An engineered palivizumab IgG2 subclass for synthetic gp130 and fas-mediated signaling

Recently, we phenocopied interleukin (IL-)6 signaling using the dimerized single-chain variable fragment (scFv) derived from the respiratory syncytial virus IgG1-antibody palivizumab (P^scFvLHFc) to activate a palivizumab antiidiotypic nanobody (AIP^VHH)-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 P^scFvLH0Fc, P^scFvLH4Fc and P^scFvLH8Fc by shortening the spacer region connecting P^scFvLH and Fc from 23 amino acids in P^scFvLHFc 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 AIP^VHHgp130 fusion proteins. Deleting the extracellular stalk region between transmembrane and AIP^VHH in the synthetic receptors AIP2^VHHgp130Δstalk and AIP3^VHHgp130Δstalk to increase rigidity and enhanced the biological activity of the short spacer P^scFvFc 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 (P^IgG2) maintained affinity while improving agonistic properties activating cells expressing AIP2^VHHgp130Δstalk and AIP3^VHHgp130Δstalk but not their full-length counterparts. Furthermore, we engineered a tetravalent palivizumab variant (P^scFvP^IgG2) 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 AIP^VHHgp130Δstalk variants opening avenues for therapeutic applications using nonphysiological targets in immunotherapy.