a complete eradication of the disease. Furthermore, a fundamental issue was to determine the safety profile of such approach against normal precursors. In untreated mice injected with cord-blood derived CD34+-cells the level of engraftment of hCD45 compartment was 42% whilst in mice treated with unmanipulated, anti-CD33-CD28-OX40-z or anti-CD123-CD28-OX40-z transduced CIK-cells the levels was 40%, 11.7% and 26.3% respectively. These experiments should offer relevant information concerning the efficacy and safety of the proposed strategy to treat transplanted and patients resistant to conventional chemotherapy. 189 SLEEPING BEAUTY SYSTEM-MEDIATED EXPRESSION OF CHIMERIC ANTIGEN RECEPTORS (CARS) IN CIK CELLS: A NOVEL TOOL TOWARDS THE CURE OF CHILDHOOD LEUKEMIA CF Magnani1, GM Giordano Attianese1, S Tettamanti1, N Turazzi1, V Marin1, LJ Cooper2, A Biondi1, E Biagi1
S53
1 Research Center “Matilde Tettamanti”, Monza (MB), Italy, 2Department of Pediatrics Patient Care, Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX
Stable gene transfer of chimeric antigen receptors (CAR) is a powerful tool to redirect antigen specificity of effector T cells for adoptive immunotherapy of tumors. The development of efficient and safe nonviral vectors would rescue the safety and manufacture concerns that limit so far their clinical application. The ex-vivo use of the latest generation Sleeping Beauty (SB) Transposon-mediated gene transfer offers a valid alternative to viral vectors for human gene therapy. Here we used nucleofection of SB system and an optimized clinical-grade stimulation protocol to generate and propagate cytokine-induced killer (CIK) cells genetically modified to express CD123-specific 3rd generation chimeric antigen receptor (CAR) to redirect antigen specificity towards the acute myelogenous leukemia (AML) CD123+ blasts. Indeed, CD123 overexpression is well known to be a poor prognosis marker in AML. With this system, the average transfection at 24hours was 68.9% (1.0, n¼6) and mean survival percentage was 34.8% (5.6, n¼5). Electroporation with the optimized stimulation protocol did not affect the phenotype of CIK cells, preserved their naïve and stem cell memory populations, and resulted in a fold increase of 15.73.3 at the end of the CIK culture at day 21. Transposed CIK cells displayed stable expression of Tr_scFvCD123.3rdCAR (39.4%8.6), efficient lysis of AML target cells, and CAR-specific cytokine secretion. Finally, long-term expansion (fold increase 423.8301.4, n¼2 at day 40) upon stimulation with AML cells in vitro promoted efficient selection of CAR+ CIK cells with potent cytotoxic activity towards AML target (92.91, n¼2), suggesting long-term persistence of functional CIK cells in vivo. Efficient transfection of CARs together with a feasible manufacturing practice (GMP) strategy to select and propagate CAR-expressing CIK cells will be instrumental in defining novel therapeutic approaches to target childhood acute myeloid leukemia.
190 GENERATION OF VIRUS-SPECIFIC T-CELLS EXPRESSING A CD19-SPECIFIC CHIMERIC ANTIGEN RECEPTOR USING THE PIGGYBAC TRANSPOSON/TRANSPOSASE GENE MODIFICATION SYSTEM AND HLA-STREPTAMERS S Ramanayake, I Bilmon, D Gottlieb, K Micklethwaite Westmead Millennium Institute, Westmead, NSW, Australia Aim: To optimise electroporation and culture conditions for the generation of virus-specific T-cells expressing CD19-specific chimeric antigen receptors (CARs) using the PiggyBac gene modification system combined with HLAstreptamer isolation. Methods: 4106 peripheral blood mononuclear cells (PBMC) were electroporated in the presence of 5ug each of PiggyBac transposase and CD19specific CAR transposon plasmids. Electroporation was carried out at a range of voltages (2000-2400v) and pulse widths (215ms and 120ms) using the Neon electroporation system (Invitrogen). Transduced cells were rested for 24 hours and then stimulated on day+1 and day+8 with CD19+ targets at a range of effector:stimulator ratios. CAR T-cells were harvested on day+15. Cells were enumerated by trypan blue exclusion and CD3, CAR expression and HLAtetramer binding were analysed by flow cytometry. Cytomegalovirus-specific Tcells were enriched from CAR expressing T-cells using HLA-streptamer technology. Results: Electroporation of PBMC with a single 20 ms pulse at 2400v produced optimal transduction efficiency with mean percentage CAR expression of 33% (n¼3, range 25.7%-47.6%). Stimulation with the CD19+ Nalm-6 cell line at an E:S ratio of 4:1 or PBMC at an E:S ratio of 1:1 produced optimal expansion. Stimulation with Nalm-6 cells led to enrichment of mean CAR expression over a 14 day culture period from starting expression of 30.4% to 78.2% (n¼4, range 62.5%-80.3%) with 12.3-fold overall cell expansion (range 3.7-20.8). Stimulation with PBMC produced enrichment of mean CAR expression to 64.75% (range 56.4%-77.9%) and 45.5-fold expansion (range 19.7-61.3). Virus-specific T-cells could be enriched with HLA-streptamers from a baseline of 2.1% to 73.4% of CAR positive cultures - generating T-cells specific for both virus and CD19. Conclusion: HLA-streptamer isolation of virus-specific T-cells, genetic modification using the PiggyBac system and stimulation with autologous PBMC provides a simple means of generating virus-specific T-cells expressing a CD19specific CAR for use in clinical trials in patients with B-cell malignancies.
