JNJ-67571244 binds to the C2 domain and mediates cytotoxicity of primary samples regardless of their SNP 12459419 genotype status. (A) Schematic showing the structure of CD33. (B) Hydrogen deuterium exchange mapping and subsequent illustration of epitope regions in IgC2 and IgV domain of CD33 extracellular domain protein for V- and C2-binder mAbs. The V epitope region is colored in blue, and the C2 epitope region is colored in red. (C) T cell–mediated cytotoxicity assays using JNJ-67368886 (CD33 V-binder) and JNJ-67571244 (CD33 C2-binder) in various CD33⁺ SNP rs12459419 CC, CT, or TT cell lines. The percent cytotoxicity was determined by flow cytometry. (D) Similar to panel B but here ex vivo assessment of cytotoxicity of CD33⁺ blasts in fresh AML patient whole blood was performed at 27 nM of bispecific antibody concentration. Mean ± standard deviation is graphed. (E) Similar to panel B but here JNJ-67571244–mediated cytotoxicity of frozen purified monocytes from 34 normal donors was assessed at 0.27 nM of bispecific antibody concentration. Mean ± standard deviation is graphed. P values were calculated by GraphPad Prism using Welch’s unequal variance t test.