Evaluating the reversible control of an engineered CAR T cell ON-switch.

Abstract

e14550 Background: CAR T cell therapy holds enormous promise for many cancer types but its application may be limited by serious toxicities. To lower this hurdle, our aim is to engineer tunable cell therapies. One of our approaches includes a “ON-switch” chimeric antigen receptor (Wu et al., Science 2015) that requires the administration of a small molecule acting as a dimerizing agent between one polypeptide chain containing the antigen recognition domain and half of an inducible heterodimerization system and another polypeptide chain containing the second half of the inducible heterodimerization motif, the CD3ζ chain and a costimulatory motif. Using an FDA approved small molecule drug, we evaluate the reversibility of ON-switch CAR T cells in preclinical models. Methods: First, we evaluated the proliferation, cytotoxicity and cytokine production of several ON-switch constructs in human primary T cells. Next, to address the reversibility of the ON-switch (ON→OFF→ON), we performed a series of co-culture experiments where the small molecule drug was added to tumor cells and ON-switch CAR T cells, then washed out, and then re-introduced back into the co-cultures. We compared CAR T cell mediated killing and cytokine production from the On-switch CAR T cells relative to a canonical CAR T control. Results: Our On-switch CAR T cells were shown to proliferate, secrete cytokines as well as mediate dose dependent cytotoxicity in the presence of the small molecule drug. Importantly, in the presence of antigen but in absence of the small molecule drug we did not measure any significant functional activity in our ON-switch CARs. Additonally, following the removal of the small molecule drug over a period several days we did not observe any significant CAR mediated cytotoxicity. Following the subsequent re-addition of the small molecule, we observed further CAR T cell mediated cytotoxicity against tumor cells. Conclusions: These results show that the small molecule inducible On-switch CARs maintain functional activity as well as reversibility allowing for the tunable control of a CAR T cell.

Overhauling CAR T Cells to Improve Efficacy, Safety and Cost

“…Here the small molecule acts as CAR activator, an “ON-switch”, which can be titrated according to patient response and can be used to control the timing of CAR activation [ 21 ]. Even in the presence of the target antigen, the CARs are not switched on if the small activator molecule is absent [ 23 ]. The major advantage of this kind of switch is that the CAR-T cells remain in the patient after treatment has been terminated and can be activated when necessary.…”