Selective positive allosteric modulation of β₂AR using antibody fragments targeting the third intracellular loop

GPCR therapeutics primarily target structured cavities formed by the seven transmembrane α-helices. However, amino acid sequence conservation in structured regions, especially among receptor subtypes, limits target selectivity. Here, we leverage the sequence divergence of the third intracellular loop (ICL3) of the receptor fold to derive a selective positive allosteric modulator for the β₂ adrenergic receptor (β₂AR). We repurpose the variable regions of a previously reported monoclonal antibody (Mab5) as single-chain (ScFv5) and single-domain (VhhL5) antibody fragments that enhance the maximal second messenger cyclic AMP signaling response downstream of the receptor. Despite ScFv5 binding a segment of ICL3 distinct to β₂AR, it also non-selectively binds and modulates cAMP signaling downstream of β₁AR and β₃AR. We find that this lack of specificity stems from multiple redundant interactions that facilitate ScFv5-ICL3 binding. In contrast, VhhL5, derived from the Mab5 light chain, selectively modulates β₂AR signaling over the β₁/β₃ subtypes. Mechanistically, VhhL5 enhances agonist-stimulated β₂AR-G protein coupling through releasing autoinhibitory ICL3 conformational states. In parallel, VhhL5 decreases β₂AR internalization promoting greater ligand-induced accumulation of cAMP. Our study demonstrates proof-of-concept for selective allosteric modulation of a GPCR by targeting a sequence divergent loop region within the receptor fold.