Understanding and Enhancing CAR T-Cell Therapies by Integrating Clinical Single-Cell Analysis with Functional Modulation

Chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment of relapsed or refractory hematologic malignancies, including large B-cell lymphoma (LBCL), B-cell precursor acute lymphoblastic leukemia (B-ALL), and multiple myeloma (MM). However, patient responses remain heterogeneous, long-term remission is inconsistent, and the molecular determinants of therapeutic durability are incompletely understood. This thesis integrates single-cell multi-omics, CAR T-cell engineering, and preclinical modeling to define how intrinsic T-cell programs and manufacturing variables shape CAR T-cell efficacy across disease settings and platforms, and to develop translational strategies to enhance therapeutic performance. Motivated by clinical single-cell omics insights, we identified type I interferon (IFN-I) signaling as a key biomarker and actionable target and developed a strategy to incorporate optimized IFN-I signaling during manufacturing to enhance CAR T-cell efficacy (Chapter 2). We then compared FDA-approved CD19-directed CAR T-cell products with distinct costimulatory domains using high-dimensional single-cell multi-modal analysis, revealing platform-specific differentiation trajectories and response-associated features (Chapter 3). Extending this analysis to BCMA-directed therapies, including a patient sequentially treated with two constructs, we uncovered construct-specific transcriptional programs and clonal dynamics linked to therapeutic outcomes (Chapter 4). Finally, we defined cellular states associated with durable versus non-durable responses in BCMA CAR T-cell therapy, identifying transcriptional programs correlated with sustained remission (Chapter 5). Collectively, this work establishes a translational pipeline that bridges clinical single-cell omics with mechanistic insight, biomarker discovery, and preclinical validation, advancing both fundamental understanding and rational optimization of CAR T-cell efficacy.