Automated, scaled, transposon-based production of CAR T cells

Abstract

Background: There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition.

Methods: We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR.

Results: We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1-3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns.

Conclusions: We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.

Keywords: Cell Engineering; Immunotherapy; Receptors, Chimeric Antigen; Translational Medical Research.

Figure 1

Small scale transfection of T cells using bi-pulse electroporation. (A) Restriction digestion analysis of conventional plasmids and MCS. Lane 1: SB100X plasmid digested with Sac I; Lane 2: SB100X MC digested with Pac I; Lane 3: PT2 CD19 CAR plasmid digested with Nhe I; Lane 4: PT2 CD19 CAR MC digested with Pac I, Lane 5: PT2 EGFP plasmid digested with Nhe I; Lane 6: PT2 EGFP MC digested with Pme I; lanes M: 1 kb DNA ladder (NE). (B) Schematic representation of applied bi-pulse system (red=first pulse, green=second pulse). (C) Viability and transfection efficiency (TE) of GFP-MC electroporated (Pulse) or non-electroporated (No pulse) T cells using the established bi-pulse system. (D) Episomal CAR-expression from MC vectors. T cells were electroporated with CD19 CAR transposon donor MC vector (CAR TP) in the presence or absence of transposase (SB100X), CAR-expression was assessed on day 6. (E–G) CD8 T cells were (co-)electroporated with mock (m, no DNA) or CD19 CAR and SB100X either encoded on plasmid (P) or MC in equimolar amounts using the two different electroporation systems. (E) viability, (F) transfection efficiency and (G) a representative flow cytometry analysis of anti-EGFRt staining 12 days after electroporation are shown. (C), (E, F) show mean+SD from N=3 donors. Statistical analysis was performed using paired t-test.

Figure 2

Large-scale automated manufacturing of CAR T cells. (A, B) Workflow of the automated manufacturing process. (A) After installing the single-use disposable tubing set (TS520 in combination with the EP-2 accessory), starting material was sterile connected and automatically processed including CD4/CD8 separation, activation, electroporation, expansion and final formulation with minimal hands-on time according to a pre-defined activity matrix. In-process controls (IPC) allow tracking of viability, cellular expansion, pH value, and glucose consumption as required. (B) Flow chart of the manufacturing process and materials required. (C) Viability of in-process control samples before and after electroporation, during the process and at day of harvest. (D) Percentage of gene-modified T cells and (E) CAR T cell expansion was tracked during the entire process.

Figure 3

Composition, phenotype and genotype of CAR T cells from automated manufacturing runs. (A) Cellular composition was analyzed before and after enrichment on day 0 as well as in the final cell product using flow cytometry. (B) CD4/CD8 ratio was analyzed before and after enrichment as well as after harvest. (C) T cell immunophenotype in final cell product. T cells were stained with antibodies against CD45RO, CD62L and CD95 and frequency of naïve T cells (T_n), stem-cell memory T cells (T_scm), central memory T cells (T_cm), effector memory T cells (T_em), and CD45-RA⁺ effector memory T cells (T_emra) was assessed in the final cell product by flow cytometric analysis. (D) Vector copy numbers were analyzed from final cell products (CAR T, N=3) or mock T cells (control, N=2). (E) Gene expression profiles of cells from final cell product (N=4) were compared by Nanostring analysis to that of freshly isolated T cells (N=10). (B, C) Show mean±SD from N=3 donors.

Figure 4

Anti-tumor reactivity of CD19 CAR T cells from automated manufacturing runs. (A) Histogram of CD19-expressing JeKo-1 wild-type (WT) or CD19 knockout (k.o.) JeKo-1 cells. (B) Effector and target cells were co-cultured for 24 hours at an effector-to-target ratio (E:T) of 1:1. Killing was analyzed using flow cytometry. (C) Cytokine release (interleukin-2 (IL-2), interferon-gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor-necrosis factor-alpha (TNF-α)) was measured after co-culturing mock or CD19 CAR T cells either in the presence or absence of CD19-positive target cells. (D) After engrafting luciferase expressing Raji^ffluc tumor cells in NSG mice for 7 days, non-electroporated mock or CD19 CAR engineered T cells were i.v. injected. Tumor burden was tracked using an in vivo tumor imaging system (IVIS). (B, C) Show mean+SD from N=3 donors. (D) Represents mean±SD from N=6 mice (CAR T) and N=4 mice (mock). Statistical analysis was performed using parametric unpaired t-test with 95% CI.