Production of functional CD19 CAR T cells under hypoxic manufacturing conditions

Isabella Micallef Nilsson^1,2Thomas Poiret^1,2Jinhye Ryu³Mohammadali Mohammadpour¹Johan Henriksson⁴Anders Österborg^1,5Jonas Mattsson^1,6Anna Schurich⁷Isabelle Magalhaes^1,2,8*

• ¹Department of Oncology-Pathology, Karolinska Insitutet, Stockholm, Sweden
• ²Center for Hematology and Regenerative Medicine, Department of Medicine Huddinge, Karolinska Insitutet, Stockholm, Sweden
• ³Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden
• ⁴Department of Molecular Biology, Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden
• ⁵Department of Hematology, Lymphoma Unit, Karolinska University Hospital, Stockholm, Sweden
• ⁶Gloria and Seymour Epstein Chair in Cell Therapy and Transplantation, Princess Margaret Cancer Centre and University of Toronto; Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada
• ⁷Department of Infectious Diseases, School of Immunology and Microbial Sciences, King's College London, London SE1 9RT, United Kingdom
• ⁸Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden

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.