Supplementary Figure 4: Complement-mediated tumor-cell killing by C1q-specific antibody variants.

(a) CDC assays with Daudi cells. 10 μg/ml of each antibody was incubated with 50 % of PHS and CD20⁺ Daudi cells for 15 mins and 30 mins. Lysed cells were detected by TO-PRO-3. (b) C1q deposition on mAb-opsonized CD20⁺ cells. Time course for C1q deposition on CD20⁺ Raji cells. Raji cells were incubated in 5% NHS and 10 μg/ml mAb for different time periods at 37°C. Cells were washed twice, incubated with FITC-conjugated anti-C1q and assayed by flow cytometry. MFI were converted to molecules of equivalent soluble fluorochrome (MESF) using calibrated beads (Spherotech). (c-d) CD20⁺ cancer cells killing activities by PBMC or PMNs. Raji (or Ramos) cells incubated with effector cells in RPMI1640 medium without serum (c) or with 25% of C9-depleted serum (d). (e) Effect of α-CR3 or α-CR4 antibodies in CDCC. Rituximab (or RA801)-opsonized Ramos cells were incubated with: 10 μg/ml of α-CR3 Ab (or α-CR4 Ab)-coated effector cells in RPMI1640 medium supplemented with 25% C9-depleted serum. (f) Cell killing of CD20⁺ cancer cells by effector cells in the presence of serum. Cell lysis of CD20⁺ cells with effector cells in RPMI1640 medium supplemented with 25% PHS. In c-f, % of cell lysis was determined 4 hours after the addition of cells and antibody. In all assays, the percent of tumor cell lysis was calculated according to the following formula: 100 × (E-S)/(M-S), where E is the fluorescence of the experimental well, S is the fluorescence in the absence of antibody (tumor cells incubated with medium and complement alone), and M is that of tumor cells with lysis buffer (Triton® X-100 at 2% v/v, SDS at 1% w/v, 100 mM NaCl, and 1 mM EDTA). In all assays, PMN cells were stimulated by incubation with 10 ng/mL GM-CSF and trastuzumab-IgG was used as a negative control. Errors bars indicate the standard deviation (s.d.) from triplicate experiments. Data are from one experiment representative of three experiments.