Figure 2.

Effect of membrane proximity of the target epitope on the anti-tumor efficacy of CD33-directed CAR T cells

(A) ML-1 cells with CRISPR-Cas9-mediated deletion of the endogenous CD33 locus and parental CD33⁻ RS4;11 cells were engineered to overexpress either CD33^FL or CD33^ΔE3-4. Relative expression of the target proteins was flow cytometrically assessed with Quantibrite-PE via a CD33^V-set antibody (P67.6). (B and C) Primary human CD8⁺ or CD4⁺ T cells expressing either (B) an My96-based CAR construct or (C) an A33-based CAR construct were incubated with different target cells at an E:T cell ratio of 1:2, 1:4, or 1:8 for 16 h before flow cytometric assessment of cytotoxicity by subtracting absolute number of live tumor cells from CD33^KO ML-1 (7.29 × 10⁶ ± 8.67 × 10⁵) or parental RS4;11 (7.77 × 10⁴ ± 2.03 × 10⁴) cells. Shown are mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns (not significant) by two-way ANOVA with post-hoc Tukey correction. Shown are technical triplicates of one representative out of two separate experiments.