iBody-mediated tuning of synthetic cytokine receptor activation via rational nanobody interface engineering.

Nanobodies are small, single-domain antibody fragments derived from heavy chain-only antibodies. They combine high binding affinity with advantages such as compact size, stability, solubility, and flexible epitope recognition, making them attractive tools in molecular biology and therapeutic applications. In this study, we engineered and optimized nanobodies for controlled activation of synthetic cytokine receptors, aiming to expand options for receptor customization. Specifically, we used nanobodies as extracellular domains of the gp130 receptor to induce dimerization upon antigen binding. To enable receptor activity, we introduced framework mutations that promote the formation of an i-shaped nanobody (iBody) dimer, adapted from i-shaped antibodies. These mutations enhanced dimerization and enabled low-level ligand-independent receptor activation. AlphaFold modeling identified the key amino acids responsible for forming the iBody interface. Additional modifications reduced intermolecular affinity, thereby minimizing background activation while preserving the structural features necessary for ligand-induced stimulation. This approach effectively broadened the receptor's activation range. Importantly, these framework mutations were not limited to the gp130-specific nanobody GP11 but were also functional in AIP3, an anti-idiotypic nanobody targeting palivizumab. Here, the modified nanobody fusion receptor could be activated by palivizumab, overcoming prior steric hindrance.