From Agonist To Antagonist: Fab Fragments of an Agonist-Like Monoclonal Anti-β2-Adrenoceptor Antibody Behave as Antagonists
- 1Laboratorio de Permeabilidad Iónica, Centro de Biofı́sica y Bioquı́mica, Instituto Venezolano de Investigaciones Cientı́ficas, Caracas, Venezuela (A.M.);2Département de Biochimie et Groupe de recherche sur le Système Nerveux Autonome, Université de Montréal, Canada (D.L.); 3Max Delbrück Center for Molecular Medicine, Berlin, Germany (G.W.); and 4UPR9021 du Centre National de la Recherche Scientifique, Institute for Molecular and Cellular Biology, Strasbourg, France (J.H.)
Abstract
We previously demonstrated that the monoclonal antibody Mab6H8 raised against the second extracellular loop of the β2-adrenoceptor (β2-AR) had an agonist-like activity, mediated by the activation of L-type Ca2+channels by protein kinase A through the adenylyl cyclase pathway. We suggested that this Mab acts by stabilizing an active dimeric conformation of the β2-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 Ca2+ channel activation. Fab fragments as such had no “agonist-like” effects in both systems but inhibited receptor activation with the β2-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.
Footnotes
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Send reprint requests to: Dr. Alfredo Mijares, Centro de Biofı́sica y Bioquı́mica, Instituto Venezolano de Investigaciones Cientı́ficas, Apartado 21827, Caracas 1020A, Venezuela. E-mail:amijares{at}cbb.ivic.ve
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This study was partially supported by Grants S1-97001675 and S3-98002244 from Consejo Nacional de Investigaciones Cientı́ficas y Technologicas-Venezuela. This work was supported by a grant from the European Community (BMH4-CT95-1008) and a convention between Consejo Nacional de Investigaciones Cientı́ficas y Technologicas and the Centre National de la Recherche Scientifique (PI-98003457).
- Abbreviations:
- β2-AR
- β2-adrenoreceptor
- ICI 118,551
- β2-antagonist (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3[(1-methylethyl)amino]-2-butanol hydrochloride
- Mab6H8
- monoclonal antibody against a peptide corresponding to the second extracellular loop of the human β2-AR
- Fab
- monomeric fragments of antibody
- β2-H19C
- peptide corresponding to the second extracellular loop of the human β2-AR (amino acids 172-190)
- Fura-AM
- Fura 2 acetoxymethyl ester
- Req
- relative response bound at equilibrium
- KA
- association constant
- kobs
- association rate
- kon
- association rate constant
- koff
- dissociation rate constant
- KI
- inhibition constant
- PKA
- protein kinase A
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- Received October 7, 1999.
- Accepted May 5, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
