Supplementary Figure 6: Mouse complement activation of C1q-specific antibody variants and FcγR-mediated internalization in CD20⁺FcγRIIb⁺ cancer cells.

(a) Binding RA801 and RA802 to mouse FcγRs. Microtiter well plates were coated with aglycosylated IgG1 (negative control), glycosylated IgG1 (Rituxan, positive control), mIgG, RA801, or RA802. His-tagged mFcγRs were added and binding was detected using anti-His IgG conjugated to HRP. Errors bars: standard deviations from triplicate experiments. (b) Lack of binding of RA801 immune complexes to mouse FcγRs expressed on CHO cells. The binding activities of performed ICs made of RA801 or Rituxan with PE-conjugated F(ab’)₂ goat anti-human IgG F(ab’)₂. (c) CDC assays with pooled mouse serum (PMS). CDC assays of CD20⁺ cells by RA801 with PMS. Experimental conditions for CDC assays of Ramos cells as in Supplementary Fig. 4. Calcein-loaded EL4-hCD20 cells were incubated with 50 % of PMS and serially diluted antibodies for 4 hrs. EC₅₀ values were presented in the plot (Orange: Rituxan, Blue: RA801). (d) α-CD20 antibody internalization in CD20⁺FcγRIIb⁺ cancer cells, HBL-1 or TMD8. CD20⁺FcγRIIb⁺ cancer cells were incubated with 100 nM of RA801 or RA802 for 0, 2, 4, or 6 h. Surface bound IgG was detected by flow cytometry using FITC conjugated goat anti-human Fc. (Abcam). In all assays, trastuzumab-IgG was used as a negative control and errors bars indicate the s.d. from triplicate experiments. Data are from one experiment representative of three experiments.