Poster abstracts
Union (EU), to facilitate their access to market and to foster European competitiveness while guaranteeing the highest level of health protection for patients. Academic GMP facilities are major contributors to the development of ATMPs. This study was designed to assess the impact of Regulation (EC) No 1394/2007 and related Directives on them. Respondents were gathered by accessibility sampling. 747 European contact points in academic and non-industrial facilities were identified. Of these, 85 responded to a first survey and 50 to a longer questionnaire. Experienced centers were selected in every member state and approached with semistructured interviews. Successful Academic and Hospital GMP facilities can be identified, independent of the country of origin. However, the implementation of EU Regulation is highly heterogeneous between member states, with evidence of substantial differences in the criteria used to define ATMPs and in the approved manufacturing environment. In ATMP Development and Manufacture, academic institutions fill an illdefined gap where commercialisation is improbable, such as rare diseases or highly patient-specific therapies. Investigator-initiated trials of ATMPs critically depend on academic GMP facilities, as does the biopharmaceutical industry which mostly uses these trials as its pipeline. However, EU legislators do not engage with Academia when considering new legislation. We have found evidence of poor harmonisation at the level of delivery across the Member States and uncertainty about the regulatory process; stifling development and commercialisation of these therapies. Most disturbing is the detrimental effect on translation to early phase trials which remain largely academic investigator-led. Academic GMP practitioners should strengthen their political visibility and contribute to the development of functional and effective EU legislation in this field.
use. The contribution of the manufacture of cell-based medicinal products (CBMPs) and gene transfer medicinal products (GTMPs) to clinical research is consistently underestimated. In addition, the establishment of an Academic GMP Facility is constrained by factors from within, such as limited GMP expertise, a lack of awareness of costs associated with drug development and manufacture, and insufficient personnel. The role of academia in the provision of potentially curative cell-based therapies is recognized but implementation of the infrastructure remains ill-defined. In Munich, an academic GMP facility for the manufacture of ATMPs has been established as a joint effort by the Technische Universität München, the Klinikum rechts der Isar and the Helmholtz Zentrum München. In doing so, the problems for GMP facilities are taken into consideration, as is the specific situation in Academia. This includes, among other factors: providing a central core facility for Academia in the region of Munich, open access for projects from various research groups, a contract-based provision of manufacturing capacity (time, equipment, personnel) according to the specific project needs, the recognition of local expertise and technical clusters, the enforcement of a ‘common trunk’ of technical platforms that lead to the GMP upscaling of selected technologies and processes, a lean, cost-effective management structure, interaction with GMP facilities in other Academic Centers in an attempt to exchange expertise and to avoid replication and the establishment of common structures in Quality Assurance for GMP and GCP in the institution. The academic GMP facility for production of ATMPs has been completed and is scheduled to be operative in the beginning of 2013. Details of the scheduled projects, management and operating structure will be presented.
