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. 2020 Jun 11;28(7):1600–1613. doi: 10.1016/j.ymthe.2020.06.004

Figure 1.

Figure 1

Fusion of the LID Domain to the CAR Allows for Dose-Dependent Shield-1-Mediated CAR Downregulation

(A) Diagram of shield-1-induced degradation of CAR-LID polypeptide. (B) Schematic of CAR constructs used in this study. SS, signal sequence; TM, transmembrane. See Figure S1A for nucleotide and amino acid sequence of the CAR-LID. (C) CAR-LID surface expression. Primary human T cells were activated ex vivo and transduced with lentivirus encoding either GD2-LID or the standard GD2 CAR. At day 7 of expansion, T cells were evaluated for CAR expression by staining with anti-mouse antibody (which binds to the murine scFvs) and analyzed by flow cytometry. Representative histograms shown from n = 6 independent experiments. (D) CAR expression across a range of shield-1 concentrations. GD2-LID T cells were incubated with shield-1 at the indicated concentrations for 24 h and evaluated for CAR expression as in (C). Values shown are percentage of GD2-LID CAR expression in a vehicle control sample that did not receive shield-1 (considered to represent “maximum” CAR expression) stained in parallel. Percent maximum expression, that is, (mean fluorescence intensity [MFI] of cells with shield-1/MFI of cells without shield-1) × 100, is plotted. The left panel depicts the dose response over a wide range of shield-1 concentrations. The non-linear curve was fit with GraphPad Prism software using one-phase exponential decay. The right panel highlights the dose response at a narrow range of shield-1 concentrations around the inflection point seen in the left panel (demarcated with a red bracket in the left panel). Values plotted are the mean, and error bars show the standard deviation (SD) from n = 3 different donors. (E) The LID system functions in additional CAR constructs. Incorporation of the LID domain into additional CAR constructs, m3F8-LID and VHH-LID, permits shield-1-mediated downregulation. The GD2 scFv was replaced by either the m3F8 scFv or VHH nanobody in the CAR-LID construct. Primary human T cells were activated ex vivo and transduced with either m3F8-LID or VHH-LID. On day 7, T cells were incubated with shield-1 (1 μM) or vehicle alone for 24 h. Surface CAR was then detected by flow cytometry. Representative histograms are shown from n ≥ 3 different T cell donors.