title: Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials.
abstract: OBJECTIVE
  Protocols for the production of CD19-specific chimeric antigen receptor (CAR19) T cells are often complex and expensive because of the use of retroviral and lentiviral vectors or the need for CAR19 T-cell enrichment. We aimed to simplify the generation of CAR19 T cells from the peripheral blood of normal donors and patients using the piggyBac transposon system of gene modification.
  
  
  METHODS
  We varied electroporation voltage, cytokines and stimulation conditions for the generation and expansion of CAR19 T cells over a 3-week culture period.
  
  
  RESULTS
  Using optimized electroporation voltage, interleukin-15 alone and co-culturing CAR T cells with peripheral blood mononuclear cells, we were able to expand CAR19 T-cell cultures by up to 765-fold over 3 weeks in normal donors and 180-fold in patients with B-cell malignancies. Final median CAR19 expression of 72% was seen in normal donors, and 81% was seen in patients with acute lymphoblastic leukaemia, chronic lymphocytic leukemia or non-Hodgkin lymphoma. CAR19 T cells produced interferon gamma on stimulation with CD19(+) cell lines and efficiently lysed both CD19(+) cell lines and primary leukemia cells. In addition, combining CAR expression with an inducible caspase safety switch allowed elimination of CAR19 T cells by the application of a small molecule dimerizer.
  
  
  CONCLUSIONS
  We have produced a simple, inexpensive and easily adoptable protocol for the generation of CAR19 T cells suitable for use in clinical trials using the piggyBac transposon system. This provides a robust platform for further enhancing the T-cell product and testing new CAR technologies.
authors.affiliation: The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia.
authors.forename: Saumya
authors.initials: S
authors.lastname: Ramanayake
authors.affiliation: The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; The Department of Haematology, Westmead Hospital, Westmead, Australia; Sydney Cellular Therapies Laboratory, Pathology West, Westmead, Australia.
authors.forename: Ian
authors.initials: I
authors.lastname: Bilmon
authors.affiliation: The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; The Department of Haematology, Westmead Hospital, Westmead, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Bishop
authors.affiliation: The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; The Department of Haematology, Westmead Hospital, Westmead, Australia.
authors.forename: Ming-Celine
authors.initials: MC
authors.lastname: Dubosq
authors.affiliation: The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; The Department of Haematology, Westmead Hospital, Westmead, Australia; Sydney Cellular Therapies Laboratory, Pathology West, Westmead, Australia.
authors.forename: Emily
authors.initials: E
authors.lastname: Blyth
authors.affiliation: The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; Sydney Cellular Therapies Laboratory, Pathology West, Westmead, Australia.
authors.forename: Leighton
authors.initials: L
authors.lastname: Clancy
authors.affiliation: The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; The Department of Haematology, Westmead Hospital, Westmead, Australia; Sydney Cellular Therapies Laboratory, Pathology West, Westmead, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Gottlieb
authors.affiliation: The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; The Department of Haematology, Westmead Hospital, Westmead, Australia; Sydney Cellular Therapies Laboratory, Pathology West, Westmead, Australia. Electronic address: kenneth.micklethwaite@sydney.edu.au.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Micklethwaite
mesh_terms: D019264:Adoptive Transfer / Q000379:methods*; D000328:Adult; D018941:Antigens, CD19 / Q000276:immunology*; D001402:B-Lymphocytes / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods*; D018920:Coculture Techniques; D004251:DNA Transposable Elements; D018274:Electroporation / Q000379:methods; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D019409:Interleukin-15 / Q000378:metabolism; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology; D008228:Lymphoma, Non-Hodgkin / Q000276:immunology; D008297:Male; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology* / Q000637:transplantation
chemical_list: D018941:Antigens, CD19; C000605956:CD19-specific chimeric antigen receptor; D004251:DNA Transposable Elements; D019409:Interleukin-15; D011948:Receptors, Antigen, T-Cell; D007371:Interferon-gamma
keywords: T cells; chimeric antigen receptor; piggyBac; transposon