Extended Data Fig. 11: Clinical applicability of CRISPR-engineered armored CAR T cells.

a-c. Anti-LeY CAR T cells derived from patients with multiple myeloma were engineered to express Gfp from either the NR4A2 or RGS16 locus and cocultured with OVCAR-3 tumor cells for 72 h before GFP expression was assessed. a. Flow cytometry plots from concatenated technical triplicates. b. Quantification of GFP percentage and c. MFI in CD8⁺ human CAR T cells, represented as mean ± SD of technical triplicates. d. Anti-LeY CAR T cells derived from patients with diffuse large B cell lymphoma were engineered to express IL-12 from the NR4A2 locus and cocultured with OVCAR-3 or MCF-7 tumor cells for 24 h before cytokine concentration in supernatants was assessed. Concentrations of IFNγ, TNF or IL-2 produced by NR4A2/IL-12 CAR T cells, represented as mean ± SD of technical triplicates. e-f. Murine anti-hHer2 CAR T cells engineered to express Gfp from either the Nr4a2 or Rgs16 locus were cocultured with MC38 tumor cells with varying Her2 expression for 24 h before GFP expression was assessed. e. Histogram overlay showing Her2 expression in MC38 tumor cells. f. Quantification of GFP MFI in CD8⁺ murine CAR T cells, represented as mean ± SD of technical triplicates. g-h. Human anti-LeY CAR T cells engineered to express Gfp from either the NR4A2 or RGS16 locus were stimulated with varying concentrations of anti-LeY idiotype antibody for 24 h before GFP and CD69 expression were assessed. g. Quantification of GFP MFI and h. CD69 percentage in CD8⁺ human CAR T cells, represented as mean ± SD of technical triplicates. i-j. Human anti-Her2 CAR T cells were engineered to express Gfp from the NR4A2 locus and assessed in vitro upon stimulation with MDA-MB-231 tumor cells (i) and in mice bearing orthotopic MDA-MB-231 tumors (j). i. Flow cytometry plots from concatenated technical triplicates (left) and quantification of GFP expression represented as mean ± SD of technical triplicates (right) in CD8⁺ human CAR T cells post 24 h of stimulation. j. Flow cytometry plots representative of n = 4 mice/group in CD8⁺ human CAR T cells 14 days post transfer. k. OT-3 T cells were engineered to express IL-12 or IL-2 from the Nr4a2 or Rgs16 locus, respectively, and cocultured with AT-3-ova tumor cells for 24 h before cytokine concentration in supernatants was assessed. Concentration of IL-12 (left) or IL-2 (right) produced by NR4A2/IL-12 or RGS16/IL-2 OT-3 T cells, respectively, represented as mean ± SD of technical triplicates. (b-d, f-g, i, k) Two-way ANOVA. n.s. not significant, ****p < 0.0001.

Source Data