Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials

Saumya Ramanayake; Ian Bilmon; David Bishop; Ming-Celine Dubosq; Emily Blyth; Leighton Clancy; David Gottlieb; Kenneth Micklethwaite

doi:10.1016/j.jcyt.2015.05.013

Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials

Cytotherapy. 2015 Sep;17(9):1251-67. doi: 10.1016/j.jcyt.2015.05.013. Epub 2015 Jul 23.

Authors

Saumya Ramanayake¹, Ian Bilmon², David Bishop³, Ming-Celine Dubosq³, Emily Blyth², Leighton Clancy⁴, David Gottlieb², Kenneth Micklethwaite⁵

Affiliations

¹ The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia.
² 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.
³ The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; The Department of Haematology, Westmead Hospital, Westmead, Australia.
⁴ The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; Sydney Cellular Therapies Laboratory, Pathology West, Westmead, Australia.
⁵ 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.

PMID: 26212611
DOI: 10.1016/j.jcyt.2015.05.013

Abstract

Background aims: 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.

Discussion: 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.

Keywords: T cells; chimeric antigen receptor; piggyBac; transposon.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adoptive Transfer / methods*
Adult
Antigens, CD19 / immunology*
B-Lymphocytes / metabolism*
Cell Separation / methods*
Coculture Techniques
DNA Transposable Elements
Electroporation / methods
Gene Transfer Techniques
Genetic Vectors
Humans
Interferon-gamma / biosynthesis
Interleukin-15 / metabolism
Leukemia, Lymphocytic, Chronic, B-Cell / immunology
Lymphoma, Non-Hodgkin / immunology
Male
Precursor Cell Lymphoblastic Leukemia-Lymphoma / immunology
Receptors, Antigen, T-Cell / genetics
Receptors, Antigen, T-Cell / immunology*
T-Lymphocytes / immunology*
T-Lymphocytes / transplantation

Substances

Antigens, CD19
CD19-specific chimeric antigen receptor
DNA Transposable Elements
Interleukin-15
Receptors, Antigen, T-Cell
Interferon-gamma