Characterization of CD20-Flex BiFP. (A) Schematic diagram of the Fab domain exchange resulting in the generation of CD20-Flex bispecific antibody when combined with the KiH technology. (B) Binding of ¹²⁵I-labeled Fc-deleted CD20-Flex BiFP, rituximab F(ab′)₂, and rituximab Fab fragment to Daudi cells. ¹²⁵I-labeled Fc-deleted CD20-Flex BiFP, F(ab′)₂, or Fab fragments of rituximab were incubated with Daudi cells for 2 h at 37°C. The saturation of CD20-Flex BiFP is ∼10 μg/mL, which is comparable with rituximab. The cell-bound and free ¹²⁵I-labeled BiFP or mAb fragments were then separated by centrifugation through phthalate oils and the cell pellets together with bound antibody counted for radioactivity. Data from saturation binding experiments were analyzed by nonlinear least-squares regression for curve-fitting and dissociation constant estimation. Data are mean ± SD (n = 5). (C) Dissociation of ¹²⁵I-labeled CD20-Flex BiFP, rituximab, and rituximab Fab from Raji cells. Cells were incubated with ¹²⁵I-labeled CD20-Flex BiFP, rituximab or rituximab Fab (10 μg/mL) at 37°C for 1 h, washed twice, and resuspended. Samples of cells were taken at time 0, 1, 2, and 4 hours and then washed and analyzed. Shown are means and SD of at least 3 experiments. (D) Validated Flex function of CD20-Flex BiFP. Both Flex-Ig and CD20-Flex BiFP exhibited dose-response curves (10 and 20 ng/mL) for purified HPC colonies, as evaluated in cultures supplemented with 2 U/mL erythropoietin, 10 ng/mL interleukin-3, 20 ng/mL GM-CSF. Mean ± SD values from 4 separate experiments.