184 ENABLING CELL BASED THERAPY MANUFACTURING SCALING UP CELL CULTURE FROM MULTITRAY STACK TO CLOSED AND CONTROLLED MULTIPLATE BIOREACTOR J Castillo, M Egloff, F Moncaubeig ATMI LifeSciences, Brussels, Belgium
186 A NEW CELL THERAPY SECTOR ARISING FROM THE CONVERGENCE OF CELL AND GENE THERAPY EJ Culme-Seymour1, S Edwards-Parton2, J Carmen3, W Folkerts3, D Smith3, C Mason2 1 London Regenerative Medicine Network, London, United Kingdom, 2Advanced Centre for Biochemical Engineering, University College London, London, United Kingdom, 3Lonza, Walkersville, MD
A successful transition from laboratory scale, which is suitable for supplying early clinical trials, to an efficient and robust good manufacturing practice (GMP) process is key and represents a major challenge for cell based products manufacturing. Implementing innovative technologies is essential to support such industrialization. Single-use bioreactors - now well established in biopharmaceutical productionrepresent a solution by minimizing manual handling, realizing economies of scale and delivering the benefits of a closed system. However process development and scale-up is challenging as stem cells are highly sensitive to the microenvironment and its physical parameters with factors such as surface material, pH, dissolved oxygen and shear stress. The XpansionÔ multiplate bioreactors (ATMI LifeSciences) have been specifically designed to enable an easy transfer from existing multitray stack process by offering the same cell growth environment -2D hydrophylized polystyrene plates- in a compact, closed system. Critical cell culture parameters as pH and dissolved oxygen are controlled in Xpansion and cell density could be automaticaly monitored via specific holographic microscopy. This presentation illustrates with different cases studies the efficient transition from traditional multitray stack systems to Xpansion bioreactor. Cell density and viability of human Mesenchymal Stem Cells were maintained after two consecutive passages for 3 different donors in Xpansion-10 plates compared to multitray stack based process. Second case demonstrates consistency of culture through scale-up. Cell density and viability were successfully maintained to the final scale Xpansion-200 plates (growth surface of 122,400 cm2 - equivalent to 20 multitray stack). Cell quality was assayed and showed equivalence between all Xpansion scales and standard multitray stack process. Trends analysis of pH and DO for several cell cultures, as well as cell confluence monitoring, highlight efficiency of controls and monitoring to develop a robust scalable process. Value and benefits are also examined through COGs analysis.
The cell therapy industry (CTI) is a rapidly growing new global healthcare sector. Encompassed within this sector is the unique offering of a combined cell and gene therapy application, which is starting to emerge as a clear focus for future product development. However, such a unique product offering presents numerous challenges that in many cases face the CTI as a whole: solving issues on mechanisms of action, manufacturing, regulation and clinical trial design. Whilst the cell therapy clinical trials pipeline has been widely published following a number of categorization strategies, the converged cell and gene therapy clinical pipeline is yet to be understood. A search was performed on the website clinicaltrials.gov using the embedded search-engine and key terms relating to ‘gene therapy’. 2,978 files were extracted (7/09/2012) encompassing all trials registered over the past decade, and individually screened for relevance to ensure the clinical procedure involved the in vivo delivery of a gene-modified cell. After screening, the resulting small number of cell and gene therapy combination trials were then categorized and core information collated including: phase, cell type, vector, recruitment status and start date. Key results from the chronology of active trials at either recruiting or non-recruiting stages highlight the progression of cell and gene therapy combinations reaching clinical development, and the data highlights current favored cell types and choice of vector, as well as the main indications targeted. This data has implications for discovery scientists, clinicians, and manufacturers looking at addressing market needs, and highlights the need for careful consideration on regulatory and product development strategy for these advanced therapies.
