Identification of a monoclonal antibody against the leptin receptor that acts as an antagonist and blocks human monocyte and T cell activation.
Nutritional status has a major impact on the immune response and this is in part mediated by leptin, a pro-inflammatory cytokine. Preliminary data suggest that antagonism of leptin may offer a therapeutic approach for the treatment of some inflammatory disorders. We have tested monoclonal antibodies (mAbs) to the human leptin receptor (ObR) for antagonist activity using a leptin signalling bioassay. We identified a mAb, 9F8, which demonstrated dose-dependent antagonist activity in the leptin bioassay. Specificity of the mAb for ObR was confirmed using a plate binding assay. The 9F8 mAb displaced leptin binding to human ObR and enzymatically generated Fab fragments of 9F8 retained antagonist activity. Therefore the Fab fragment of 9F8 was cloned and recombinant 9F8 Fab (rFab) was purified from E. coli periplasmic fraction using a C-terminal His tag. Purified 9F8 rFab bound to human ObR and exhibited leptin antagonist activity. In vitro studies demonstrated that the 9F8 mAb inhibited leptin induced TNF-alpha production from human monocytes and anti-CD3 mAb induced proliferation of human T cells in PBMC culture. In conclusion, this study has identified a mAb to the human leptin receptor which inhibits leptin signalling and acts as a leptin antagonist in vitro.
Fazeli
Mehdi
M
Division of Clinical Sciences (North), University of Sheffield, Sheffield S5 7AU, UK.
Zarkesh-Esfahani
Hamid
H
Wu
Zida
Z
Maamra
Mabrouka
M
Bidlingmaier
Martin
M
Pockley
A Graham
AG
Watson
Philip
P
Matarese
Giuseppe
G
Strasburger
Christian J
CJ
Ross
Richard J M
RJ
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D004306:Dose-Response Relationship, Immunologic; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics; D008213:Lymphocyte Activation / Q000187:drug effects; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors* / Q000276:immunology*; D054411:Receptors, Leptin; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; C515448:LEPR protein, human; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha