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. 2010 Apr;21(4):427-37.
doi: 10.1089/hum.2009.114.

piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies

Pallavi V Raja Manuri  1 , Matthew H Wilson, Sourindra N Maiti, Tiejuan Mi, Harjeet Singh, Simon Olivares, Margaret J Dawson, Helen Huls, Dean A Lee, Pulivarthi H Rao, Joseph M Kaminski, Yozo Nakazawa, Stephen Gottschalk, Partow Kebriaei, Elizabeth J Shpall, Richard E Champlin, Laurence J N Cooper
Affiliations

piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies

Pallavi V Raja Manuri et al. Hum Gene Ther. 2010 Apr.

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(+) 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.

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Figures

FIG. 1.
FIG. 1.
Schematic of the two PB DNA plasmids electrotransferred. (A) CoOpCD19RCD28/pXLBacIIUbnls (pPB-CAR, Transposon): polyubiquitin promoter; CoOpCD19RCD28, codon-optimized CD19RCD28 CAR; pBac3′ and pBac5′, PB-inverted/direct repeats; BGH-polyA, polyadenylation signal from bovine growth hormone; AmpR, ampicillin resistance gene. (B) pCMV-hpB (Transposase): hpB, codon-optimized PB-transposase; CMV IE, CMV enhancer/promoter; pUC ori, minimal E. coli origin of replication. (C) Scheme for electroporation with PB plasmids and propagation on CD19+ K562-derived artificial antigen-presenting cells (aAPCs). Electroporation with transposon (blue) provides only transient expression unless incorporated into a transposon vector that can be cleaved from the plasmid and integrated into a host genome by a source of transposase (red). On the day after electroporation, T cells are cocultured with γ-irradiated K562 genetically modified to coexpress CD19, CD64, CD86, CD137L (4-1BBL), and cell surface membrane-bound IL-15 (fusion of IL-15 cytokine peptide and human Fc region), with the addition of IL-2, resulting in expansion of stably transfected CAR+ T cells to clinically significant numbers.
FIG. 2.
FIG. 2.
CAR expression on T cells after electrotransfer of PB vector(s) and selected outgrowth of CAR+ T cells upon coculture with aAPCs. (A) Expression of CD19RCD28 CAR on CD3+ T cells by flow cytometry with anti-Fc antibody after electrotransfer of PB transposon with or without PB transposase at 24 hr and 3 weeks of coculture on γ-irradiated K562-derived aAPCs (clone 4). (B) Kinetics of T cell growth on coculture with aAPCs. (C) CAR expression over time. Percentage expression of CAR and MFI (surrogate for density) on T cells cotransfected with PB transposon and transposase upon coculture with K562-aAPCs.
FIG. 3.
FIG. 3.
Redirected specificity of PBMCs genetically modified with the PB system. (A) GFP+ U251T targets were transfected with truncated CD19-expressing plasmid and stable transfectants were analyzed for CD19 expression by flow cytometry. (B) Killing of CD19+ target cells (CD19-expressing human Burkitt's lymphoma or Daudi cells, U251T CD19– glioblastoma cells, and U251T cells transfected to express truncated CD19) in a standard 4-hr CRA. Points represent mean specific lysis of triplicate wells at two effector-to-target (E:T) cell ratios; error bars represent the SD. (C) VTLM to evaluate tumor killing by PB-modified CAR+ T cells. (i) To distinguish GFP+CD19+ from GFP+CD19– U251T cells, the red fluorescent dye PHK-26 was preloaded onto CD19+ target cells, which resulted in cells appearing orange (a merging of GFP [green] with PHK [red]). The CD19-negative and -positive targets mixed at a 1:1 ratio were plated overnight. PB-modified CAR+ T cells were added to these targets after overnight plating at an E:T ratio of 10:1. Cells were cocultured for 4 hr and imaged by VTLM. CD19+ tumor targets, which were engaged, disengaged, and killed by the T cells, imploded and lysed and are shown as greenish-yellow irregular cells, whereas live CD19– tumor targets remained flat and spread out (green). (ii) Two movies, one at low power (movie 1) and one at high power (movie 2), show tumor cell killing by PB-modified CAR+ T cells. In each case the killing events measured over 2 hr were condensed to 12–14 sec for visualization.
FIG. 4.
FIG. 4.
Characterization of CAR+ T cells on PBMCs after electrotransfer of PB vectors. (A) Immunophenotype of memory cell markers (CD27, CD28, CD62L, and CCR7) on PB-modified T cells generated after 4 weeks of coculture on aAPCs. Histograms presented as solid black lines reveal the percentage of T cells expressing CD27, CD28, CD62L, and CCR7 in the lymphocyte-gated population. T cells expressing the memory cell markers were analyzed for coexpression of CAR and CD4 or CD8. (B) The central memory phenotype (TCM) of T cells generated after coculture. CD45RO and CCR7 double-positive T cells were analyzed for the expression of CD62L. In addition, TCM cells, defined as CD45RO+CCR7+CD62L+, were analyzed for coexpression of CAR.
FIG. 5.
FIG. 5.
Lack of autonomous proliferation after electrotransfer with PB vectors and safety issues. (A) T-cell proliferation analyses directly imaged with a Cellometer in the absence/presence of K562-aAPCs and IL-2. Data show primarily dead T cells (shriveled) when K562-aAPCs and IL-2 are removed compared with healthy (refractile, rounded) T cells when K562-aAPCs and IL-2 are present. (B) Lack of integration of PB transposase by genomic PCR from genetically modified and propagated peripheral blood-derived T cells. DNA was isolated from T cells after mock electroporation (lanes 2 and 4, 50 and 100 ng of genomic DNA, respectively), from T cells 28 days after electroporation with the two-plasmid PB system (lanes 3 and 5, 50 and 100 ng of genomic DNA, respectively). Lane 1, pCMV-hpB plasmid DNA (1 ng) loaded as a positive control. PCR was carried out with transposase-specific primers and GAPDH-specific primers in the same reaction. (C) Fluorescence in situ hybridization (FISH) analysis of PB-modified CAR+ T cells. Number of copies of the CD19RCD28 transgene integrated on electroporation with PB vectors and propagation on CD19-specific K562-derived aAPCs was determined by FISH analysis as described in Materials and Methods. Data shown are a representation after analyzing 40–50 individual metaphase spreads. Twenty-three pairs of chromosomes are shown and the arrow indicates the integration sites. (D) Idiogram of a Giemsa-banded karyotype of PB-modified T cells, showing no apparent numerical or structural chromosome alterations.
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