Figure 3.
The effects of FcγRIIB crosslinking on the agonistic activities of engineered SF2 antibodies by HEK-Blue NF-κB reporter assay. (A) Increasing concentrations (10 ng/mL to 1000 ng/mL) of SF2IgG1_RE were incubated with HEK-Blue: OX40 cells with or without co-culturing with Raji cells. Another set of assays were set up in which Raji cells were pre-incubated with 5 μg/mL 2B6 antibody before co-culturing with HEK-Blue: OX40 cells to test the effect of blocking FcγRIIB-mediated crosslinking. Agonistic activities of the antibodies were assessed by the HEK-Blue NF-κB reporter assay. Agonistic activities of SF2 antibodies, normalized as percent activity relative to that driven by 1 μg/mL OX40 ligand, were plotted vs. the concentrations of test antibodies (Data expressed as mean ± SEM, n). (B) Similar assays were set up to study the effects of co-culturing with Raji cells on the agonistic activities of SF2IgG2σ_RE (Data expressed as mean ± SEM, n).