Structure-guided disulfide engineering restricts antibody conformation and flexibility to elicit TNFR agonism

A promising strategy in cancer immunotherapy is activation of immune signalling pathways through antibodies that target co-stimulatory receptors. hIgG2, one of four human antibody isotypes, is known to deliver strong agonistic activity, and modification of hIgG2 hinge disulfides can influence immune-stimulating activity. This was shown for antibodies directed against the hCD40 receptor, where cysteine-to-serine exchange mutations caused changes in antibody conformational dynamics and flexibility. Here we demonstrate that the principles of increasing agonism by restricting antibody conformation through hinge disulfide modification can be translated to the co-stimulatory receptor h4-1BB, another member of the tumour necrosis factor receptor superfamily. Furthermore, we explore structure-guided design of the anti-hCD40 antibody ChiLob7/4 and show that engineering additional disulfides can elicit even greater conformational restriction, concomitant with enhanced agonism. These results reveal the impact of modulating conformational flexibility to tune immunostimulatory activity and provide strategies for the rational design of more powerful antibody therapeutics.