Chimeric Antigen Receptor (CAR) T cells on demand: developing potent CAR T cells in less than 24 hr for adoptive immunotherapy

Background & Aim The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic “drugs” for cancer therapy. CAR T therapies generally relies upon viral transduction of T cell-receptor (TCR) activated T cell followed by ex vivo expansion of patient-derived T cells over days to weeks prior to infusion. In addition to the required time and labor, we previously reported that TCR/CD3 activation and ex vivo expansion leads to progressive differentiation of the CAR T cells with associated loss of CAR T cell potency. Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that elimination of the CD3/CD28 activation step would yield a T cell product with high functional potency. Methods, Results & Conclusion Here, we show that functional CD19-specific CAR T cells (CART19) can be generated in as little as 24 hours using lentiviral vectors without the need for prior T cell activation. We further demonstrate that some of the restriction factors that limit the infection of quiescent T cells can be overcome by changes to the medium formulation. Most importantly, we show that non-activated T cells exhibit potent antitumor function with deep and durable control of leukemia achieved in vivo with greater than 1-log fewer CD19-specific CAR T cells compared with a 9-day process currently used by tisagenlecleucel (Fig. 1). These findings confirmed that as few as an estimated 12,000-32,000 non-activated CAR T cells produced within 24 hours from mononuclear cell collection could eradicate leukemia. These results demonstrate the potential for vastly reducing the time, materials and labor required to generate CAR T cells, which could be especially beneficial in patients with rapidly progressive disease and in resource-poor health care environments.