Structural and pharmacological characterization of novel potent and selective monoclonal antibody antagonists of glucose-dependent insulinotropic polypeptide receptor - PubMed (original) (raw)

. 2013 Jul 5;288(27):19760-72.

doi: 10.1074/jbc.M112.426288. Epub 2013 May 20.

Chaithanya Madhurantakam, Susan Kunze, Evelyn Matthews, Claire Priest, Siobhan O'Brien, Andie Collinson, Monika Papworth, Maria Fritsch-Fredin, Lutz Jermutus, Lambertus Benthem, Markus Gruetter, Ronald H Jackson

Affiliations

Structural and pharmacological characterization of novel potent and selective monoclonal antibody antagonists of glucose-dependent insulinotropic polypeptide receptor

Peter Ravn et al. J Biol Chem. 2013.

Abstract

Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that, upon binding to its receptor (GIPr; a class B G-protein-coupled receptor), stimulates insulin secretion by beta cells in the pancreas. There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure to support studies on GIP biology. Here we describe the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog, and human GIP receptors with a Ki of 7 nm for the human GIPr. Gipg013 antagonizes the GIP receptor and inhibits GIP-induced insulin secretion in vitro and in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr extracellular domain (ECD) shows that the antibody binds through a series of hydrogen bonds from the complementarity-determining regions of Gipg013 Fab to the N-terminal α-helix of GIPr ECD as well as to residues around its highly conserved glucagon receptor subfamily recognition fold. The antibody epitope overlaps with the GIP binding site on the GIPr ECD, ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP.

Keywords: Antagonist; Antibody Engineering; Crystal Structure; Diabetes; G-Protein-coupled Receptors (GPCR); GIP Receptor (GIPr); Glucose-dependent Insulinotropic Polypeptide (GIP); Incretin; Phage Display.

PubMed Disclaimer

Figures

FIGURE 1.

FIGURE 1.

Antagonism of GIP-induced cAMP production in GIPr-overexpressing cell lines. Gipg013 and Gipg133 were characterized for GIPr antagonism in a cell-based cAMP HTRF assay, and data were plotted using nonlinear regression. A and B show antagonistic profiles from HEK293 cells overexpressing human, mouse, rat, and dog GIPr using Gipg013 and Gipg133 IgGs, respectively. C and D show antagonistic and agonistic profiles in the human GIPr assay for GIP(7–30), Pro3GIP, and GIP. Values have been normalized to the maximum activity of GIPr, which is defined by total cellular cAMP produced in the agonism assay or in the absence of peptide/IgG in the antagonism assay. Values shown are the mean ± S.E. (error bars) from duplicate wells, and data shown are representative of at least three separate experiments. Values shown are the mean ± S.E. from duplicate wells. ●, human; ■, mouse; ▾, rat; ▴, dog; ♦, isotype control IgG1; *, GIP; +, GIP(7–30); ×, Pro3GIP.

FIGURE 2.

FIGURE 2.

Antibody cell binding and inhibition of ligand binding. A, binding of Gipg013 to GIPr and related receptors on overexpressing cells. Shown is direct binding of 0.25 μg/ml Gipg013 IgG to human (h), dog (d), mouse (m), or rat (r) orthologs of GCGr, GLP-1r, or GIPr. Control for nonspecific binding on GIPr orthologs was NIP228_TM at 0.25 μg/ml. Values shown are the mean ± S.E. from duplicate wells, and data shown are representative of two separate experiments for the human receptors and a single experiment for the rodent and canine receptors. B, receptor ligand competition assay showing IC50 determination of Gipg013 IgG binding to GIPr-overexpressing cells. □, Gipg013; ▴, isotype control IgG1. Values shown are the mean ± S.E. (error bars) from duplicate wells, and data shown are representative of four separate experiments.

FIGURE 3.

FIGURE 3.

Analysis of Gipg013 antagonism of GIPr. A, GIP dose-response curves in the presence of Gipr013. The nonlinear regression plot of the dose-effect curve for GIP was determined in the presence of various concentrations of Gipg013: 3750 n

m

(▿), 1250 n

m

(▵), 417 n

m

(□), 139 n

m

(○), 46 n

m

(♦), 15 n

m

(▾), 5 n

m

(▴), 1.7 n

m

(■), and 0 n

m

(●). B, Schild plot analysis of dose-response curves. The Schild plot intersects the abscissa at p_A_2 (= KD). Values shown are the mean ± S.E. (error bars) from duplicate wells, and data shown are representative of three separate experiments.

