Functional GLP-1R antibodies identified from a synthetic GPCR-focused library demonstrate potent blood glucose control

Abstract

G protein-coupled receptors (GPCRs) are a group of seven-transmembrane receptor proteins that have proven to be successful drug targets. Antibodies are becoming an increasingly promising modality to target these receptors due to their unique properties, such as exquisite specificity, long half-life, and fewer side effects, and their improved pharmacokinetic and pharmacodynamic profiles compared to peptides and small molecules, which results from their more favorable biodistribution. To date, there are only two US Food and Drug Administration-approved GPCR antibody drugs, namely erenumab and mogamulizumab, and this highlights the challenges encountered in identifying functional antibodies against GPCRs. Utilizing Twist's precision DNA writing technologies, we have created a GPCR-focused phage display library with 1 × 10 10 diversity. Specifically, we mined endogenous GPCR binding ligand and peptide sequences and incorporated these binding motifs into the heavy chain complementarity-determining region 3 in a synthetic antibody library. Glucagon-like peptide-1 receptor (GLP-1 R) is a class B GPCR that acts as the receptor for the incretin GLP-1, which is released to regulate insulin levels in response to food intake. GLP-1 R agonists have been widely used to increase insulin secretion to lower blood glucose levels for the treatment of type 1 and type 2 diabetes, whereas GLP-1 R antagonists have applications in the treatment of severe hypoglycemia associated with bariatric surgery and hyperinsulinomic hypoglycemia. Here we present the discovery and creation of both antagonistic and agonistic GLP-1 R antibodies by panning this GPCR-focused phage display library on a GLP-1 R-overexpressing Chinese hamster ovary cell line and demonstrate their in vitro and in vivo functional activity.

Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8

“…Intriguingly, our mAb1 recognizes CCR8 via a similar CDRH3-ECL2 interaction. This emerging theme could be further leveraged to build new in vitro antibody display libraries with long, structured CDRH3s to better engage the ECLs of class A GPCRs 65 . Additionally, the convex paratope of heavy chain only antibodies (VHHs), including their long CDRH3, appears ideally suited to engage the orthosteric pocket of GPCRs.…”

Structure elucidation of a human melanocortin-4 receptor specific orthosteric nanobody agonist

“…Only one clear example of an antibody with full agonistic pharmacology for a GPCR has been reported in peer-reviewed literature 28 . The agonistic nanobodies described in this study, including pN162, are not obtained by structure-informed design 27 or (repertoire) engineering 24,25,26 but are enriched by phage display from an in vivo matured repertoire. The immune repertoires were induced by genetic immunization of camelids with an engineered active state MC4R conformation 37,38 .…”

“…Indeed, despite multiple reports describing positive allosteric modulating nanobodies that increase agonist a nity or signaling potency or e cacy at low ligand tonus 19,20,21,22,23 , only a few are reported to agonize the GPCR in the absence of any ligand. Some of the reported agonistic antibodies have been designed by ligand tethering (genetically fusing or grafting agonist ligand motifs into the antibody hypervariable complementarity determining region (CDR)) 24,25,26 or by structure-informed engineering 27 . De novo identi ed agonistic antibodies towards class A GPCRs are scarce and have only been reported for β1AR (bivalent IgG Mab1) 28 , ChemR23 (bivalent IgG) 29 and APJ (monovalent nanobody JN300) 30 .…”

Evolution of phage display libraries for therapeutic antibody discovery

“…As a result, this GPCR-focused library successfully led to discovery of a panel of antagonistic antibodies targeting glucagon-like peptide-1 receptor with high affinity. 15 …”

“… 4–6 Moreover, due to fully controlled selection conditions, phage display can be tailored for selection for desired properties that may not be achievable by in vivo approaches, e.g., selection for a specific epitope recognition, 7 , 8 pH-dependent binding, 9 , 10 antibody internalization, 11 , 12 and even catalytic activity. 13 Furthermore, phage display libraries have successfully led to antibody discovery against the most challenging targets or epitopes, e.g., the stem region of influenza hemagglutinin, 14 G-protein-coupled receptors (GPCRs), 15 and specific conformations of ion channels. 16–18 A recently developed new generation of phage libraries with protein quality control steps in the library construction will further close the developability gap between antibodies derived in vivo and in vitro.…”