Engineered CD40 agonist antibodies displayed divergent FcγRIIa and FcγRIIb-dependent agonistic activity. (A) Different concentrations of CD40 agonist antibody variants were added to CD40 reporter cells in the presence of human FcγRIIb, FcγRIIaR131 or FcγRIIaH131 -expressing HEK293T cells, the activation of CD40 after 24 h stimulation was indicated by the expression of GFP of the reporter cell and analyzed by flow cytometry. (B) B cells were stimulated by different concentrations of NK003 variants and the upregulation of CD23, CD86 or HLA-DR was analyzed by flow cytometry. MFI: Mean Fluorescence Intensity. (C) Schematic of OVA-specific CD8+ T cell response model. FcγR/CD40-humanized mice were adoptively transferred with OT-I cells, and then immunized with DEC-OVA in the presence of CD40 antibody variants, expansion of OT-I cells was analyzed by flow cytometry. (D) Quantification of OT-I cells (left) and the percentage of OT-I cells among CD8+ T cells (right) as in OVA-specific CD8+ T cell response model. Each circle represents an individual mouse. Bars represent the mean ± SEM. ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001; One-way ANOVA Tukey's multiple comparisons test was used.