FIGURE 4.

FIGURE 4.

Crystal structure of GIPr ECD and Gipg013 Fab complex. A, overall structure of the complex where GIPr ECD and Gipg013 Fab heavy chain and light chains are represented in gray, cyan, and magenta schematics, respectively (PDB code 4HJ0). B, GIPr ECD-GIP(1–42) (PDB code 2QKH) superposed on GIPr ECD-Gipg013 Fab crystal structure. GIPr ECDs overlap with a root mean square deviation of 0.799 Å2. GIP(1–42) and GIPr ECD (2QKH) are shown in orange and salmon schematics. C, glucagon family recognition fold is highly conserved. The three clusters (viz cluster 1 (Trp71, Val99, Arg101, and Trp109 in orange atomic color mode), cluster 2 (Trp39, Tyr42, and Phe65 in yellow atomic color mode), and cluster 3 (Tyr68, Pro85, Tyr87, Leu88, and Trp90 in cyan atomic color mode)) are a characteristic feature of class B GPCR N-terminal extracellular domain. Asp66 in green atomic color mode is a highly conserved residue involved in stabilizing the structure. The three disulfide links are shown in magenta atomic color mode. D, CDRs of heavy chain play a vital role in complex formation. H-CDR1 (Tyr32) creates a network of hydrogen bond interactions with Arg113 and His115 of GIPr ECD. It is further aided by Ser31 (H-CDR1) interacting with Glu119 and Asp107 (H-CDR3) with Tyr68 of GIPr ECD. E, L-CDR1 and L-CDR2 of Gipg013 Fab light chain make a series of hydrogen bond interactions with N-terminal α-helix of GIPr ECD. In D and E, GIPr ECD, Gipg013 Fab heavy chain (H-CDRs) and light chains (L-CDRs) are represented in white, cyan, and magenta atomic color modes, respectively.

FIGURE 5.

FIGURE 5.

Inhibition of GIP-induced insulin secretion. A and B show inhibition of GIP stimulation in a glucose-stimulated insulin secretion assay with dispersed rat islets. A, effect of GIP on top of the glucose-stimulated insulin secretion (open bars) and the inhibition of this effect with 600 n

m

Gipg013 (filled bars). B, the GIP enhanced insulin secretion was inhibited with Gipg013 at concentrations of both 300 and 600 n

m

(*, p < 0.05; **, p < 0.01; ***, p < 0.001). C, inhibition of GIP-induced insulin secretion in vivo. Insulin response was measured as the relative insulin AUC0–30 min between period 1 and 2 for GIP-induced insulin secretion in anesthetized rats, AUC1 before and AUC2 30 min after Gipg013 infusion. Insulin response = AUC2/AUC1 × 100. n = 6 for vehicle and n = 3 for Gipg013. *, p < 0.05 between vehicle and Gipg013. Error bars, S.E.

FIGURE 6.

FIGURE 6.

Gipg013 Fab exhibits high affinity toward GIPr ECD. Shown are footprints of GIP(1–42) and Gipg013 Fab on GIPr ECD domains. The buried surface area is greater in the GIPr ECD-Gipg013 Fab complex (see Table 4), and the interacting residues are labeled and shown on a green shaded surface. Only four residues are involved in hydrogen bond interactions with GIP(1–42).

References

    1. Brubaker P. L. (2007) Incretin-based therapies. Mimetics versus protease inhibitors. Trends Endocrinol. Metab. 18, 240–245 - PubMed
    1. Holst J. J., Vilsbøll T., Deacon C. F. (2009) The incretin system and its role in type 2 diabetes mellitus. Mol. Cell. Endocrinol. 297, 127–136 - PubMed
    1. Nauck M. A. (2009) Unraveling the science of incretin biology. Eur. J. Intern. Med. 20, S303–S308 - PubMed
    1. Hinke S. A., Manhart S., Pamir N., Demuth H., Gelling R. W., Pederson R. A., McIntosh C. H. (2001) Identification of a bioactive domain in the amino-terminus of glucose-dependent insulinotropic polypeptide (GIP). Biochim. Biophys. Acta 1547, 143–155 - PubMed
    1. Tseng C. C., Kieffer T. J., Jarboe L. A., Usdin T. B., Wolfe M. M. (1996) Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat. J. Clin. Invest. 98, 2440–2445 - PMC - PubMed

MeSH terms

Substances

LinkOut - more resources