Abstract
Recently, we phenocopied interleukin (IL-)6 signaling using the dimerized single-chain variable fragment (scFv) derived from the respiratory syncytial virus IgG1-antibody palivizumab (PscFvLHFc) to activate a palivizumab antiidiotypic nanobody (AIPVHH)-gp130 receptor fusion protein. Palivizumab was unable to activate STAT3 signaling, so we aimed to create a similar ligand capable of triggering this pathway. Here, we created three variants of the ligand called PscFvLH0Fc, PscFvLH4Fc and PscFvLH8Fc by shortening the spacer region connecting PscFvLH and Fc from 23 amino acids in PscFvLHFc to 0 amino acids or expanding it by rigid linkers of four or eight alpha helical loops, respectively. The rigid-linker ligands had completely altered cellular activation patterns via AIPVHHgp130 fusion proteins. Deleting the extracellular stalk region between transmembrane and AIPVHH in the synthetic receptors AIP2VHHgp130Δstalk and AIP3VHHgp130Δstalk to increase rigidity and enhanced the biological activity of the short spacer PscFvFc ligands. Since scFv constructs are less stable than antibodies and have not been Food and Drug Administration approved, we looked for different antibody backbones. Transferring palivizumab's variable region to a more rigid and hence more agonistic IgG2 backbone (PIgG2) maintained affinity while improving agonistic properties activating cells expressing AIP2VHHgp130Δstalk and AIP3VHHgp130Δstalk but not their full-length counterparts. Furthermore, we engineered a tetravalent palivizumab variant (PscFvPIgG2) capable of inducing higher-order receptor clustering, activating Fas-induced apoptosis. In summary, we engineered a fully-synthetic cytokine/cytokine receptor pair based on the IgG2-variant of palivizumab and the AIPVHHgp130Δstalk variants opening avenues for therapeutic applications using nonphysiological targets in immunotherapy.