Sleeping Beauty transposon-mediated engineering of human primary T cells for therapy of CD19+ lymphoid malignancies - PubMed (original) (raw)

doi: 10.1038/sj.mt.6300404. Epub 2008 Jan 29.

Hongfeng Guo, Johnthomas Kang, Suet Choi, Tom C Zhou, Syam Tammana, Christopher J Lees, Zhong-Ze Li, Michael Milone, Bruce L Levine, Jakub Tolar, Carl H June, R Scott McIvor, John E Wagner, Bruce R Blazar, Xianzheng Zhou

Affiliations

Sleeping Beauty transposon-mediated engineering of human primary T cells for therapy of CD19+ lymphoid malignancies

Xin Huang et al. Mol Ther. 2008 Mar.

Abstract

We have reported earlier that the non-viral Sleeping Beauty (SB) transposon system can mediate genomic integration and long-term reporter gene expression in human primary peripheral blood (PB) T cells. In order to test whether this system can be used for genetically modifying both PB T cells and umbilical cord blood (UCB) T cells as graft-versus-leukemia effector cells, an SB transposon was constructed to coexpress a single-chain chimeric antigen receptor (CAR) for human CD19 and CD20. PB and UCB were nucleofected with the transposon and a transposase plasmid, activated and then expanded in culture using anti-CD3/CD28 beads. Stable dual-gene expression was confirmed in both T-cell types, permitting enrichment by positive selection with Rituxan. The engineered CD4(+) T cells and CD8(+) T cells both exhibited specific cytotoxicity against CD19(+) leukemia and lymphoma cell lines, as well as against CD19 transfectants, and produced high-levels of antigen-dependent Th1 (but not Th2) cytokines. The in vivo adoptive transfer of genetically engineered T cells significantly reduced tumor growth and prolonged the survival of the animal. Taken together, these data indicate that T cells from PB and UCB can be stably modified using a non-viral DNA transfer system, and that such modified T cells may be useful in the treatment of refractory leukemia and lymphoma.

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Figures

Figure 1

Figure 1. Cell surface expression of chimeric antigen receptor (CAR) and CD20 after transfection

(a) Schematic representation of the CAR for CD19 and the Sleeping Beauty (SB) transposon system used in this study. PGK, phosphoglycerate kinase. mCMV, human cytomegalovirus (CMV) minimal core promoter element, (b) Expression of CAR and CD20 in 293T cells. 293T cells (8 × 105 per well in 12-well plates) were transfected with 2 μg of the SB transposon (19BBζ/CD20) without the SB10, using a standard calcium phosphate precipitation method. Twenty-four to forty-eight hours after transfection, the cells were stained with CAR [goat anti-mouse immunoglobulin G F(ab′)2] and CD20 (Rituxan) antibodies and analyzed by flow cytometry. Untransfected 293T cells were used as mock control. (c) Expression of CAR and CD20 in PBL from two donors (PBL1 and PBL2). (d) CAR and CD20 expression in umbilical cord blood (UCB) T cells. Note: neither mock treated PBL cells nor mock-treated UCB cells were sorted, and they were used for staining control only. A background CAR staining was observed in mock UCB cells. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 2

Figure 2. Sleeping Beauty-engineered T cells specifically kill CD19+ leukemia and lymphoma cells

(a) Cytotoxicity against CD19+ target cells shown by engineered PBL from two donors (PBL1 and PBL2). (b) Expression of CD19 on K562 transfectants. (c) Cytotoxicity shown by the engineered PBL against K562 transfectants. (d) CD19 specificity of the engineered umbilical cord blood (UCB) T cells, (e) Engineered T cell killing of target cells inhibited by the perforin pathway inhibitors. Similar results were obtained in at least three independent assays. EGTA, ethylene glycol tetraacetic acid; E/T ratio, effector/target ratio; GFP, green fluorescent protein.

Figure 3

Figure 3. Selection of the Sleeping Beauty-engineered T cells using Rituxan

(a) PBL. The optimal amount of biotin-Rituxan used for selection of transfected PBL and umbilical cord blood (UCB) T cells was predetermined by staining T cells of PBL (PBL2-19BB_ζ_/CD20) and UCB (UCBCD3+-19BB_ζ_/CD20) expressing 19BB_ζ_/CD20 with a series of biotin-Rituxan antibody dilutions. Two to four weeks after nucleofection, the T cells were harvested, washed once with magnetic cell sorting buffer (Miltenyi), resuspended, and incubated with human FcR blocking reagent (Miltenyi) and biotin-Rituxan (1–2 μg per 106 cells). The cells were washed and mixed with antibiotin microbeads (Miltenyi). After being washed, the cell pellet was resuspended and run through MS or LS columns (Miltenyi). (b) UCB. This experiment was repeated seven times and two times, respectively, with PBL and UCB, yielding similar results. CAR, chimeric antigen receptor; E/T ratio, effector/target ratio; FITC, fluorescein isothiocyanate; Ig, immunoglobulin; SSC, side scatter.