185 TUMCELLS: MANUFACTURE OF ADVANCED THERAPIES IN ACADEMIA A Slobodianski, M Hildebrandt, M Neuenhahn TUMCells, Munich, Germany
CELLULAR
The availability of dedicated GMP (Good Manufacturing Practice) facilities is crucial for the development and provision of cell-based therapies for clinical
187 GRB10 PROTEIN IN DELAYING PROGRESSION OF DUCHENNE MUSCULAR DYSTROPHY (DMD) WITH COMBINED THERAPY OF GLUCOCORTICOID CORTICOSTEROIDS TC Chang University of California, San Diego, La Jolla, CA Duchenne Muscular Dystrophy (DMD) is the most severe form of muscular dystrophy. Patients experience rapid muscle deterioration/wasting, motor
19th Annual ISCT Meeting
function loss, and eventually myocardium abnormality and death. The pathological cause of DMD is the production of non-functional dystrophin from mutated gene. Dystrophin, a vital component that connects cytoskeleton of muscle fiber to surrounding extracellular matrix (ECM), shield muscle fibers from injury, and promoting stable cell signaling. With dysfunctional dystrophin, muscle fibers are damaged under routine contraction and relaxation; intracellular signaling is disrupted (ICS), and eventually leads to myofiber necrosis. Recent study of glucocorticoid corticosteroids therapy (GCT) may strengthen and retains functionality of muscle to delay progression of condition. However, strengthening muscle alone might not be sufficient to prevent premature death caused by myocardium deterioration or diaphragm collapse. Recent study on protein Grb10 implicates possible treatment for DMD. Growth factor receptor-bound protein 10 (Grb10), originally responsible for signaling of insulin/insulin-like growth factor 1 (IGF1) is found to actively influences the growth of muscle fibers. In recent study, global inhibition or deletion of Grb10 in rodents shows substantial and permanent growth in muscle . Although the exact pathway of affect in human is not yet understood, with enhanced growth of muscle fibers, patient with DMD may benefit from increased amount of functional muscles and allows more physical-related muscle strengthening therapy to be performed. This research is aimed at combining GCT and Grb10 inhibition to determine the efficacy on patient with DMD and also any possible contraindication. 188 CHIMERIC ANTIGEN RECEPTOR FOR SPECIFIC TARGETING OF ACUTE MYELOID LEUKEMIA I Pizzitola1,2, F Anjos-Afonso1, K Rouault-Pierre1, F Lassailly1, A Biondi2, E Biagi2, D Bonnet1 1 Cancer Research UK, London Research Institute, London, United Kingdom, 2M.Tettamanti Research Center, Clinica Pediatrica, Università Milano-Bicocca, Monza, Italy Chimeric antigen receptors (CARs) have recently emerged as a powerful tool to redirect T-cell activity against tumors. Acute myeloid leukemia (AML) is a good target for a CAR strategy due the over-expression of CD33 and CD123 molecules. Since CD33 is also expressed on hematopoietic stem/progenitors cells (HSPCs), CD123 has emerged as new attractive molecules being still overexpressed by AML population, and at the same time less expressed on HSPCs. We describe the in-vivo efficacy and the safety of this approach based on Cytokine-Induced-Killers (CIK)-cells genetically modified to express CAR specific for the CD33 or CD123 antigen. Once injected into low-level AML engrafted mice, genetically modify T-cells had a potent antitumor effect. Bone-marrow of untreated animals or mice treated with un-manipulated CIK-cells, was infiltrated by leukemic cells (86% and 81% leukemic engraftment), while in 7/8 anti-CD33-CD28-OX40-z and 8/ 10 anti-CD123-CD28-OX40-z treated mice we couldn’t detect any AML-cells. Similar results have been obtained with high AML burden. One week after the last CIK’s injection the level of AML engraftment was 96%, 87%, 0.35% and 0.34% for untreated mice, mice treated with un-manipulated CIK-cells and with anti-CD33-CD28-OX40-z and anti-CD123-CD28-OX40-z transduced CIK-cells respectively. With residual AML-cells we performed secondary transplantation and mice were treated again with transduced CIKcells. AML-cells were still sensitive to CARs approach, leading to a complete eradication of the disease. Furthermore, a fundamental issue was to determine the safety profile of such approach against normal precursors. In untreated mice injected with cord-blood derived CD34+-cells the level of engraftment of hCD45 compartment was 42% whilst in mice treated with unmanipulated, anti-CD33-CD28-OX40-z or anti-CD123-CD28-OX40-z transduced CIK-cells the levels was 40%, 11.7% and 26.3% respectively. These experiments should offer relevant information concerning the efficacy and safety of the proposed strategy to treat transplanted and patients resistant to conventional chemotherapy. 189 SLEEPING BEAUTY SYSTEM-MEDIATED EXPRESSION OF CHIMERIC ANTIGEN RECEPTORS (CARS) IN CIK CELLS: A NOVEL TOOL TOWARDS THE CURE OF CHILDHOOD LEUKEMIA CF Magnani1, GM Giordano Attianese1, S Tettamanti1, N Turazzi1, V Marin1, LJ Cooper2, A Biondi1, E Biagi1
