Induction of a central memory and stem cell memory phenotype in functionally active CD4+ and CD8+ CAR T cells produced in an automated good manufacturing practice system for the treatment of CD19+ acute lymphoblastic leukemia

Abstract

Relapsed/refractory B-precursor acute lymphoblastic leukemia (pre-B ALL) remains a major therapeutic challenge. Chimeric antigen receptor (CAR) T cells are promising treatment options. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to promote sustained proliferation and persistence after T-cell therapy, constituting essential preconditions for treatment efficacy. Therefore, we set up a protocol for anti-CD19 CAR T-cell generation aiming at high Tcm/Tscm numbers. 100 ml peripheral blood from pediatric pre-B ALL patients was processed including CD4⁺/CD8⁺-separation, T-cell activation with modified anti-CD3/-CD28 reagents and transduction with a 4-1BB-based second generation CAR lentiviral vector. The process was performed on a closed, automated device requiring additional manual/open steps under clean room conditions. The clinical situation of these critically ill and refractory patients with leukemia leads to inconsistent cellular compositions at start of the procedure including high blast counts and low T-cell numbers with exhausted phenotype. Nevertheless, a robust T-cell product was achieved (mean CD4⁺ = 50%, CD8⁺ = 39%, transduction = 27%, Tcm = 50%, Tscm = 46%). Strong proliferative potential (up to > 100-fold), specific cytotoxicity and low expression of co-inhibitory molecules were documented. CAR T cells significantly released TH1 cytokines IFN-γ, TNF-α and IL-2 upon target-recognition. In conclusion, partly automated GMP-generation of CAR T cells from critically small blood samples was feasible with a new stimulation protocol that leads to high functionality and expansion potential, balanced CD4/CD8 ratios and a conversion to a Tcm/Tscm phenotype.

Keywords: CAR T cells; GMP production; Pediatric ALL; Tscm/cm.

Fig. 1

Schematic representation of T-cell transduction (TCT) process on the CliniMACS Prodigy: PBMCs from pediatric ALL patients were thawed and immediately transferred to the device on day 0. After automated CD4/8 separation and CD3/28 activation, transduction with the lentiviral anti-CD19 CAR vector was performed on day 1. CAR T cells were expanded for 12 days until final CAR T-cell product was harvested from the device. For the clinical setting, the CliniMACS Prodigy is currently located in GMP grade B or C. Open steps (GMP grade A), spiking (↑) and sealing () procedures are indicated in the lower part of the figure. Cells are cultured in safety level S2. Due to multiple washing and dilution steps, the final product is downscaled to safety level S1. PBMC peripheral blood mononuclear cells, PB peripheral blood, CAR chimeric antigen receptor, GMP good manufacturing practice

Fig. 2

Activation, expansion and viability during TCT process. a Exemplary microscope pictures of the T-cell culture before, 24 and 48 h after activation with CD3/CD28-based TransAct T Cell Reagent showed clustering of T cells as a sign of T cell activation (400-fold magnification, taken with microscope included in the CliniMACS Prodigy). b T cells expressed various activation markers 48 h after CD3/CD28 activation (n = 2). c A mean expansion rate of 47.9-fold was achieved during the process (overall cell count in culture chamber, n = 4). d Mean viability was higher than 95% throughout the protocol (n = 4). For CAR002, cell count decreased below detection threshold on day 2 of the process and survival of T cells was only confirmed by re-concentration through additional centrifugation of the material provided in the sampling pouch. e A trend towards decreased T-cell expansion in the presence of blasts was observed (gated on CD4⁺/CD8⁺ T cells, n = 2 for each group)

Fig. 3

T-cell transduction with anti-CD19 CAR lentiviral vector. a Transduction rate was analyzed by flow cytometric stain for Biotin-Protein L and secondary stain for anti-Biotin-PE or -APC. Cells stained only with the secondary antibody (FMO) or untransduced T cells were used as controls. b Transduction rate among different cell subsets was analyzed by flow cytometry and showed no significant difference between the subsets. Mean T-cell transduction rate was 26.95% (n = 4). Two-tailed unpaired t test was performed to determine statistical significance. c Cellular composition of CAR⁺ cells was analyzed by flow cytometry (n = 4). Transduced T cells consisted of CD4⁺ T cells, CD8⁺ T cells and NKT cells. No transduced B cells or blasts were detected. Two-tailed unpaired t test was performed to determine statistical significance. FMO: fluorescence minus one

Fig. 4

Cellular composition and T-cell phenotype of CAR T cells. a Despite broad variety in cellular composition of the initial blood sample and after CD4/8 separation, a robust final product was achieved consisting of CD4⁺, CD8⁺ T cells and NKT Cells. No blasts or B cells were detectable on day 5 of the process and in the final product. b Despite a rather exhausted phenotype of the initial blood product, a Tcm and Tscm T-cell phenotype of the final product was reached. Effector T cells: CD62L⁻, CD45RO⁻, CD95⁺; effector memory T cells: CD62L⁻, CD45RO⁺, CD95⁺; central memory T cells: CD62L⁺, CD45RO⁺, CD95⁺; stem cell-like memory T cells: CD62L⁺, CD45RO⁻, CD95⁺; naïve T cells: CD62L⁺, CD45RO⁻, CD95⁻. c, d Anti-CD19 CAR T cells were stained for extracellular expression of T-cell exhaustion/senescence markers and co-inhibitory molecules. No significant differences in expression between CAR⁺ and CAR⁻ T cells were detectable. Wilcoxon signed rank test was performed to determine statistical significance

Fig. 5

Functionality analysis of CAR T cells. a CAR T cells showed dose-dependent killing of the CD19⁺ leukemic target cell line Raji determined by flow cytometry-based functionality assay (n = 4 CAR T-cell products, technical duplicates or triplicates). CD19⁻ cell lines U-266 and Molm-13 served as control (n = 3 CAR T-cell products, technical duplicates or triplicates). Two-tailed unpaired t test was performed to determine statistical significance. b Proliferation results of patient-derived CAR T cells: CAR T cells or untransduced T cells were co-cultured with CD19⁺ cell line Jeko and re-stimulated with the same cell line every 24 h. Proliferation assay was performed before stimulation and every 24 h after stimulation. The figure shows fold change of proliferating CAR T cells after co-culture compared to T cells only. c Proliferation in percentage of proliferating cells 24 h after stimulation 1–4 compared to CAR T cells without stimulation

Fig. 6

Functionality and target cell-dependent TH1 cytokine release of CAR T cells. a CAR T cells were co-cultured with the CD19⁺ target cell line Raji. After 24 h, supernatant was analyzed for cytokine concentrations using a flow cytometry-based assay. Untransduced T cells of the same donor and CD19⁻ cell lines (U-266 and Molm-13) served as negative, stimulation with PMA/ionomycine as positive control (n = 3 CAR T-cell products, technical duplicates or triplicates). b Exemplary cytokine release assay of run CAR003: CAR T cells were co-cultured with the negative fraction of CD4/CD8 separation performed on day 0 of the process. After 24 h, supernatant was analyzed for cytokine concentrations using a flow cytometry-based assay. Untransduced T cells of the same patient co-cultured with the same autologous target-cell fraction served as negative, stimulation with PMA/ionomycine as positive control (technical triplicates). c Intracellular cytokine stain of anti-CD19 CAR T cells for IFN-γ and TNF-α was performed 24 h after contact to the CD19⁺ cell line Raji. A significant increase of IFN-γ and TNF-α positivity was detected for CAR⁺ cells (n = 4 CAR T-cell products). Ratio paired two-tailed t test was performed to determine statistical significance