Development and characterization of CD19/CD3 and CD22/CD3 bsAbs. (A-B) Schematic structures of CD19/CD3 and CD22/CD3 bsAbs. HNT: FMC63 fused to the N-terminus of SP34 HC; HG: FMC63 fused to S184-L187 of SP34 HC; HCT: FMC63 fused to the C-terminus of SP34 HC; LNT: Nb25 fused to the N-terminus of SP34 LC; LG: Nb25 fused to S171-D173 of SP34 LC; LCT: Nb25 fused to the C-terminus of SP34 LC. (C-D) Confocal representative synapse images from 3 independent experiments. Nalm6 cells (transduced with green fluorescent protein [GFP]) were cocultured with Jurkat cells in the presence of CD19/CD3 or CD22/CD3 bsAbs (100 nM) for 3 hours, and cell‒cell conjugations were imaged at 100 × oil objective magnification under a laser scanning confocal microscope (Nikon, A1R). Hoechst (blue), anti-LFA-1 (red), GFP (green), and an overlay of all stains are shown. Scale bar, 10 μm. (E-F) Data on the accumulation of LFA-1 molecules at the IS between Nalm6 and Jurkat cells. The graphic shows the mean ± SD of the LFA-1 fluorescence intensities for 15 cell‒cell conjugations from 3 independent experiments. Asterisks indicate statistical significance using the Newman‒Keuls multiple comparison test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001; ns, not statistically significant (≥ .05). (G-H) Cytotoxicity assays of CD19/CD3 and CD22/CD3 bsAbs were performed with expanded T cells and the indicated target cells at an E:T ratio of 1:1 for 24 hours in triplicate. Error bars on the graph indicate the mean ± SD obtained from 3 independent experiments.