Figure 1.

Optimization of CD33^V-set CAR My96

(A) Schematic of different types of CD33 antibodies binding full-length CD33 (CD33^FL), a naturally occurring isoform lacking the exon 2-encoded V-set domain (CD33^ΔE2), and an artificial CD33 molecule with deletion of exons 3 and 4 that results in membrane proximal relocation of the V-set domain (CD33^ΔE3-4). (B) Schematic of CAR constructs with short, intermediate, and long extracellular spacers. Note the mutated CH2 domain to prevent Fc binding. Illustrations created with BioRender.com. (C) Human myeloid CD33⁺ ML-1, K562, or ML-1 CD33^KO (by CRISPR-Cas9) cells were incubated with primary human T cells transduced with a CD33^V-set My96-based CAR construct with either a short, intermediate, or long spacer at various E:T cell ratios as indicated. After 4 h, cytotoxicity was assessed by chromium-51 release. (D) Geometric mean fluorescence intensity of transduction marker tCD19 expression by flow cytometry. Experiments performed in technical triplicates. Shown are mean ± SEM in triplicate from two representative experiments from different healthy donors. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns (not significant) by two-way ANOVA with post-hoc Tukey correction.