manufacturing-chimeric-antigen-receptor-car-t-cells-for-adoptive

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Perelman School of Medicine at the University of Pennsylvania

Etomoxir (ETO) is a widely used small-molecule inhibitor of fatty acid oxidation (FAO) through its irreversible inhibitory effects on the carnitine palmitoyl-transferase 1a (CPT1a). We used this compound to evaluate the role of fatty acid oxidation in rapidly proliferating T cells following costimulation through the CD28 receptor. We show that ETO has a moderate effect on T cell proliferation with no observable effect on memory differentiation, but a marked effect on oxidative metabolism. We show that this oxidative metabolism is primarily dependent upon glutamine rather than FAO. Using an shRNA approach to reduce CPT1a in T cells, we further demonstrate that the inhibition of oxidative metabolism in T cells by ETO is independent of its effects on FAO at concentrations exceeding 5 μM. Concentrations of ETO above 5 μM induce acute production of ROS with associated evidence of severe oxidative stress in proliferating T cells. In aggregate, these data indicate that ETO lacks specificity for CTP1a above 5 μM, and caution should be used when employing this compound for studies in cells due to its non-specific effects on oxidative metabolism and cellular redox.

The CPT1a inhibitor, etomoxir induces severe oxidative stress at commonly used concentrations.

The success of chimeric antigen receptor (CAR)-mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells  for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of  culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function  when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with  culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice-compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. .

Saba Ghassemi

Center for Cellular Immunotherapies

Perelman School of Medicine at the University of Pennsylvania

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Saba Ghassemi

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Perelman School of Medicine at the University of Pennsylvania

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Center for Cellular Immunotherapies