Clinical outcomes and spatial transcriptomic profiles of CD19/20 CAR-T therapy in relapsed or refractory B-cell non-Hodgkin's lymphoma

Background: Relapsed or refractory (R/R) B-cell non-Hodgkin's lymphoma (B-NHL) remains a major therapeutic challenge despite CD19-directed chimeric antigen receptor (CAR) T-cell therapies, with treatment failure often driven by antigen escape and limited CAR-T persistence. Dual targeting of CD19 and CD20 may mitigate antigen loss. We conducted an expanded phase I/II study of bispecific CD19/20 CAR-T cells and incorporated spatially resolved single-cell transcriptomics to evaluate tumor-intrinsic and microenvironmental factors of clinical response. Methods: Patients with R/R B-NHL received CD19/20 CAR-T cells following lymphodepletion. Efficacy, safety, CAR-T expansion/persistence, and B-cell reconstitution were assessed. Pretreatment tumor biopsies from five patients with divergent outcomes underwent spatial single-cell transcriptomic profiling. Results: 32 patients were treated, including 24 with diffuse large B-cell lymphoma. Among 31 evaluable patients, the best overall response rate was 74%, including 58% achieving complete remission. Median progression-free and overall survival were 6.8 and 22.1 months, respectively. Response rates were higher in patients with normal lactate dehydrogenase. CAR-T expansion peaked on days 7-17, and persistence exceeded 500 days in long-term responders. Cytokine release syndrome occurred in 53% (12% grade ≥3) and immune effector cell-associated neurotoxicity syndrome in 9% (all grade 3), with no lasting deficits. Spatial profiling identified two dominant tumor architectures: (1) a B-cell-dominant phenotype, in which durable remission was associated with heightened apoptotic competence in malignant B cells, and (2) a fibroblast-enriched and monocyte/macrophage-enriched phenotype, in which response correlated with a chemokine-rich, T-cell-permissive microenvironment. Conclusions: Bispecific CD19/20 CAR-T therapy produced durable clinical activity with manageable toxicity. Spatial single-cell analysis reveals distinct tumor-intrinsic and microenvironmental features associated with CAR-T responsiveness, providing a spatially informed framework for understanding heterogeneous therapeutic outcomes. Trial registration number: NCT04723914.