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. 2013 May 31;8(5):e64138.
doi: 10.1371/journal.pone.0064138. Print 2013.

Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells

Harjeet Singh  1 , Matthew J Figliola, Margaret J Dawson, Simon Olivares, Ling Zhang, Ge Yang, Sourindra Maiti, Pallavi Manuri, Vladimir Senyukov, Bipulendu Jena, Partow Kebriaei, Richard E Champlin, Helen Huls, Laurence J N Cooper
Affiliations

Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells

Harjeet Singh et al. PLoS One. .

Abstract

Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR VSports手机版. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10) T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Harvest and characterization of aAPC.
(A, B) Sepax volume reduction. aAPC clone #4 grown in VueLife bags were harvested using CS-490.1 kit in Sepax II. The Sepax harvest (S, n = 4) was compared to manual (M, n = 1) procedure. The mean pre/post-processing cell-counts (4.9×108 vs 5×108) were similar using the Sepax system. (C) Phenotype of aAPC (clone #4). Flow cytometry analysis showing expression of CD19, CD64, CD86, CD137L and mIL-15 (expressed with EGFP) (mIL-15-EGFP) on K562 aAPC and K562 parental controls.
Figure 2
Figure 2. Schematic of the process of generating clinical grade CD19-specific T cells.
A MCB (PACT) and WCB (MDACC) were generated for K562-derived aAPC (clone #4). For the generation of CAR+ T cells, aAPC were numerically expanded in bags, harvested using the Sepax II system, irradiated (100 Gy), and cryopreserved for later use. CD19-specific T cells were manufactured as follows; PBMC were isolated from normal donor apheresis products using the Sepax II system and cryopreserved. The PBMC were later thawed, electroporated with the SB DNA plasmids (CD19RCD28 CAR transposon, SB11 transposase) using the Nucleofector System, co-cultured with thawed irradiated aAPC along with cytokines (IL-2 and IL-21) for a culture period of 28 days and cryopreserved.
Figure 3
Figure 3. Phenotype of CAR+ T cell.
(A) Expression of CD19RCD28 CAR on T cells day after electroporation (culture day 1) and after 28 days of co-culture on aAPC clone #4 along with lack of CD19+ aAPC. (B) CAR expression by western blot analysis using CD3-ζ specific antibody. Whole cell lysates were run on SDS-PAGE under reducing conditions. Molecular weight marker (M), Parental Jurkat cells (Lane 1), CD19RCD28+ Jurkat cells (Lane 2), CARneg control T cells (Lane 3) and CD19RCD28+ T cells (Lane 4). (C) Percent expression of CD3+, CD4+CAR+ and CD8+CAR+ T cells with in a lymphocyte gate in cultures over time. Each symbol represents a separate experiment; the solid lines are mean of the three validation experiments. (D) Immunophenotype of memory/naïve, adhesion, activation, cytolytic and exhaustion markers on CAR+ T cells at the end (d28) of co-culture.
Figure 4
Figure 4. Expansion kinetics and redirected specificity of CAR+ T cells.
Genetically modified T cells were co-cultured with aAPC clone #4 for 28 days. At the end of each stimulation cycle (7 days), cells were counted and stained for expression of CAR and CD3. Three validation runs (V1, V2, and V3) were performed and the graphs represent inferred (A) CAR+ T cells, (B) CD3+ T cells, (C) Total viable cells over time. Arrows indicate addition of aAPC to the culture. (D) Lysis of CD19+ targets (Daudiβ2m, NALM-6, CD19+ EL-4) as compared to background lysis of CD19neg EL-4 using 4-hr chromium release assay by CAR+ T cells. Mean ± SD of three validation runs is represented.
Figure 5
Figure 5. Safety profile associated with the SB system.
(A) Telomere length of cells was measured using fluorescence in situ hybridization and flow cytometry (Flow-FISH) assay. Predominant T cell population at day 28 (V1 and V2, CD8+ T cells; V3, CD4+ T cells) was compared to respective miltenyi column purified subset of T cells from day 0. Mean ± SD of triplicates for each validation run is represented. (B) Genomic DNA from CAR+ T cells at day 28 was amplified using primers and probes specific for CD19RCD28 CAR. Relative Quantity (RQ) analyses of the CD19RCD28 target copy number was determined using normal donor PBMC as reference and endogenous RNaseP as a normalizer. Mean ± SD of triplicates for each validation run is shown. (C) TCR Vβ analysis of day 28 and day 35 CAR+ T cells. Data shows mean ± SD of three validation run CAR+ T cells as compared to day 0 unmanipulated controls. (D) A representative genomic PCR showing lack of SB11 transposase integration. Genomic DNA (20 ng) was amplified using SB11 or GAPDH primers. CARneg control T cells (lane 5) and CAR+ T cells (lane 7) amplified using SB11 primers; CARneg control T cells (lane 6), CAR+ T cells (lane 8) and Jurkat stably expressing SB11 (lane 4) amplified using GAPDH primers. Jurkat stably expressing SB11 (Jurkat/SB11-IRES2-EGFP) (lane 3) and the linearized plasmid, pKan-CMV-SB11 (lane 2) amplified using SB11 primers were used as positive controls. (E) G-banded karyotypes of CAR+ T cells from the three validation runs reveal no structural or numeric alteration. A representative spread from validation 2 is shown.
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