From Agonist To Antagonist: Fab Fragments of an Agonist-Like Monoclonal Anti-β₂-Adrenoceptor Antibody Behave as Antagonists

Abstract

We previously demonstrated that the monoclonal antibody Mab6H8 raised against the second extracellular loop of the beta(2)-adrenoceptor (beta(2)-AR) had an agonist-like activity, mediated by the activation of L-type Ca(2+) channels by protein kinase A through the adenylyl cyclase pathway. We suggested that this Mab acts by stabilizing an active dimeric conformation of the beta(2)-AR. To substantiate this hypothesis, we prepared monomeric Fab fragments of Mab6H8. Comparison of the physicochemical parameters of antigen interaction with both the Mab and its Fab fragments were determined by surface plasmon resonance, showing a 5- to 10-fold lower affinity of the fragments compared with the bivalent antibody. We determined the biological activity of antibody and Fab fragments in two systems: spontaneous beating neonatal rat cardiomyocytes to study the chronotropic effects and isolated guinea pig cardiomyocytes to study L-type Ca(2+) channel activation. Fab fragments as such had no "agonist-like" effects in both systems but inhibited receptor activation with the beta(2)-specific agonist clenbuterol. Addition of a cross-linking rabbit anti-mouse IgG restored the agonist-like effect of the Fab fragments. These results suggest that Fab fragments induce a conformational change in the receptor, inhibiting the accessibility of the pharmacophore pocket to clenbuterol. Dimerization of this receptor conformation induces an agonist-like effect. Antireceptor antibodies can thus act both as agonist in the dimeric state and as antagonist in the monomeric state.

Monitoring Receptor Oligomerization Using Time-resolved Fluorescence Resonance Energy Transfer and Bioluminescence Resonance Energy Transfer

“…Indeed, a monoclonal anti-␤ 2 -adrenoreceptor antibody with agonist-like properties has been described in which Fab fragments behave as antagonists (35). This is at least consistent with the idea that clustering of receptors might be required for signal transduction.…”

Ligation of cancer cell surface GRP78 with antibodies directed against its COOH-terminal domain up-regulates p53 activity and promotes apoptosis

“…Similar behavior also has been reported for antibodies to other cell surface resident proteins (38)(39)(40)(41)(42)(43). We do not precisely understand the mechanism by which antibodies against the two terminal domains of GRP78 elicit two opposite cellular responses.…”

Geldanamycin stimulates internalization of ErbB2 in a proteasome-dependent way

“…Even monovalent Fab-fragments could potentially induce conformational changes or affect protein interactions (Agus et al, 2002;Hipfner et al, 1999;Mijares et al, 2000). This emphasizes the need for non-invasive assays such as the one we have described here when studying receptor internalization.…”