Molecular Therapy: Methods & Clinical Development (Mar 2024)
Optimization of anti-CD19 CAR T cell production for treatment of patients with chronic lymphocytic leukemia
Abstract
T cells expressing anti-CD19 chimeric antigen receptors (CARs) have activity against chronic lymphocytic leukemia (CLL), but complete response rates range from 18% to 29%, so improvement is needed. Peripheral blood mononuclear cells (PBMCs) of CLL patients often contain high levels of CLL cells that can interfere with CAR T cell production, and T cells from CLL patients are prone to exhaustion and other functional defects. We previously developed an anti-CD19 CAR designated Hu19-CD828Z. Hu19-CD828Z has a binding domain derived from a fully human antibody and a CD28 costimulatory domain. We aimed to develop an optimized process for producing Hu19-CD828Z-expressing T cells (Hu19-CAR T) from PBMC of CLL patients. We determined that supplementing Hu19-CAR-T cultures with interleukin (IL)-7 + IL-15 had advantages over using IL-2, including greater accumulation of Hu19-CAR T cells during in vitro proliferation assays. We determined that positive selection with anti-CD4 and anti-CD8 magnetic beads was the optimal method of T cell purification because this method resulted in high T cell purity. We determined that anti-CD3/CD28 paramagnetic beads were the optimal T cell activation reagent. Finally, we developed a current good manufacturing practices-compliant clinical-scale protocol for producing Hu19-CAR T from PBMC of CLL patients. These Hu19-CAR T exhibited a full range of in vitro functions and eliminated leukemia from mice.
