Identification of a monoclonal antibody against the leptin receptor that acts as an antagonist and blocks human monocyte and T cell activation

Mehdi Fazeli, Hamid Zarkesh-Esfahani, Zida Wu, Mabrouka Maamra, Martin Bidlingmaier, A. Graham Pockley, Philip Watson, Giuseppe Matarese, Christian J. Strasburger, Richard J M Ross

Research output: Contribution to journalArticle

Abstract

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-α 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.

Original languageEnglish
Pages (from-to)190-200
Number of pages11
JournalJournal of Immunological Methods
Volume312
Issue number1-2
DOIs
Publication statusPublished - May 30 2006

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Leptin Receptors
Leptin
Monocytes
Monoclonal Antibodies
T-Lymphocytes
Immunoglobulin Fab Fragments
Biological Assay
Nutritional Status
Cytokines
Escherichia coli

Keywords

  • Antagonist
  • Antibodies
  • antigen binding fragment
  • c-sis inducible element
  • Cytokine receptors
  • Fab
  • Jak/STAT
  • Janus kinase/signal transducers and activators of transcription
  • leptin
  • leptin receptor
  • mAb
  • MAPK
  • mitogen activated protein kinase
  • monoclonal antibody
  • Monocytes
  • ob
  • ObR
  • PBMC
  • peripheral blood mononuclear cell
  • SIE
  • T cells
  • TNF-α
  • tumor necrosis factor alpha.

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Identification of a monoclonal antibody against the leptin receptor that acts as an antagonist and blocks human monocyte and T cell activation. / Fazeli, Mehdi; Zarkesh-Esfahani, Hamid; Wu, Zida; Maamra, Mabrouka; Bidlingmaier, Martin; Pockley, A. Graham; Watson, Philip; Matarese, Giuseppe; Strasburger, Christian J.; Ross, Richard J M.

In: Journal of Immunological Methods, Vol. 312, No. 1-2, 30.05.2006, p. 190-200.

Research output: Contribution to journalArticle

Fazeli, M, Zarkesh-Esfahani, H, Wu, Z, Maamra, M, Bidlingmaier, M, Pockley, AG, Watson, P, Matarese, G, Strasburger, CJ & Ross, RJM 2006, 'Identification of a monoclonal antibody against the leptin receptor that acts as an antagonist and blocks human monocyte and T cell activation', Journal of Immunological Methods, vol. 312, no. 1-2, pp. 190-200. https://doi.org/10.1016/j.jim.2006.03.011
Fazeli, Mehdi ; Zarkesh-Esfahani, Hamid ; Wu, Zida ; Maamra, Mabrouka ; Bidlingmaier, Martin ; Pockley, A. Graham ; Watson, Philip ; Matarese, Giuseppe ; Strasburger, Christian J. ; Ross, Richard J M. / Identification of a monoclonal antibody against the leptin receptor that acts as an antagonist and blocks human monocyte and T cell activation. In: Journal of Immunological Methods. 2006 ; Vol. 312, No. 1-2. pp. 190-200.
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AU - Fazeli, Mehdi

AU - Zarkesh-Esfahani, Hamid

AU - Wu, Zida

AU - Maamra, Mabrouka

AU - Bidlingmaier, Martin

AU - Pockley, A. Graham

AU - Watson, Philip

AU - Matarese, Giuseppe

AU - Strasburger, Christian J.

AU - Ross, Richard J M

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N2 - 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-α 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.

AB - 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-α 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.

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