Unraveling cellular and molecular mechanisms of relapse in CD19/CD22 dual-targeting chimeric antigen receptor T-cell therapy for B-cell acute lymphoblastic leukemia.

CD19/CD22 dual-target chimeric antigen receptor (CAR19/22) T cell therapies mark a key advance over single-target options for refractory B-cell malignancies. However, over 20% of patients relapse and underlying mechanisms remain less understood. This study evaluated relapse in 91 B-cell acute lymphoblastic leukemia (B-ALL) patients treated with CAR19/22 T cells a clinical trial (ChiCTR-OPN-16008526). The complete remission rate was 91.9%, and 32.9% (26/79) of responders relapsed at a median of approximately 7 months. Among late relapses (>7 months), 92.3% was CAR T functional insufficiency showing lack of persistence or recovery of CD19⁺ B lymphocytes. Early relapses showed greater heterogeneity with 30.8% attributable to CAR T functional insufficiency and 30.8% to antigen insufficiency. Notably, two early relapses (15.4%) exhibited concurrent CD19/CD22 downregulation and harbored a preexisting PAX5 deletion or frameshift insertion. These PAX5-mutated subclones persisted and expanded at relapse, with one patient acquiring additional CD19 mutations. PAX5 knockout in leukemic cells reduced CD19/CD22 expression and proximal enhancer activities, causing in vitro resistance to CAR19/22 T cells. Collectively, CAR T functional insufficiency and antigen insufficiency are the most (61.5%) and second (15.4%) frequent drivers of relapse, respectively. Disruptive PAX5 mutations define a distinct molecular mechanism of early relapse, guiding rational targeted strategies.