Fig. 2: Structure-guided design of anti-hCD40 hIgG2 mAb with additional engineered disulfides.
From: Structure-guided disulfide engineering restricts antibody conformation to elicit TNFR agonism
a Schematic of the parental agonistic hIgG2 C232S κC214S variant (cross-over) showing experimentally determined disulfides as solid yellow lines. Disulfides not resolved in the structure (PDB: 6TKE) are shown as dashed yellow lines. Cysteine amino acid residues are labeled. b Structure-based design and predicted disulfides for C232S + K228C κC214S (cross-over + K228C) and C232S + T222C κE123C + κC214S (cross-over + T222C κE123C) shown in yellow, with disulfides from the parent cross-over variant in gray. c Experimentally determined crystal structures of the new F(ab’)2 variants shown as surface representation, with disulfides as sticks. d Sulfur single-wavelength anomalous dispersion (Sulfur SAD) crystallography reveals the position of sulfur atoms and confirms disulfides between neighboring chains. The anomalous electron density is shown as green mesh (anomalous difference Fourier map, contoured at 5 σ). Disulfides shown in yellow as sticks. Engineered antibody variants labeled by color: blue C232S κC214S (cross-over), teal cross-over + K228C, orange cross-over + T222C κE123C.
