Hypoxia during GPC2-CAR T-cell manufacturing compromises cell expansion and cytotoxic capacity

Hypoxia is a hallmark of the solid tumor microenvironment and is known to impair CAR T-cell function. In order to condition CAR T-cells to this microenvironment during manufacturing, we evaluated whether varying oxygen tension during the manufacturing of GPC2-CAR T-cells improves cellular characteristics and function against neuroblastoma. Cell products were produced over 8-9 days under normoxic (21% oxygen), transitional (21%, then transitioned to 1% on Day 2), or maintained hypoxic conditions (1% from Day 0 onward). RNA-sequencing, metabolic assays, phenotyping, and functional studies were performed. Sustained hypoxia induced universal changes in CAR T-cells, including increased surface CAR expression, enrichment of T N /T SCM subsets, and a shift toward glycolysis. However, manufacturing fully in hypoxic conditions yielded markedly reduced cell expansion relative to normoxic and transitional conditions. Normoxia-produced CAR T-cells were most potent in eliminating neuroblastoma tumor cells in vitro and in vivo. In parallel, hypoxia also altered neuroblastoma biology, including downregulation of GPC2 expression and induction of transcriptional programs associated mesenchymal transition and hypoproliferation. Together, these findings indicate that oxygen tension substantially alters both CAR T-cell products and tumor cell state. Sustained hypoxic manufacturing does not improve GPC2-CAR T-cell efficacy and instead compromises yield and antitumor function. Thus, for this GPC2-CAR T-cell product, normoxic manufacturing remains the most favorable approach for clinical translation.