Transition from serum-supplemented monolayer to serum-free suspension lentiviral vector production for generation of chimeric antigen receptor T cells |
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| Institution: | 1. Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil;2. Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil;1. Krembil Research Institute, Toronto, ON, Canada;2. Princess Margaret Cancer Centre, Toronto, ON, Canada;3. Institute of Medical Science, University of Toronto, Toronto, ON, Canada;4. Faculty of Medicine, University of Toronto, Toronto, ON, Canada;1. Bone Therapeutics, Gosselies, Belgium;2. Newborn Research Centre, Royal Women''s Hospital, Melbourne, Victoria, Australia;3. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada;4. Stanford University School of Medicine, Stanford, California, USA;5. Interdepartmental Division of Critical Care, Department of Medicine and the Keenan Center for Biomedical Research, St. Michael''s Hospital, University of Toronto, Toronto, Canada;6. Medical Center Groningen, Department of Pathology and Medical Biology, University of Groningen, Groningen, the Netherlands;7. Mesoblast, Melbourne, Australia;8. Department of Laboratory Medicine, Karolinska Institutet, Sweden;9. Department of Cellular Therapy and Allogeneic Stem Cell Transplantation, Karolinska University Hospital, Stockholm, Sweden;10. University of San Francisco, San Francisco, California, United States;11. Wellcome-Wolfson Institute for Experimental Medicine, Queen''s University Belfast, NI, UK;12. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada;13. Sinclair Centre for Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada;14. Sentien Biotechnologies, Lexington, Massachusetts, USA;15. Rutgers University, Piscataway, New Jersey, USA;16. Laboratory of Pulmonary Investigation, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;17. Cystic Fibrosis Foundation, Bethesda, Maryland, USA;18. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada;19. University of Vermont College of Medicine, Burlington, Vermont, USA;1. Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada;2. Centre for Journalology, Clinical Epidemiology Program, The Ottawa Hospital Research Institute, Ottawa, Canada;3. School of Epidemiology and Public Health, Faculty of Medicine, University of Ottawa, Ottawa, Canada;4. Department of Anesthesiology and Pain Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Canada;5. Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada;6. Neonatology, Department of Pediatrics, Children''s Hospital of Eastern Ontario (CHEO) and CHEO Research Institute, Ottawa, ON, Canada;1. Center for Cancer and Immunology Research, Children''s National Hospital, Washington, DC, USA;2. Department of Neurosurgery, Georgetown University Medical Center, Washington, DC, USA;3. Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer Center, Buffalo, New York, USA;4. Genomics Shared Resource, Department of Cancer Genetics and Genomics, Roswell Park Comprehensive Cancer Center, Buffalo, New York, USA;5. George Washington University Cancer Center, George Washington University, Washington, DC, USA;1. Jack Copland Centre, Scottish National Blood Transfusion Service, Edinburgh, UK;2. Cell and Gene Therapy Catapult, Guy''s Hospital, London, UK;3. Autolus Therapeutics plc, London, UK;4. National Health Service Blood and Transplant, Filton, UK;5. Christie National Health Service Foundation Trust, Manchester, UK;6. GlaxoSmithKline, Stevenage, UK;7. Cell and Gene Therapy, Novartis Pharmaceuticals UK Limited, London, UK;8. eXmoor Pharma Concepts Ltd, Stoke Gifford, UK;9. Kite Pharma EU B.V., Hoofddorp, The Netherlands;10. King''s College Hospital National Health Service Foundation Trust, London, UK;11. University College London Hospital National Health Service Foundation Trust, London, UK;12. Terumo BCT Europe NV, Zaventem, Belgium;13. bluebird bio Inc, Cambridge, Massachusetts, USA;14. Great Northern Children''s Hospital, Royal Victoria Infirmary, Newcastle upon Tyne, UK;15. National Marrow Donor Program/Be The Match, Minneapolis, Minnesota, USA;16. Cytiva, Cambridge, UK |
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| Abstract: | Background aimsLentiviral vectors (LVs) have been used extensively in gene therapy protocols because of their high biosafety profile and capacity to stably express a gene of interest. Production of these vectors for the generation of chimeric antigen receptor (CAR) T cells in academic and research centers is achieved using serum-supplemented static monolayer cultures. Although efficient for pre-clinical studies, this method has a number of limitations. The main hurdles are related to its incompatibility with robust and controlled large-scale production. For this reason, cell suspension culture in bioreactors is desirable. Here the authors report the transition of LV particle production from serum-supplemented monolayer to serum-free suspension culture with the objective of generating CAR T cells.MethodsA self-inactivating LV anti-CD19 CAR was produced by transient transfection using polyethylenimine (PEI) in human embryonic kidney 293 T cells previously adapted to serum-free suspension culture.ResultsLV production of 8 × 106 transducing units (TUs)/mL was obtained in serum-supplemented monolayer culture. LV production in the serum-free suspension conditions was significantly decreased compared with monolayer production. Therefore, optimization of the transfection protocol was performed using design of experiments. The results indicated that the best condition involved the use of 1 μg of DNA/106 cells, 1 × 106 cells/mL and PEI:DNA ratio of 2.5:1. This condition used less DNA and PEI compared with the standard, thereby reducing production costs. This protocol was further improved with the addition of 5 mM of sodium butyrate and resulted in an increase in production, with an average of 1.5 × 105 TUs/mL. LV particle functionality was also assessed, and the results indicated that in both conditions the LV was capable of inducing CAR expression and anti-tumor response in T cells, which in turn were able to identify and kill CD19+ cells in vitro.ConclusionsThis study demonstrates that the transition of LV production from small-scale monolayer culture to scalable and controllable bioreactors can be quite challenging and requires extensive work to obtain satisfactory production. |
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