title: Selection of non-competitive leptin antagonists using a random nanobody-based approach.
abstract: The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. Accumulating evidence suggests that leptin plays a role in human pathologies, such as autoimmune diseases and cancer, thus providing a rationale for the development of leptin antagonists. In the present study, we generated and evaluated a panel of neutralizing nanobodies targeting the LR (leptin receptor). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family. We identified three classes of neutralizing nanobodies targeting different LR subdomains: i.e. the CRH2 (cytokine receptor homology 2), Ig-like and FNIII (fibronectin type III) domains. Only nanobodies directed against the CRH2 domain inhibited leptin binding. We could show that a nanobody that targets the Ig-like domain potently interfered with leptin-dependent regulation of hypothalamic NPY (neuropeptide Y) expression. As a consequence, daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. All of these characteristics resemble the phenotype of leptin and LR-deficient animals. The results of the present study support proposed models of the activated LR complex, and demonstrate that it is possible to block LR signalling without affecting ligand binding. These nanobodies form new tools to study the mechanisms of BBB (blood-brain barrier) leptin transport and the effect of LR inhibition in disease models.
authors.affiliation: Flanders Institute for Biotechnology, Department of Medical Protein Research, Ghent University, Faculty of Medicine and Health Sciences, A. Baertsoenkaai 3, 9000 Ghent, Belgium.
authors.forename: Lennart
authors.initials: L
authors.lastname: Zabeau
authors.forename: Annick
authors.initials: A
authors.lastname: Verhee
authors.forename: Dominiek
authors.initials: D
authors.lastname: Catteeuw
authors.forename: Liesbeth
authors.initials: L
authors.lastname: Faes
authors.forename: Sylvie
authors.initials: S
authors.lastname: Seeuws
authors.forename: Tine
authors.initials: T
authors.lastname: Decruy
authors.forename: Dirk
authors.initials: D
authors.lastname: Elewaut
authors.forename: Frank
authors.initials: F
authors.lastname: Peelman
authors.forename: Jan
authors.initials: J
authors.lastname: Tavernier
mesh_terms: D000273:Adipose Tissue; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000494:pharmacology*; D002161:Camelids, New World; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D006946:Hyperinsulinism; D020738:Leptin / Q000037:antagonists & inhibitors*; D008099:Liver / Q000033:anatomy & histology; D051379:Mice; D008810:Mice, Inbred C57BL; D049329:Nanostructures / Q000737:chemistry*; D009478:Neuropeptide Y / Q000235:genetics / Q000378:metabolism; D009929:Organ Size; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054411:Receptors, Leptin / Q000037:antagonists & inhibitors*; D015398:Signal Transduction; D015430:Weight Gain
chemical_list: D000906:Antibodies; D020738:Leptin; D009478:Neuropeptide Y; D054411:Receptors, Leptin