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 scFv LHFc) 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 scFv LH0Fc, P scFv LH4Fc and P scFv LH8Fc by shortening the spacer region connecting P scFv LH and Fc from 23 amino acids in P scFv LHFc 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 VHH gp130 fusion proteins. Deleting the extracellular stalk region between transmembrane and AIP VHH in the synthetic receptors AIP2 VHH gp130Δstalk and AIP3 VHH gp130Δstalk to increase rigidity and enhanced the biological activity of the short spacer P scFv Fc 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 VHH gp130Δstalk and AIP3 VHH gp130Δstalk but not their full-length counterparts. Furthermore, we engineered a tetravalent palivizumab variant (P scFv P 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 VHH gp130Δstalk variants opening avenues for therapeutic applications using nonphysiological targets in immunotherapy.