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 18-29%, so improvement is needed. Peripheral blood mononuclear cells (PBMC) 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 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 (cGMP)-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.