Effector functions of 22-20 and 20-22. (A) Complement-dependent cytotoxicity. Daudi cells were incubated with serial dilutions (3.33 × 10−8 to 2.6 × 10−10 M) of 22-20 (△), 20-22 (▲), e-mab (▼), v-mab (■), CH3-AD2-IgG-v-mab (○), or labetuzumab (●) in the presence of human complement. The percentage complement control (number of viable cells in the test sample compared with cells treated with complement only) was plotted versus the log of the nM concentration. (B) Antibody-dependent cellular cytotoxicity. Daudi cells (target) were incubated with 22-20, 20-22, v-mab, e-mab, or h734 at 5 μg/mL in the presence of freshly isolated peripheral blood mononuclear cells (effector). A 100% lysis reference was generated by the addition of detergent to wells only containing target cells. The percentage lysis obtained for each of 2 effector cell donors is represented in the bar graph. (C) Evaluation of membrane distribution of CD22 after treatment of Raji (top 2 panels) or Daudi (bottom 2 panels) with untreated (lane 1), e-mab (lane 2), v-mab (lane 3), 22-20 (lane 4), or 20-22 (lane 5). Cells were lysed in buffer containing 1% Triton X-100 and fractionated by sucrose density-gradient ultracentrifugation. The lipid rafts were collected from the interface of 5% and 30% sucrose, and the soluble fractions from the 40% sucrose at the bottom of the tubes. The lipid raft and soluble fractions were analyzed by anti-CD22 immunoblot. Vertical lines have been inserted to indicate a repositioned gel lane.
