Canonical antibodies are formed with two identical polypeptide heavy chains and light chains. They consist of antigen-binding (Fab’) regions for specificity and the Fc domain for effector function and structural stability. Antibody therapies, checkpoint blockers like PD-L1, PD-1, and CTLA-4, can transform cancer treatment. Immunostimulatory antibodies activate immune cells by targeting the co-stimulatory receptors like CD40, 4-1BB, OX40, and CD27 from the tumor necrosis factor receptor superfamily (TNFRSF). Early clinical trial antibodies like Utomilumab, Urelumab, and 4-1BB mAb have raised concerns regarding toxicity and efficacy.
This study is focused on determining how the structural modifications, specifically disulfide engineering, inside and outside the human IgG2 hinge region (hIgG2) antibody. These modifications can enhance conformational rigidity, increase immunostimulatory activity, and develop more effective and potent antibody treatments for cancer immunotherapy. This study analyzes the functional and structural efficiency of the therapeutic antibodies with a focus on how modification to the hinge region, especially in the human hIgG2, affects it. It can improve immunostimulatory activity against cancer.
This study followed the ethical guidelines of the University of Southampton by using blood cones from healthy adult donors with informed consent. Antibodies were produced by using site-directed mutagenesis and protein production in the ExpiCHO-S cells. It is purified by using size exclusion chromatography and protein A affinity chromatography. The antibodies were low in aggregation and endotoxin content. F(ab’)2 fragments were produced and purified, and their purity was analyzed. Immunostimulatory activity was determined by NF-ÎşB activation by using primary human B cells and Jurkat cells.
Cell activation and adhesion markers were analyzed. B-cell proliferation was determined by using scintillation counting. Binding of the antibody to the cell surface receptors was assessed by flow cytometry. Structural properties were analyzed by small-angle X-ray scattering (SAXS). In silico, the design of the antibody variant was analyzed using Disulfide by Design 2.0 and manual inspection. Molecular dynamics (MD) simulations were used to study the structural behavior of the antibody variant with production and equilibration simulations by GROMACS. Residuals were examined to assess the model accuracy. GraphPad Prism software was used for statistical analyses, with detailed test details and reproducibility information displayed in figure legends.
This research investigates how structural flexibility and agonistic activity in human IgG2 monoclonal antibodies, specifically anti-hCD40 and anti-h4-1BB, are affected by hinge disulphide modifications. Researchers used a cysteine-to-serine mutation in the area around the hinge to produce antibody variants with different levels of conformational stiffness. Functional tests found that the rigid form (C232S κC214S) had maximum agonistic activity, and the flexible variant (C232S + C233S) had minimal activity.
Structural analysis showed that more agonistic variants acquire compact conformations and preserve high receptor binding affinity. The researchers used structure-guided disulphide engineering to insert more disulphide bonds, which led to two novel variants: cross-over + K228C and cross-over + T222C κE123C. The modified versions had equivalent agonistic activity to the parent variant at high levels, but significantly increased activity at lower doses, with ~3-fold reduced EC50 levels.
The study investigates the link between agonism and conformation in the anti-hCD40 hIgG2 mAb ChiLob7/4 and the effect of C-to-S exchange mutations on the conformational freedom. It shows that structure and conformation are important to develop therapeutic agonistic mAb with engineered variants that show greater biological activity.
Reference: Elliott IG, Fisher H, Chan HTC, et al. Structure-guided disulfide engineering restricts antibody conformation to elicit TNFR agonism. Nat Commun. 2025;16:3495. doi:10.1038/s41467-025-58773-8
