Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy

Abstract

On the basis of promising preclinical data demonstrating the eradication of systemic B-cell malignancies by CD19-targeted T lymphocytes in vivo in severe combined immunodeficient-beige mouse models, we are launching phase I clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia. We present here the validation of the bioprocess which we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semiclosed culture system using the Wave Bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in severe combined immunodeficient-beige mice bearing disseminated tumors. The validation requirements in terms of T-cell expansion, T-cell transduction with the 1928z CAR, biologic activity, quality control testing, and release criteria were met for all 4 validation runs using apheresis products from patients with CLL. Additionally, after expansion of the T cells, the diversity of the skewed Vbeta T-cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemorefractory CLL and in patients with relapsed acute lymphoblastic leukemia. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any CAR or T-cell receptor.

FIGURE 1

Scheme of the manufacturing process. The semi-closed system relies on the use of the Cytomate™ to wash the apheresis product prior to freezing and after thawing, the capture of CD3⁺CD28⁺ T cells with Dynabeads^® and subsequent selection on the ClinExVivo magnetic particle concentrator (MPC), expansion in the Wave™ bioreactor, debeading on the ClinExVivo MPC and formulation using the Cytomate™.

FIGURE 2

T cell expansion, T cell concentration, glucose and lactate levels in the Wave Bioreactor during the course of VR1, VR2, VR3 and VR4. A, Total cumulative fold expansion of patient T cells. Data is shown from initial activation until completion of the expansion runs. B, Viable cell concentrations of patient T cells during activation and within the WAVE bioreactor. C, Glucose and D, Lactate levels were measured daily during the course of the validation runs.

FIGURE 3

In vitro and in vivo cytotoxic activity of the 1928z⁺ T cells. A, In vitro chromium release assays performed on 1928z+ EOP T cells derived from VR1, VR2, VR3 and VR4. Both autologous patient tumor B-CLL cells and Raji lymphoma cell line were used as target cells. B, Survival of mice bearing established disseminated Raji tumors after infusion of EOP 1928z+ T cells. Six days after intravenous injection of 5 × 10⁵ Raji tumor cells, mice received 10⁷ fresh 1928z CD3+ transduced T cells from VR4 run. Untransduced T cells from the same donor grown in parallel served as control. Note: we previously demonstrated that untransduced T cells and T cells transduced with an irrelevant CAR are equally unable to eradicate Raji tumors in vivo (data not shown).

FIGURE 4

Spectratyping analysis in T cells from the apheresis product and in the EOP 1928z T cells from VR1 and VR2. Representative spectratype analysis of Vβ1, Vβ3, Vβ9 and Vβ17 shows a skewed TCR repertoire in the T cells from the apheresis product and restored TCR repertoire in the EOP 1928z T cells after expansion with Dynabeads^® (final product).