Production of functional CD19 CAR T cells under hypoxic manufacturing conditions

Chronic lymphocytic leukemia (CLL) has proven difficult to treat with chimeric antigen receptor (CAR) T cell therapy. CLL cells can negatively alter T cell fitness and induce a pseudohypoxic state. We hypothesized that production of CAR T cells under restricted oxygen conditions resembling physiological oxygen levels that can be encountered in tissues (i.e. 2% O₂) could promote outgrowth of hypoxia-tolerant CAR T cells.

We performed in vitro phenotypic and functional assessments of CD19-directed CAR T cells produced in either 21% (^NorCAR) or 2% (^HypCAR) O₂ derived from healthy donors (HDs) or patients with CLL.

Production of HD-derived CAR T cells in 2% O₂ promoted the enrichment of a naïve-like subset. ^HypCAR and ^NorCAR cells were functionally distinct; CD4+ ^HypCAR cells produced more IL-2 and tumor necrosis factor than CD4+ ^NorCAR cells. Production in 2% O₂ was not detrimental to viability or proliferation upon cognate antigen-stimulation and led to increased activation. After chronic stimulation in hypoxia, ^HypCAR-product remained enriched in naïve-like cells, and demonstrated cytotoxic and cytokine production capacity. In CAR T cells derived from patients with CLL, ^NorCAR and ^HypCAR subsets were functionally and phenotypically comparable, but displayed different mitochondrial metabolism.

We demonstrated that production in 2% O₂ is not detrimental, confers subtle but lasting functional and phenotypic changes in CAR T cells warranting further research on the impact of hypoxic production on CAR T cell functionality in hypoxic tumor microenvironments.