piggyBac Transposon/Transposase System to Generate CD19-Specific T Cells for the Treatment of B-Lineage Malignancies

Abstract

Nonviral integrating vectors can be used for expression of therapeutic genes. piggyBac (PB), a transposon= transposase system, has been used to efficiently generate induced pluripotent stems cells from somatic cells, without genetic alteration. In this paper, we apply PB transposition to express a chimeric antigen receptor (CAR) in primary human T cells. We demonstrate that T cells electroporated to introduce the PB transposon and transposase stably express CD19-specific CAR and when cultured on CD19 þ artificial antigen-presenting cells, numerically expand in a CAR-dependent manner, display a phenotype associated with both memory and effector T cell populations, and exhibit CD19-dependent killing of tumor targets. Integration of the PB transposon expressing CAR was not associated with genotoxicity, based on chromosome analysis. PB transposition for generating human T cells with redirected specificity to a desired target such as CD19 is a new genetic approach with therapeutic implications.

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

“…56 Suitability of the PiggyBac system for generation of CAR-T cells has been demonstrated recently. [57][58][59] In summary, we developed a novel antibody discovery platform that allows the establishment of full-length IgG antibody libraries in mammalian B cells by transposition using simple protocols. Rapid isolation of top-binding candidates from these cellular antibody libraries is facilitated by display of antibodies on the cell surface and FACS-sorting, followed by direct analysis of soluble antibody molecules secreted into the supernatant of sorted single cell clones using analytical and functional assays of choice.…”

Infusing CD19-Directed T Cells to Augment Disease Control in Patients Undergoing Autologous Hematopoietic Stem-Cell Transplantation for Advanced B-Lymphoid Malignancies

“…An advantage of combining the SB system with aAPC is efficient integration of plasmid DNA and associated shortened time in tissue culture to recover clinically sufficient numbers of T cells that stably express CAR (Ivics et al, 1997;Geurts et al, 2003;Hackett et al, 2005Hackett et al, , 2010Singh et al, 2008;Manuri et al, 2009;Izsvák et al, 2010). DNA plasmids used for the human application of SB transposition can be manufactured at approximately one-tenth the cost of recombinant retrovirus used to transduce clinical grade T cells, which facilitates implementation of early-phase gene therapy trials.…”

“…The two SB DNA plasmids (manufactured by Waisman Biomanufacturing, Madison, WI), encoding the CAR (designated CD19RCD28) transposon (Cooper, 2007;Singh et al, 2008;Jena et al, 2010) and SB11 transposase (Singh et al, 2008;Manuri et al, 2009), will be simultaneously electrotransferred into T cells with Nucleofector device (Lonza Group, Basel, Switzerland). The genetically modified T cells will be selectively propagated in a CAR-dependent manner on c-irradiated K562 cells that have been genetically modified by transduction with lentivirus (in collaboration with C. June at the University of Pennsylvania, Philadelphia, PA) and cloned by limiting dilution to function as CD19 + aAPC.…”

Clinical Application of <em>Sleeping Beauty</em> and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

“…The plasmids used in the current study were produced commercially by Waisman Clinical Biomanufacturing Facility (Madison, WI). The aAPC (clone #4), derived from K562 cells (parental line obtained from American Type Culture Collection), co-express desired T cell co-stimulatory molecules (each introduced molecule at 3 90% on cell surface of aAPC), as previously described 12 .…”

“…To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb) 12 . In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR + T cells suitable for human application.…”