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. 2021 Dec 2;10(1):2003532. doi: 10.1080/2162402X.2021.2003532

Figure 2.

Figure 2.

Specificity and sensitivity of the AdCAR-T system

A) AdCAR-T were incubated for 24 h with NALM6 at an E:T ratio of 1:1 in the presence of 10 ng/mL LLE-CD19 mAb and indicated concentrations of free biotin or human serum. Target-cell lysis was determined by LCA. Comparison of conditions without LLE-CD19 mAb was not significant. Comparison of conditions with LLE-CD19 mAb was not significant. Conditions without versus with LLE-CD19 mAb were highly significant (n = 4, p < .0001). B) AdCAR-T or non-transduced activated T cells with or without 10 ng/mL LLE-CD19 mAb or conventional CD19-CAR-T were incubated with NALM6 at indicated E:T ratios. Target-cell lysis was determined by LCA after 6 h and 12 h (n = 4, p = .0055 at E:T 0.15:1, 12 h). C) AdCAR-T or nontransduced activated T cells with or without 10 ng/mL LLE-GD2 mAb or conventional GD2-CAR-T were incubated with LS at an E:T ratio of 2:1. Target-cell lysis was determined by xCELLigence an impedance-based real-time cytotoxicity assay (ICA), that provides a real-time analysis. Comparison of conditions without LLE-GD2 mAb was not significant. Comparison of AdCAR-T with LLE-GD2 mAb was not significant. Comparison of AdCAR-T without versus with LLE-GD2 mAb was highly significant (n = 4, p < .0001). D) AdCAR-T were incubated with NALM6 at an E:T ratio of 1:1 for 24 h in the presence of LLE-CD19 mAb at indicated concentrations. Target-cell lysis was determined by LCA. Titration curve is shown. EC50 = 7.9 pg/mL. E) AdCAR-T were incubated with NALM6 at an E:T ratio of 1:1 for 24 h at 1 ng/mL LLE-CD19 mAb and indicated concentrations of CD19 mAb (without LLE-tag). Target-cell lysis was determined LCA (upper panel). Concentrations of LLE-CD19 mAb and CD19 mAb are schematically illustrated (lower panel). Data shown in A–C) and E) represent mean ± SEM of four independent experiments in triplicates from four different donors. Data shown in D) represent mean ± SEM of (n = 5) independent experiments in triplicates from different donors. In A) and C) significance was determined by one-way ANOVA and Tukey post hoc test, in B) by unpaired, two-tailed Mann-Whitney test. (n.s.) not significant.