A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR T cells undergo dynamic expansion and contraction in vivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and T cell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression in vivo and demonstrate imaging of the cells with TMP radiotracers. In vitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.

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Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of interests S.A. and E.P. have received sponsored research awards from Tmunity Therapeutics. M.A.S. is a cofounder of Vellum Biosciences and inventor on intellectual property (IP) related to the PET imaging of genetic therapies. In addition, the University of Pennsylvania has filed IP on PROTAC compounds related to this work, on which M.A.S., I.K.L., N.S., and J.M.E. are inventors (WO2022217295A1). M.A.S. is supported by the NIH Office of the Director Early Independence Award (DP5-OD26386), R01GM150804, and the Burroughs Wellcome Fund Career Award for Medical Scientists. S.M.A., E.P., B.M.C., and G.P.L. are supported by the NIH (NCI P01-CA217805). The other authors declare no competing interests.