Engineering and clinical translation of CD19-CAR T cell therapy for pediatric BCP-ALL: from safety optimization to single-cell dissection of in vivo persistence

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B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common malignancy in childhood and young adulthood. CD19-directed chimeric antigen receptor (CAR) T-cell therapy has transformed outcomes in the relapsed/refractory setting, yet important challenges remain: manufacturing failures and long vein-to-vein times, toxicity management, relapse (including CD19− or “masked” relapse), and limited mechanistic insight into how clinical CAR T-cell products expand and persist in vivo. In this thesis, we aimed to develop a safe, scalable, and mechanistically informed CD19-CAR T-cell platform for pediatric and young adult BCP-ALL at Ospedale Pediatrico Bambino Gesù (OPBG), spanning the full translational path from vector engineering to clinical implementation and deep biological characterization. First, we optimized a second-generation iC9.CAR.CD19 construct incorporating an inducible caspase-9 (iC9) suicide switch and a ΔCD34 tracking tag. We demonstrated potent, antigen-specific antitumor activity and showed that rimiducid rapidly and selectively ablates CAR+ cells in vitro and in vivo, including models of inadvertent leukemic blast transduction, thereby providing a clinically actionable safety layer. In parallel, we dissected how CAR architecture (VL–VH linker and hinge design) influences cis CD19 epitope masking on CAR+ blasts and identified short-linker designs that preserve target visibility without compromising function. Building on these preclinical and clinical data (NCT03373071), we implemented point-of-care (POC) manufacturing of fresh CD19-CAR T-cells using closed, GMP-compliant systems. In the autologous lentiviral POC trial (NCT04787263), we characterized CAR T-cell kinetics, phenotype, and patient’s immune reconstitution associated with rapid production and durable MRD-negative remissions. We then extended this approach to healthy donor–derived allogeneic CD19-CAR T-cells under hospital exemption, showing feasibility, manageable toxicity, deep medullary responses, and low incidence of graft-versus-host disease—evidence that directly supported the design and initiation of the allogeneic prospective trial NCT06080191. Finally, using single-cell RNA-seq and TCR-seq, we performed a longitudinal study of OPBG CD19-CAR T-cells in vivo, linking drug product composition to clonal dynamics and 3 transcriptional programs after infusion. We identified oligoclonal CD8+ expansions at the peak of response and metabolically adapted memory-like states associated with long-term persistence and sustained B-cell aplasia. Taken together, this work integrates vector design, safety engineering, Point of Care manufacturing, clinical immunomonitoring, and single-cell profiling to advance CD19-CAR T-cell therapy for children and young adults with high-risk BCP-ALL, and provides a framework for rational optimization of next-generation CAR therapies.

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