Fig. 3: Glyco-bridge activates CARs in a target antigen density-dependent manner.
From: Tuning CAR-T cells by targeting cancer-associated glycan in pancreatic cancer
a Schematic showing the mechanism of glyco-bridge depending on the target antigen density of the glyco-bridge. b Surface level of Tn-MUC1 on RPMI 8226 cells following itraconazole incubation for 48 h. c Real-time cytotoxicity assay against RPMI 8226 cells with itraconazole with indicated bridges at a 1:1 E:T ratio. Results are mean ± s.d. of n = 3 independent measurements. d–f, Real-time cytotoxicity assay with mesothelin-targeted CAR-T cells with indicated bridges at a 1:1 E:T ratio. Panels show RPMI 8226 (d), low CD19 (e), and medium CD19 (f). Results are mean ± s.d. of n = 3 independent measurements. g log of the number of CD19 molecules versus log of the normalized target cell GFP signal from d–f at time point 60 h; best-fit line to a power-law model with slope = −0.16 and R2 = 0.6568. h Surface level of CD19 on Jeko-1 clones. i, j Real-time cytotoxicity assay with mesothelin-targeted CAR-T cells with indicated bridges at a 1:1 E:T ratio. Panels show Jeko-1 (i) and Jeko-1 clone B (j). Results are mean ± s.d. of n = 3 independent measurements. k log of the number of CD19 molecules versus log of the normalized target cell GFP signal from (i, j) at time point 60 h; best-fit line to a power-law model with slope = −0.74 and R2 = 0.6392. l Schematic showing ΔSS1 scFv on CAR molecules. m log of the number of CD19 molecules versus log of the normalized GFP signals from Jeko-1 cell lines from (h) using ΔSS1 scFv CAR at time point 60 h; best-fit line to a power-law model for each donor; R2 for 0.7719 (donor #1) and 0.7235 (donor #4). Results are mean ± s.d. of n = 3 independent experiments per donor. n Proposed relationship between CAR and bridge molecules. o Proposed the mechanism between glyco-bridge-mediated killing, target antigen density for the glyco-bridge, and binding affinity. In b, h representative flow cytometry data from three independent experiments. In e, f, and i statistical analysis was performed by two-way ANOVA with correction for multiple comparisons.
