Unraveling Cellular and Molecular Mechanisms of Relapse in CD19/CD22 Dual-Targeting CAR T Therapy for B-ALL

CD19/CD22 dual-target chimeric antigen receptor (CAR) T cell therapies mark a key advance over single-target options, offering strong potential for patients with refractory B-cell malignancies. Over 20% of patients still experience relapse, and the factors driving this remain less understood. This study evaluated relapse after CAR19/22 T cell therapy in a clinical trial (ChiCTR-OPN-16008526) involving 91 patients with B-cell acute lymphoblastic leukemia (B-ALL). The complete remission rate was 91.9%, 32.9% (26/79) patients relapsed, with a median relapse time of approximately 7 months. 92.3% of all 13 late relapses (after 7 months) linked to CAR T cell exhaustion. Among 13 early relapses, 23.1% involved CAR T exhaustion and 30.8% CD19 antigen downmodulation. Two early relapses (15.4%) exhibited simultaneous CD19/CD22 antigen reductions. These patients had subclonal PAX5 mutations prior to treatment—these subclones survived and dominated at relapse, with one patient acquiring additional CD19 mutations. Functional studies demonstrated that PAX5 knockout in leukemia cells reduced CD19/CD22 expression and proximal enhancer activity, causing resistance to CAR19/22 T cells. The findings highlight CAR T exhaustion (61.5%) as the primary, CD19 downmodulation as the secondary relapse driver in CAR19/22 T therapy for B-ALL. Disruptive PAX5 mutations represent a unique early relapse mechanism informing targeted strategies.