Abstract
Antibodies conjugated to bioactive compounds allow targeted delivery of therapeutics to cell types of choice based on that antibody’s specificity. Here we develop a new type of conjugate that consists of a nanobody and a peptidic ligand for a G protein-coupled receptor (GPCR), fused via their C-termini. We address activation of parathyroid hormone receptor-1 (PTHR1) and improve the signaling activity and specificity of otherwise poorly active N-terminal peptide fragments of PTH by conjugating them to nanobodies (VHHs) that recognize PTHR1. These C-to-C conjugates show biological activity superior to that of the parent fragment peptide in vitro. In an exploratory experiment in mice, a VHH-PTH peptide conjugate showed biological activity, whereas the corresponding free peptide did not. The lead conjugate also possesses selectivity for PTHR1 superior to that of PTH(1-34). This design approach, dubbed “conjugation of ligands and antibodies for membrane proteins” (CLAMP), can yield ligands with high potency and specificity.
Highlights
Antibodies conjugated to bioactive compounds allow targeted delivery of therapeutics to cell types of choice based on that antibody’s specificity
The association between the extracellular domain of parathyroid hormone receptor-1 (PTHR1) and residues 15–34 of PTH provides the bulk of the binding energy and specificity for this interaction
To mimic receptor association exhibited by PTH(1-34), we used either wild-type PTHR1 or PTHR1 variants modified to carry an epitope in the extracellular domain recognized by a VHH of choice
Summary
Antibodies conjugated to bioactive compounds allow targeted delivery of therapeutics to cell types of choice based on that antibody’s specificity. We address activation of parathyroid hormone receptor-1 (PTHR1) and improve the signaling activity and specificity of otherwise poorly active N-terminal peptide fragments of PTH by conjugating them to nanobodies (VHHs) that recognize PTHR1 These C-to-C conjugates show biological activity superior to that of the parent fragment peptide in vitro. The conjugation of a ligand for a surface receptor to an antibody that recognizes that same receptor should increase the effective concentration of the ligand and so increase its potency and specificity, provided appropriate spatial constraints are maintained This method could be used with an antibody that directly targets the receptor of interest to enable application without the need for genetic modification of the target cells or organism. G protein-coupled receptor (GPCR) family of proteins is an attractive class of targets to pursue using this approach
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