This study reports the results of a phase I clinical trial evaluating a novel chimeric antigen receptor (CAR) T-cell therapy targeting CD22 in children and adults with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). CD22 is a surface antigen expressed on most B-ALL cells and is often retained even after loss of CD19, which is a common cause of resistance to CD19-targeted immunotherapies. The trial included 21 patients, 17 of whom had received prior CD19-directed immunotherapy. Dose-dependent anti-leukemic activity was observed, with complete remission in 73% (11/15) of patients receiving ≥1 × 10^6 CD22-CAR T cells/kg, including 5/5 patients with CD19dim/neg B-ALL. The median remission duration was 6 months. Relapses were associated with diminished CD22 site density, suggesting that this could be a mechanism for escape from CD22-CAR T-cell killing. These findings establish the clinical activity of a CD22-CAR in pre-B ALL and suggest that dual-targeted immunotherapeutics targeting multiple antigens may enhance the durability of remissions.This study reports the results of a phase I clinical trial evaluating a novel chimeric antigen receptor (CAR) T-cell therapy targeting CD22 in children and adults with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). CD22 is a surface antigen expressed on most B-ALL cells and is often retained even after loss of CD19, which is a common cause of resistance to CD19-targeted immunotherapies. The trial included 21 patients, 17 of whom had received prior CD19-directed immunotherapy. Dose-dependent anti-leukemic activity was observed, with complete remission in 73% (11/15) of patients receiving ≥1 × 10^6 CD22-CAR T cells/kg, including 5/5 patients with CD19dim/neg B-ALL. The median remission duration was 6 months. Relapses were associated with diminished CD22 site density, suggesting that this could be a mechanism for escape from CD22-CAR T-cell killing. These findings establish the clinical activity of a CD22-CAR in pre-B ALL and suggest that dual-targeted immunotherapeutics targeting multiple antigens may enhance the durability of remissions.