Neutralizing and nonneutralizing monoclonal antibodies to the human granulocyte-macrophage colony-stimulating factor receptor alpha-chain.
A panel of monoclonal antibodies was raised against the low-affinity human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor alpha-chain expressed as recombinant protein on murine FDC-P1 cells. All the selected antibodies were of the IgG2A isotype and bound to protein A. They each recognized both native and recombinant receptors by indirect surface immunofluorescence and by immunoprecipitation. Several of the antibodies also recognized presumably denatured receptors as detected by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three different epitopes on the extracellular domain of the GM-CSF receptor alpha-chain were defined by these antibodies, and two of the epitopes did not appear to be involved in binding hGM-CSF or in interactions with the beta-chain of the GM-CSF receptor that are required for high-affinity binding of GM-CSF. On the other hand, the epitope recognized by antibody 2B7-17-A appeared to be critically involved in the binding of GM-CSF because this antibody completely abrogated both high- and low-affinity binding of GM-CSF to native and recombinant receptors. Antibody 2B7-17-A had a relatively high affinity for the GM-CSF receptor alpha-chain (kd = 3 nmol/L) and slow dissociation kinetics (kd = 0.002 min-1). These properties made the 2B7-17-A antibody a potent inhibitor of hGM-CSF biologic action in several different bioassays, with a half-maximal inhibitory dose of about 6 nmol/L (1 microgram/mL). This antibody could prove useful in alleviating any pathologic states mediated by excess GM-CSF levels and in defining the domains of the GM-CSF receptor required for ligand binding.
Walter and Eliza Hall Institute of Medical Research, Ludwig Institute for Cancer Research-Melbourne Tumour Biology Branch, Parkville, Australia.
N A
NA
Nicola
K
K
Wycherley
A W
AW
Boyd
J E
JE
Layton
D
D
Cary
D
D
Metcalf
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D004558:Electric Stimulation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D009500:Neutralization Tests; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins