Vitamin C Conditioning Generates Tumor-Targeting CAR T Cells with Superior Cytotoxicity and Fitness in a Posttransplant Lymphoproliferative Disorder Tumor Model

CAR T-cell therapy has shown efficacy in hematological malignancies but faces challenges in solid tumors and virus-associated malignancies such as post-transplant lymphoproliferative disorder (PTLD). Various strategies, including optimization of receptor design, genetic modifications addressing immunomodulatory mechanisms and refining the manufacturing process have been explored to overcome limited in vivo persistence and tumor infiltration, antigen escape and the immunosuppressive tumor microenvironment (TME). This study investigated the effect of vitamin C (vitC) conditioning on CD19-targeting CAR T cells (vitC-CAR19-Ts) to improve efficacy of CAR T-cell therapy. VitC has been shown to influence immune responses through epigenetic regulation and oxidative stress reduction. Enhanced transduction efficiency and proliferative capacity by vitC conditioning resulted in a higher yield of CD4+ and CD8+ CAR19-Ts. VitC-CAR19-Ts exhibited faster and improved cytotoxic response towards CD19+ Nalm-6 cells and Epstein-Barr virus (EBV)-infected B-lymphoblastoid cell lines (B-LCLs), the in vitro model of PTLD. Increased demethylation was observed in TBX21 regions, which was in line with a type 1-like phenotype and higher expression of effector molecules such as Granulysin in both CD4+ and CD8+ in vitC-CAR19-Ts, providing insights into the effects of vitC conditioning. Importantly, vitC-CAR19-Ts outperformed CAR19-Ts in long-term antigen stress assays and 3D multicellular spheroid models, indicating a potentially improved in vivo functionality and tumor infiltration capacity. In summary, vitC conditioning represents a promising strategy to enhance CAR T-cell yield, cytotoxic potential and durability, complementing existing approaches to overcome the limitations of CAR T cells in the treatment of hematological malignancies and solid tumors.