Enhancing immunostimulatory antibodies via targeted disulfide-bond-driven rigidity

Antibody-based therapies have transformed oncology by recruiting the immune system to selectively eliminate tumour cells. Immunostimulatory antibodies are particularly promising as they engage and activate immune cells directly, rather than solely marking cancer cells for destruction. This activation is commonly mediated through receptors of the tumour necrosis factor receptor superfamily (TNFRSF), potent modulators of anti-tumour immune responses. However, the therapeutic efficacy of this approach depends on achieving a careful balance: insufficient receptor activation fails to trigger an effective response, while excessive stimulation risks inducing systemic inflammation. The present study illustrates how structure-guided design combined with experimental validation can be employed to fine-tune this balance.

Antibodies are Y-shaped proteins composed of flexible and rigid domains; their conformational dynamics influence antigen binding and signalling. The hinge region, connecting the two antigen-binding fragments to the constant region, is a key determinant of molecular flexibility. Previous research indicated that the human IgG2 subtype, known for its potent immunostimulatory activity, might be further optimized through modifications to hinge architecture [1]. Building on these findings, this work explored whether altering hinge flexibility systematically enhances antibody function across multiple TNFRSF targets.

The hypothesis that increased molecular rigidity improves antibody performance was tested using disulfide engineering, which introduces or removes cysteine residues to form stabilizing disulfide bonds (Figure 1). By strategically modifying cysteine positions within the hinge region, the researchers fine-tune the antibodies’ conformational flexibility.

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Fig. 1: Antibody architecture and hinge engineering strategy. Antibodies are Y-shaped proteins composed of an Fc region, hinge region, and two antigen-binding (F(ab’)) regions. Disulfide engineering in and around the hinge region aims to reduce molecular flexibility, thereby enhancing biological activity and receptor signalling.

Direct measurement of antibody rigidity presents significant challenges, but synchrotron X-ray techniques provided critical insights. High-resolution crystal structures obtained at beamline ID30A-3 and at Diamond Light Source confirmed the precise architecture of engineered disulfide bonds. Complementary small-angle X-ray scattering (SAXS) experiments at beamline BM29 revealed the antibodies’ conformations in solution, showing more compact and rigid structures (Figure 2). Together, these methods highlight the essential role of synchrotron-based techniques in enabling rational antibody design.
 

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Fig. 2: Integrative structural characterization of engineered antibody variants using macromolecular crystallography, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations. a) Crystal structure of the engineered F(ab’)2 fragment (PDB: 8PUL) shown as a surface representation (top), with engineered disulfide bonds indicated as yellow sticks. Sulfur single-wavelength anomalous dispersion crystallography confirms the positioning of engineered disulfide bonds between Cys127, Cys228, and Cys233 residues (bottom), with anomalous difference Fourier density shown as a green mesh (contoured at 5σ). b) SAXS analysis indicates increased molecular rigidity of the engineered cross-over + K228C variant. Top: Dimensionless Kratky plot suggests a compact, folded conformation. Middle: Fit of the experimental SAXS profile (grey) to the best-fitting theoretical model calculated from MD trajectories (blue), with corresponding χ² value and residuals shown. Bottom: Surface representation of the best-fitting model, with heavy and light chains in darker and lighter shades, respectively.

The most active antibody variants exhibited more compact solution conformations in the SAXS analysis and enhanced signalling activity in cell-based assays. Molecular dynamics simulations showed these findings, demonstrating that increased rigidity correlates with improved receptor activation. Functional assays – including NF-κB reporter activation, B cell adhesion, expression of activation markers, and immune cell proliferation – consistently confirmed the superior activity of the engineered antibodies. 

Notably, this approach was effective across multiple TNFRSF receptors, improving activity against both CD40 and 4-1BB. These results suggest that disulfide engineering represents a broadly applicable strategy for designing next-generation immunostimulatory antibodies with improved precision and potency. By introducing targeted structural modifications, antibody function can be significantly enhanced while maintaining safety profiles. 

This approach may serve as a framework for the rational engineering of antibody therapeutics, with potential implications for cancer immunotherapy and other biomedical applications. The study exemplifies how interdisciplinary integration of structural biology, molecular engineering, and advanced synchrotron methods can facilitate transformative advances in drug development. 

Principal publication and authors
Structure-guided disulfide engineering restricts antibody conformation to elicit TNFR agonism, I.G. Elliott (a,b,c,d), H. Fisher, (a,b,c,d,e), H.T.C. Chan (c), T. Inzhelevskaya (c), C.I. Mockridge (c), C.A. Penfold (c), P.J. Duriez (c), C.M. Orr (f), J. Herniman (a), K.T.J. Müller (c), J.W. Essex (a,d), M.S. Cragg (c,d), I. Tews (b,d), Nat. Commun. 16, 3495 (2025); https://doi.org/10.1038/s41467-025-58773-8
(a) School of Chemistry and Chemical Engineering, Faculty of Engineering and Physical Sciences, University of Southampton, Southampton (UK)
(b) School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, Southampton (UK)
(c) Antibody & Vaccine Group, Centre for Cancer Immunology, School of Cancer Sciences, Faculty of Medicine, Southampton General Hospital, University of Southampton, Southampton (UK)
(d) Institute for Life Sciences, University of Southampton, Southampton (UK)
(e) ESRF
(f) Diamond Light Source, Didcot (UK)

References
[1] C.M. Orr et al., Sci. Immunol. 7, 73 (2022).