Fig. 3: Conditional CAR T cells are as effective as conventional CAR T cells and can be modulated by MTX.
From: Direct control of CAR T cells through small molecule-regulated antibodies
a Schematic of CAR constructs. b Antigen-specific cytotoxicity was determined after a 48 h co-incubation of MV4-11mut cells with conventional CAR or condCAR T cells ± MTX (n = 3 biologically independent samples, mean ± SD). Effector:target (E:T) = 1:1. c MTX- and CD33-dependent T cell activation was quantified by measuring the surface expression of CD69. Partial (yellow) or complete (red) inhibition of CD69 expression is achieved at or above 10 μM MTX, respectively. E:T = 1:2. d Antigen-specific secretion of the IL-2 and IFN-γ was quantified after a 24-h co-culture with either MV4-11mut cells or anti-CD3/anti-CD28-coated beads ± MTX (n = 3 independent experiments, mean ± SD). E:T = 1:2. e Antigen-specific proliferation was analyzed after 7 days of co-culture with either MV4-11mut (E:T = 1:5) or anti-CD3/anti-CD28-coated beads ± MTX (n = 3 biologically independent samples, mean ± SD). f Antigen-specific cytotoxicity of MV4-11mut cells by condCAR T cells can be modulated in a reversible manner. Kinetic analysis of cytotoxicity ± MTX was quantified every 2 days. The presence of MTX is shown by regions in gray (n = 3 technical triplicates, mean ± SD). E:T = 1:1. g Left panel: workflow used to assess the extent of condCAR T cell control by MTX in vivo. Two-day continuous infusion of MTX is indicated by the gray bar, and bioluminescence (BLI) analysis time points are indicated by blue dots. A parallel study was utilized for blood collection for CAR T cell enumeration with time points indicated by red dots. Middle panel: BLI analysis of MV4-11mut tumor model infused with condCAR T cells, conventional CAR T cells, or NTD cells ± 250 mg/day MTX (gray region) (n = 5 animals, mean ± SEM). Right panel: Circulating CAR T cell enumeration. One mouse was sacrificed at each time point for blood analysis by flow cytometry. P values in b, d, and e were calculated by paired two-tailed t test. P values in g were calculated by two-way ANOVA with Bonferroni posttest. N.S. non-significant (P > 0.01), ***P < 0.001, ****P < 0.0001.
