Improved GPCR ligands from nanobody tethering

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.

Detection of genome‐edited and endogenously expressed G protein‐coupled receptors

“…These parameters become important when considering phenomena such as receptor reserve, which can dramatically impact the signalling responses measured in a particular assay [4]. GPCR overexpression therefore CXCR4 [37] FPR2 [36] Nanobodies Easily genetically or chemically modified Can be purified from bacterial cultures in large quantities Improved tissue penetration compared to full-length antibodies due to smaller size Can be conjugated to other proteins for improved binding characteristics or retention Low retention in vivo due to glomerular filtration and excretion Could be challenging to find noncompetitive extracellular nanobodies for peptidergic receptors due to overlap between extracellular epitopes and the ligand binding site Few GPCRs have extracellular-binding nanobodies ACKR3 (CXCR7) [54] CXCR2 [53] CXCR4 [55][56][57][58]60,62] PTH1R [52] US28 [65,66] [104] µ opioid receptor [108] removes the subtlety inherent in a physiological system.…”

Comparable Initial Engagement of Intracellular Signaling Pathways by Parathyroid Hormone Receptor Ligands Teriparatide, Abaloparatide, and Long‐Acting PTH

“…Receptor internalization was assessed in the HEK293 cell line stably transfected with human PTH1R‐pHluorin2‐GFP (GPG‐10 cells). ( 33,34 ) Confluent monolayers of cells in black‐walled, 96‐well plates were incubated in HBSS with BSA (0.1% wt/vol) and HEPES buffer (pH 7.4, 10mM). Peptides were added and wells were analyzed by recording fluorescence readouts with excitation at 485 or 405 nm and emission at 535 nm.…”

“…Recruitment of β ‐ arrestin was assessed in HEK293 cells stably transfected to express β ‐ arrestin2‐YFP ( 36 ) and glosensor (GBR‐24 cells) ( 34 ) and transiently transfected to express the hPTH1R or mDsRed‐hPTH1R in which the monomeric Discosoma sp . red fluorescent protein is inserted in the E2 region of the receptor's extracellular domain.…”

A multivalent biparatopic EGFR-targeting nanobody drug conjugate displays potent anticancer activity in solid tumor models

“…Enhanced internalization and lysosomal trafficking could benefit the uptake of ADC, thus improving its efficacy. 4 The internalization of 97m and S7 ADC were two times as much as cetuximab (Fig. 1f ).…”