Environmental conditioning during CD19-CAR T cell manufacturing impacts antitumor potency in a subcutaneous NALM6 tumor model

Chimeric Antigen Receptor (CAR) T cell therapy has transformed outcomes for hematologic malignancies, yet optimizing manufacturing conditions remains critical to improving potency and consistency. We previously demonstrated that culturing CAR-T cells under physiological oxygen and pressurized conditions improved persistence and efficacy in a disseminated NALM6 human ALL model.In this study, we investigated whether these optimized manufacturing conditions similarly improve antitumor activity in a subcutaneous NALM6 xenograft model. CD19-targeted CAR-T cells were expanded under three conditions: standard CO 2 incubation (21% O 2 + 0 PSI), mild hypoxia with hyperbaric pressure (15% O 2 + 5 PSI), reflecting arterial and peripheral blood environments, and hypoxia with hyperbaric pressure (5% O 2 + 5 PSI), modeling the solid tumor and bone marrow microenvironments. After a 10-day tumor stage, a dose of 2.5e6 CAR+ T cells achieved complete tumor clearance in all conditions at 20 days post T cell dose, with faster tumor regression under 5% O 2 + 5 PSI conditions (7 days post T cell dose) compared to normoxia or mild hypoxia (10 days post T cell dose). These in vivo outcomes were consistent with enhanced in vitro functional activity, characterized by greater cytoxicity at CAR-T-to-effector ratios under 1:30 and increased cytokine production. This consistency mirrors our previous findings in the disseminated model, where CAR-T cells cultured under physiologically relevant conditions demonstrated the in vitro characteristics predictive of in vivo efficacy. Preliminary findings show enhanced potency and persistence under 5% O 2 + 5 PSI conditions. To determine whether these manufacturing advantages are sustained at lower CAR-T doses, a dose titration study is currently in progress, with complete results forthcoming.These data highlight the potential of physiological manufacturing with the GMP AVATAR Foundry to enhance CAR-T function and lower effective doses, supporting scalable next-generation cell therapies.