S53
1 Research Center “Matilde Tettamanti”, Monza (MB), Italy, 2Department of Pediatrics Patient Care, Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX
Stable gene transfer of chimeric antigen receptors (CAR) is a powerful tool to redirect antigen specificity of effector T cells for adoptive immunotherapy of tumors. The development of efficient and safe nonviral vectors would rescue the safety and manufacture concerns that limit so far their clinical application. The ex-vivo use of the latest generation Sleeping Beauty (SB) Transposon-mediated gene transfer offers a valid alternative to viral vectors for human gene therapy. Here we used nucleofection of SB system and an optimized clinical-grade stimulation protocol to generate and propagate cytokine-induced killer (CIK) cells genetically modified to express CD123-specific 3rd generation chimeric antigen receptor (CAR) to redirect antigen specificity towards the acute myelogenous leukemia (AML) CD123+ blasts. Indeed, CD123 overexpression is well known to be a poor prognosis marker in AML. With this system, the average transfection at 24hours was 68.9% (1.0, n¼6) and mean survival percentage was 34.8% (5.6, n¼5). Electroporation with the optimized stimulation protocol did not affect the phenotype of CIK cells, preserved their naïve and stem cell memory populations, and resulted in a fold increase of 15.73.3 at the end of the CIK culture at day 21. Transposed CIK cells displayed stable expression of Tr_scFvCD123.3rdCAR (39.4%8.6), efficient lysis of AML target cells, and CAR-specific cytokine secretion. Finally, long-term expansion (fold increase 423.8301.4, n¼2 at day 40) upon stimulation with AML cells in vitro promoted efficient selection of CAR+ CIK cells with potent cytotoxic activity towards AML target (92.91, n¼2), suggesting long-term persistence of functional CIK cells in vivo. Efficient transfection of CARs together with a feasible manufacturing practice (GMP) strategy to select and propagate CAR-expressing CIK cells will be instrumental in defining novel therapeutic approaches to target childhood acute myeloid leukemia.
190 GENERATION OF VIRUS-SPECIFIC T-CELLS EXPRESSING A CD19-SPECIFIC CHIMERIC ANTIGEN RECEPTOR USING THE PIGGYBAC TRANSPOSON/TRANSPOSASE GENE MODIFICATION SYSTEM AND HLA-STREPTAMERS S Ramanayake, I Bilmon, D Gottlieb, K Micklethwaite Westmead Millennium Institute, Westmead, NSW, Australia Aim: To optimise electroporation and culture conditions for the generation of virus-specific T-cells expressing CD19-specific chimeric antigen receptors (CARs) using the PiggyBac gene modification system combined with HLAstreptamer isolation. Methods: 4106 peripheral blood mononuclear cells (PBMC) were electroporated in the presence of 5ug each of PiggyBac transposase and CD19specific CAR transposon plasmids. Electroporation was carried out at a range of voltages (2000-2400v) and pulse widths (215ms and 120ms) using the Neon electroporation system (Invitrogen). Transduced cells were rested for 24 hours and then stimulated on day+1 and day+8 with CD19+ targets at a range of effector:stimulator ratios. CAR T-cells were harvested on day+15. Cells were enumerated by trypan blue exclusion and CD3, CAR expression and HLAtetramer binding were analysed by flow cytometry. Cytomegalovirus-specific Tcells were enriched from CAR expressing T-cells using HLA-streptamer technology. Results: Electroporation of PBMC with a single 20 ms pulse at 2400v produced optimal transduction efficiency with mean percentage CAR expression of 33% (n¼3, range 25.7%-47.6%). Stimulation with the CD19+ Nalm-6 cell line at an E:S ratio of 4:1 or PBMC at an E:S ratio of 1:1 produced optimal expansion. Stimulation with Nalm-6 cells led to enrichment of mean CAR expression over a 14 day culture period from starting expression of 30.4% to 78.2% (n¼4, range 62.5%-80.3%) with 12.3-fold overall cell expansion (range 3.7-20.8). Stimulation with PBMC produced enrichment of mean CAR expression to 64.75% (range 56.4%-77.9%) and 45.5-fold expansion (range 19.7-61.3). Virus-specific T-cells could be enriched with HLA-streptamers from a baseline of 2.1% to 73.4% of CAR positive cultures - generating T-cells specific for both virus and CD19. Conclusion: HLA-streptamer isolation of virus-specific T-cells, genetic modification using the PiggyBac system and stimulation with autologous PBMC provides a simple means of generating virus-specific T-cells expressing a CD19specific CAR for use in clinical trials in patients with B-cell malignancies.