Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies

Abstract

Here, we identify the importance of molecular crowding agents in the functional stabilization of scFv antibodies. Antibodies were tethered through an engineered calmodulin (CaM)-binding peptide into a stimulus-responsive hydrogel composed of poly(ethylene glycol) (PEG)-functionalized CaM. Macromolecular crowding is modulated by transient heating, which decreases effective pore sizes. Using a fluorescent ligand bound to the scFv, frequency-domain fluorescence spectroscopy was used to assess the structural coupling between the V and the V domains and relationships with functional stabilization. There is minimal structural coupling between the V and the V domains in solution, as is apparent from the substantial rotational mobility for the bound ligand, that is suggestive of an independent mobility for the V and the V domains. In comparison, the hydrogel matrix acts to structurally couple the V and the V domains, resulting in a reduction in rotational mobility and a retention of ligand binding in the presence of 8.0 M urea. Under these same conditions, ligand binding is disrupted for scFv antibodies in solution. Increases in the stabilization of scFv antibodies in hydrogels is not simply the result of molecular crowding because decreases in pore size act to destabilize ligand binding. Rather, our results suggest that the functional stabilization of the scFv antibody within the PEG hydrogel matrix includes important factors involving protein solvation that stabilize interdomain interactions between the V and the V domains necessary for ligand binding.