Mouse Anti-GCGR Recombinant Antibody (clone mAb7)

A, mapping of amino acids required for mAb7 binding by alanine-scanning mutagenesis of GCGR ECD. F values (21) for individual amino acids in the αA helix (residues 27–46 (i)) and L4 (residues 72–94 (ii)) are graphed. The horizontal line represents the cutoff for F values (logF> 3) considered meaningful using this method. B, i, linear epitope map for mAb7 interactions with the ECD. Residues that have a calculated F values between 3 and 5 or>5 are labeled in wheat or magenta, respectively. *, phage libraries held Ala constant at this position; the natural residue at this position (Asp) is shown. ii, GCGR alanine mutations that impact mAb7 binding are mapped onto the surface of the GCGR ECD, colored as described in i and labeled. The boundary of the predicted glucagon-binding cleft (6) is highlighted with blue dots. C, mAb7 (blue) fails to inhibit glucagon-induced activation of full-length, human, G40S GCGR in 293 cell-based CRE-luciferase assays. Inhibition of GCGR by mAb1 (red) is shown as a control. D, Alphascreen competition assay measuring the ability of soluble WT or G40S GCGR ECD to compete with WT ECD bound to donor beads for binding to mAb7 (blue) or mAb1 (red) on acceptor beads.

Mukund, S., Shang, Y., Clarke, H. J., Madjidi, A., Corn, J. E., Kates, L.,... & Zhang, Y. (2013). Inhibitory mechanism of an allosteric antibody targeting the glucagon receptor. Journal of Biological Chemistry, jbc-M113.

(A) Antibodies block glucagon-induced PEPCK gene expression in human hepatocytes. Average IC50s (nM) from two experiments are 0.15, 1.5, and 0.4 for mAb1, mAb7, and mAb23, respectively. (B) Antibodies block 125I-glucagon binding to cells expressing GCGR. Kis (nM) are 5, 47, and 10 for mAb1, mAb7, and mAb23, respectively. The EC50 of glucagon binding is 70 nM. (C) Reduction of basal GCGR activity in cells expressing human GCGR by mAb23. (D) Alphascreen assay measuring the ability of ECD to compete with mAbs bound to acceptor beads for binding to full-length GCGR (dashed lines) or ECD (solid lines) bound to donor beads. IC50s (nM) of mAbs on full-length GCGR are 1.2 ± 0.2, 2.9 ± 1.0, and 0.2 ± 0.1, and on ECD are 1.9 ± 0.3, 3.6 ± 1.4, and 0.6 ± 0.2, for mAb1, mAb7, and mAb23, respectively. Data shown are from a single representative of three (A and C) or two (B and D) independent experiments. Error bars represent SD of duplicate or triplicate determinations.

Koth, C. M., Murray, J. M., Mukund, S., Madjidi, A., Minn, A., Clarke, H. J.,... & Rondon, J. (2012). Molecular basis for negative regulation of the glucagon receptor. Proceedings of the National Academy of Sciences, 201206734.