Figure 6.

CD40 activation by reconstituted anti-CD40 agonist. CD40-expressing HEK293 cells were treated with different concentrations between 200 nM and 2 pM of reconstituted anti-CD40 IgG1, anti-CD40 IgG2, reference antibody APX005M (used as a positive control) and trastuzumab (used as a negative control) at 37°C, 5% CO₂ for 6 h. LPS is known to influence certain cell assays and was used as an assay control up to a concentration of 500 nM. TCEP and DHAA were added in dose concentrations to evaluate false luminescence signal detection. CD40 was activated after treatment with anti-CD40 mAbs and BioGlo Luciferase substrate was added for luminescence readout. Luminescence signal was measured at 0.5 s per well using a sensitivity of 170. The IC50 values were calculated by fitting the dose-response curves using a 4PL with Graphpad Prism software 7.