Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: 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

Introduction CD19-directed CAR T-cell therapy has transformed the management of relapsed/refractory B-cell precursor acute lymphoblastic leukemia (r/r BCP-ALL), yet major obstacles remain: toxicities, relapse due to CAR T-cell failure or antigen escape, and manufacturing limitations in immune-compromised patients. To address these challenges, the Ospedale Pediatrico Bambino Gesù (OPBG) has developed a fully integrated translational program encompassing preclinical engineering, point-of-care (POC) GMP manufacturing, bedside safety control, and deep biological profiling of CD19.CAR T-cells in pediatric and young-adult patients. Aim This PhD project aimed to: (i) Optimize and validate a second-generation CD19.CAR incorporating the inducible caspase-9 (iC9) suicide switch; (ii) implement and optimize fresh, POC lentiviral CAR T-cell manufacturing in autologous and allogeneic settings; (iii) establish a comprehensive immunomonitoring platform for CD19.CAR-T clinical trials open at OPBG; (iv) elucidate transcriptional and clonal mechanisms of CAR T-cell expansion and persistence through single-cell RNA/TCR sequencing. Results and methods Preclinical engineering: We generated and optimized a second generation CD19(FMC63).CAR-4.1BB-CD3ζ containing ΔCD34 and iC9 elements. Functional assays demonstrated potent cytotoxicity, robust expansion, and complete, rimiducid-induced ablation of CAR+ cells within hours. The iC9 mechanism prevented survival of inadvertently transduced CD19+ leukemic blasts and provided a rapid safety tool in case of toxicities or CAR-positive relapse. Moreover, we refined the CAR.CD19 architecture by engineering the VH–VL linker and spacer domains to prevent cis masking of the CD19 epitope on blasts, improving target recognition and augmenting the overall safety profile and effectiveness of CD19.CAR T cells. POC manufacturing and clinical application: We established a closed-system, fresh POC CAR T-cell workflow on CliniMACS Prodigy®, leading to rapid drug-product release and reduced vein-to-vein time to 21 days. Single-cell multi-omics: Using 10x Genomics 5' scRNA-seq and scTCR-seq from infusion product (DP), day+9, and day+28 samples of treated patients, we reconstructed the trajectory of in vivo CAR T-cell responses. Analysis of 48,001 high-quality CAR T-cells revealed: • a shift from CD4-enriched, memory-like drug products to CD8-dominant proliferative states at peak of expansion of CAR-T in peripheral blood; • emergence of metabolically adapted, cytotoxic CD8+ memory populations at day+28; • oligoclonal expansion of a limited number of TCR clonotypes that persisted across timepoints; • gene programs defining activation, integrin signaling, mitochondrial fitness, and long-term survival. These results provide mechanistic insight into why OPBG POC CD19.CAR T-cells achieve durable persistence and remission. Conclusions This work integrates engineering, clinical translation, and systems immunology to advance safer and more accessible CAR T-cell therapy for pediatric BCP-ALL. The iC9 safety switch offers immediate, controllable CAR T-cell ablation; POC manufacturing improves feasibility and equity of access; and single-cell dissection reveals cellular states and clonal architectures that govern therapeutic success. Together, these findings lay the foundation for next-generation CAR products, optimized manufacturing strategies, and predictive biomarkers to guide precision cell-therapy interventions.