From agonist to antagonist: Fab fragments of an agonist-like monoclonal anti-beta(2)-adrenoceptor antibody behave as antagonists.

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.