Design and characterization of CD19/CD22/CD3 (CC). (A) Schematic structure of CD19/CD22/CD3 (CC) by fusing FMC63 to the C-terminus of SP34 HC and Nb25 to the C-terminus of SP34 LC based on the CD19/CD3 HCT and CD22/CD3 LCT formats. (B) Binding profiles of CD19/CD22/CD4 (CC) and the corresponding bsAbs to CD19- and CD22-expressing K562 cells, parental K562 cells, and T cells were detected by flow cytometry in triplicate. The mean ± SD of the mean fluorescence intensity (MFI) obtained from 3 independent experiments is indicated. ND, not determined. (C) Cytotoxicity comparison of CD19/CD22/CD3 (CC) and the corresponding bsAbs against Nalm6, Nalm6-KO19, and Nalm6-KO22 cell lines after 24 hours of incubation with expanded T cells at an E:T ratio of 1:1. Experiments were performed in triplicate and repeated 3 times with similar results. (D) Inflammatory cytokines (IL-2, IFN-γ, and TNF-α) released from T cells cocultured with the indicated target cells in the presence of antibodies (100 pM) for 24 hours at an E:T ratio of 1:1 in triplicate. Data are presented as the means ± SDs from 3 independent experiments, and statistical significance was calculated using the Newman‒Keuls multiple comparison test: ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001; ns, not statistically significant (≥.05).