Fig. 1: C-S disulfide variants exhibit the same trend in agonistic activity and conformation in anti-hCD40 and anti-h4-1BB mAb.
From: Structure-guided disulfide engineering restricts antibody conformation to elicit TNFR agonism
a Model of disulfide arrangements in hIgG2 mAb affecting agonistic activity and flexibility, derived from studies of the hIgG2 anti-hCD40 ChiLob7/420. Disulfides shown in yellow, and cysteines involved in disulfide bonds labeled (with the Kabat numbering system). hIgG1 and hIgG2 wildtypes are shown inset above. b NF-κB/Jurkat/GFP reporter cells expressing hCD40 or h4-1BB were stimulated with serially diluted ChiLob7/4 or SAP1.3 mAb, respectively. NF-κB activation triggers GFP expression and was quantified after 24 h by flow cytometry (inset histograms representative from n = 3 at 0.04 μg/ml for ChiLob7/4 and 5 μg/mL for SAP1.3). Graphs show dose-response curves of the percentage of GFP-positive cells. n = 3–7 independent biological experiments; mean ± SEM, taken from technical triplicates for each independent experiment. hIgG1 (gray) and hIgG2 (black) shown as controls. c F(ab’)2 of anti-hCD40 and anti-h4-1BB C-S disulfide variants were analyzed by SEC-SAXS. Graphs show dimensionless Kratky plots derived from SEC-SAXS data, with the Guinier-Kratky point (√3, 1.103) indicated by the pale gray crosshairs. hIgG1 (gray) and hIgG2 (black) shown as controls. Errors calculated following standard BioXTAS RAW software procedures63. Disulfide C-S antibody variants labeled by color: red C232S + C233S, purple C233S κC214S, blue C232S κC214S. Source data are provided as a Source Data file.
