ALBERT

Description: A large number of drugs can induce prolongation of cardiac repolarization and life-threatening cardiac arrhythmias. The prediction of this side effect is however challenging as it usually develops in some genetically predisposed individuals with normal cardiac repolarization at baseline. Here, we describe a platform based on a genetically diverse panel of induced pluripotent stem cells (iPSCs) that reproduces susceptibility to develop a cardiotoxic drug response. We generated iPSC-derived cardiomyocytes from patients presenting in vivo with extremely low or high changes in cardiac repolarization in response to a pharmacological challenge with sotalol. In vitro, the responses to sotalol were highly variable but strongly correlated to the inter-individual differences observed in vivo. Transcriptomic profiling identified dysregulation of genes (DLG2, KCNE4, PTRF, HTR2C, CAMKV) involved in downstream regulation of cardiac repolarization machinery as underlying high sensitivity to sotalol. Our findings offer novel insights for the development of iPSC-based screening assays for testing individual drug reactions.

Description: Autologous T-cells engineered to express chimeric antigen receptors (CARs) that target specific tumor antigens are known to be of high potential in treating different kinds of cancer. However, they must be generated on a “per patient” basis, thereby limiting the population of patients that could benefit from this approach. In particular, immune homeostasis may be affected in heavily pre-treated patients, such that autologous T-cells may be low in number, not fully functional, or unable to expand, thereby restricting the amount of cells that could be manufactured. The use of allogeneic T-cells isolated from healthy third party donors could constitute an easy-to-scale-up alternative, producible in advance, with potential for standardized quality controls, better batch consistency, and immediate availability for administration to a larger number of patients. In this context, we have established a highly efficient, 18-day, good manufacturing practice (GMP)–compatible process to produce CAR T-cells from healthy donor peripheral blood mononuclear cells (PBMCs). To circumvent the potential of allogeneic T-cells inducing graft-versus-host disease (GvHD) in recipient patients, the TCR alpha constant (TRAC) gene was inactivated using a proprietary transcription activator-like effector nuclease (TALEN™)-mediated gene editing technology. The CD52 gene was also disrupted using another specific TALEN™ to allow the administration of engineered T-cells following an alemtuzumab-based lymphodepleting therapy. The antitumor activity of these double-knockout CAR T-cells was shown to be as potent as non-nuclease-edited cells expressing the same CAR in vitro. The current manufacturing process is highly reproducible, making it suitable for use in a larger scale manufacturing platform for administration as “off-the-shelf” immunopharmaceuticals. We estimate that a single production run, starting from a healthy volunteer leukapheresis product containing 109 PBMCs, would allow the production of up to 500 doses of CAR double-knockout T-cells, at 2x107 cells per dose, allowing the extension of CAR therapies to a larger number of patients. Our results provide the proof of concept for the general applicability of this approach as a platform for large-scale GMP–compliant manufacturing of allogeneic, off-the-shelf, non-alloreactive, frozen CAR T-cells. From this manufacturing platform, we produced UCART19 cells, which are TCR/CD52-deficient, RQR8+ (as a safety attribute), and anti-CD19 CAR+, to investigate their potential in the treatment of CD19+ B cell leukemias. We believe this adaptable manufacturing platform offers multiple opportunities to improve CAR T-cell therapies through multiplex genome editing, such as rendering UCART cells resistant to standard chemotherapy or to tumor evasion mechanisms. Disclosures Derniame: Cellectis SA: Employment. Poirot:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Galetto:Cellectis SA: Employment. Beurdeley:Cellectis SA: Employment. Reynier:Cellectis SA: Employment. Arnould:Cellectis SA: Employment. Smith:Cellectis SA: Employment.