Figure 4

Figure 4. Sleeping Beauty-engineered CD4+ and CD8+ T cells kill CD19+ target cells

(a) Cytotoxicity assay of sorted subsets of CD4 and CD8 T cells after SB engineering of umbilical cord blood (UCB) and PBL with the SB 19BB_ζ_/CD20 transposon. (b) Expression of cytolytic molecules in subsets of CD4 and CD8 T cell subsets. The mean fluorescence intensity was displayed inside the histograms. Similar results were obtained from at least two independent assays. E/T ratio, effector/target ratio.

Figure 5

Figure 5. Cytokine profiling of the engineered T cells

Cytokine release assays were performed by coculture of 5 × 104 T cells with 2 × 104 target cells per well in duplicate in 96-well flat-bottom plates at a final volume of 200 μl T-cell media. After 24 hours, the supernatant was assayed for cytokine production using Fluorokine MAP Immunoarray. Similar data were also obtained from another assay. CM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-γ, interferon-γ; IL-2, interleukin-2; TNF-α, tumor necrosis factor-α; UCB, umbilical cord blood.

Figure 6

Figure 6. In vivo antitumor responses by the engineered T cells

(a) The experimental schedule. (b) Bioluminescent imaging of tumor growth in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice (three groups, n = 5 each) treated with engineered PBL expressing 19BB_ζ_/CD20 with or without interleukin-2 (IL-2) injections. The mice in the first group (n = 5) received no treatment. The mice in the second group (n = 5) received three intraperitoneal infusions of PBL2-19BB_ζ_/CD20 T-cells (5 × 106/mouse) on days 0, 4, and 8. A third group (n = 5) of mice received three intraperitoneal infusions of PBL2-19BB_ζ_/CD20 T cells (5×106/mouse) on days 0, 4, and 8 along with human IL-2 (Chiron, 2.5 × 104 IU/mouse) intraperitoneally on days 0, 2, 4, 6, and 8. Note: the third group of mice treated with the engineered PBL and IL-2 died on day 17 after the first T cell infusion because of an accidental malfunction of the drinking water equipment. The level of tumor in one mouse from each of the T-cell groups was measured as 61,336 and 84,799 photons/s/cm2/steradian (p/s/cm2/sr). (c) Bioluminescence intensity of the mice treated with engineered PBL. (d) Animal survival after T-cell therapy. One group (n = 5) received no treatment. The other group (n = 5) received three intraperitoneal infusions of PBL2-19BB_ζ_/CD20 T-cells (5×106/mouse) on days 0, 4, and 8. The mice were not imaged, but monitored for survival instead. The P value from log-rank test is 0.0026. The median time to death for untreated tumor-bearing mice, and for mice treated with engineered PBL, was 60 days [95% confidence interval (CI) is (51, 61)] and 105 days [95% CI is (91, 110)], respectively, (e) Bioluminescent imaging of tumor growth in mice treated with the engineered umbilical cord blood (UCB) T cells with or without IL-2. On day -6, the NOD/SCID mice were γ_-irradiated (2.5 Gy, Cesium-137) and then injected intraperitoneally with 106 Daudi-LVhfflucN on the following day. On day -3, mice receiving Daudi-LVhfflucN were examined for tumor engraftment by bioluminescent imaging (BLI). On day 0, the first group of mice (n = 7) received no treatment. The second and third groups (n = 7 each) received three intraperitoneal infusions of UCBCD3+-19BB_ζ/CD20 (10×106/mouse) or UCB mock on days 0, 4, and 8, respectively. The fourth and fifth groups (n = 7 each) received three intraperitoneal infusions of UCBCD3+-19BB_ζ_/CD20 or UCB mock (10×106/mouse) on days 0, 4, and 8, plus human IL-2 on those same days. Tumor BLI was performed every 4 days after the first T-cell infusion. Note: Tumor-bearing mice mock-treated with UCB (the third group), and those mock-treated with UCB and receiving IL-2 (the fifth group), showed early tumor regression after the first T-cell infusion. However, the tumors rebounded rapidly to the level of those in the untreated mice (data not shown), (f) Bioluminescence intensity of the mice treated with the engineered UCB T cells. PB, peripheral blood.

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