Comparison of different formats of CD19/CD22/CD3 tsAbs. (A) Schematic structures of different CD19/CD22/CD3 tsAbs. NN: FMC63 and Nb25 fused to the N-terminus of SP34 HC and LC, respectively; NC: FMC63 fused to the N-terminus of SP34 HC and Nb25 fused to the C-terminus of LC; CN: FMC63 fused to the C-terminus of SP34 HC and Nb25 fused to the N-terminus of LC; CC: FMC63 fused to the C-terminus of SP34 HC and Nb25 fused to the C-terminus of LC; (B, D) Cytotoxicity comparison of CD19/CD22/CD3 tsAbs against various CD19⁺ and/or CD22⁺ tumor cell lines after 24 hours of incubation with expanded T cells at an E:T ratio of 1:1 in triplicate. Results from 1 of more than 3 independent experiments are shown. (C, E) Inflammatory cytokines (IL-2, IFN-γ, and TNF-α) released from T cells cocultured with the indicated target cells in the presence of tsAbs (100 pM) for 24 hours at an E:T ratio of 1:1 in triplicate. Values indicate the mean ± SD from the data of 3 independent experiments. Asterisks indicate statistical significance using the Newman‒Keuls multiple comparison test. ∗P < .01, ∗∗P < .05, ∗∗∗P < .001; ns, not statistically significant (≥.05).