Inhibition of STAT5 Phosphorylation by Anti-GM-CSF Monoclonal Antibodies: Validation of a New Whole Blood Assay

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates survival, proliferation, and differentiation of monocytes/macrophages. The GM-CSF receptor (CD116) is expressed on primary monocytes and mediates cell signaling through STAT5 phosphorylation. Therefore STAT5 is central to GM-CSF action. BioCytex has developed a new semi-quantitative flow cytometry (FC) assay to measure the phosphorylation status of STAT5 in human primary monocytes and lymphocytes in whole blood samples. The assay was used to investigate the potency of recombinant monoclonal antibodies (MAbs) for the neutralization of human GM-CSF.

Engineered anti-GM-CSF MAbs were generated and characterized by Merck Serono. Several clones and preparation were tested, including MAbs 343 (low and high LPS), 467; 340; 339; 366, and MAb 285 as isotype control. Their different in vitro potencies for blocking recombinant GM-CSF activity in stable cell lines was determined using the TF-1 proliferation and IL-8 secretion in U937 cell assays. Whole blood FC assay was designed and validated at BioCytex using either a rabbit MAb or a rabbit polyclonal antibody specific for phospho-STAT5 (Tyr694). Monocytes were gated using a PE-labeled anti-CD14 probe. Inhibition of STAT5 phosphorylation by anti-GM-CSF MAbs was performed using recombinant GM-CSF at EC90.

EC90 of GM-CSF was determined at 2.3 pM from n=5 healthy volunteers. All GM-CSF specific MAbs inhibited STAT5 phosphorylation in monocytes, whereas the control (MAb 285) did not. More interestingly, there was a marked effect of LPS content since inhibition of STAT5 phosphorylation was not completed in presence of high LPS load (MAb 343). IC50 and IC90 for MAbs 339 and 366 were 10-fold increased as compared to MAbs 467 and 340. IC50 were ranging from 0.12 to 0.19 nM for clones 343 (low LPS), 467 and 340. IC50 and IC90 for clone 339 were 0.016 and 0.034 nM, respectively.

Inhibition of GM-CSF mediated monocyte activation by neutralizing MAbs can be monitored in a whole blood assay. STAT5 phophorylation could be viewed as a potential biomarker for GM-CSF receptor activation. The cell-signaling inhibition potency in primary cells correlated with in vitro potency using stable cell lines. In summary, the new validated FC could be used for research and clinical development purposes.

Grivel:LFB Biotechnologies: Consultancy. Magnenat:Merck Serono S.A.: Employment. Moulard:BioCytex: Consultancy, Employment.