The triple variant effective kills RR cells in vitro. (A) Surface expression of CD20 on RR cells. Raji or Raji-R cells (2 × 10⁶) were incubated with different concentrations of FITC-labeled rituximab or triple variant for 1 hour on ice. Then, the cells were washed and then analyzed by FCM. The intensity of surface CD20 expression was measured by the mean fluorescence intensity. (B-C) Rituximab-resistant cells derived from Raji and Daudi cells were exposed to different rituximab variants (10 μg/mL), followed by the addition of NHS (B) or PBMCs at an E/T ratio of 40:1 (C), respectively. A standard LDH assay was performed. (D) Cross-linked triple mutant induced more apoptosis relative to rituximab in RR cells in vitro. Cells (2 × 10⁶) were treated with isotype control antibody, rituximab, or triple variant (10 μg/mL) with or without cross-linker (20 μg/mL) for 16 hours. The apoptotic cells were determined by single staining of annexin V–Fluos on FCM. Columns represent mean (n = 3); bars represent SD.