Identification of two potential receptor-binding sites for hGM-CSF

Abstract

Two receptor-binding sites for hGM-CSF are described. Competitive binding ELISA using four monoclonal antibodies (MAbs) showed different epitope recognitions. The antibody combining sites were mapped using sets of overlapping peptides and hexapeptide libraries prepared by the SPOT synthesis technique. We identified the conformationally dependent epitopes A18E21R23R24F119 and R23E21N17W13 bound by MAb CC5B5 and the nonlinear epitope P118F119W13E14 bound by MAb M1B8. The epitopes recognized by these two MAbs are very closely located on the native protein surface. The peptide L61YKQGKLRGSLTK72 was recognized by MAb M7E10 and the peptide A1PAR4, representing the N-terminal sequence of the protein, was bound by the nonneutralizing MAb CC1H7. Inhibition assays of the GM-CSF biological activity demonstrated that MAb M1B8, CC5B5 and M7E10 bind to domains which are responsible for the interaction of the cytokine with the GM-CSF receptor

Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti-human GM-CSF antibody

“…17 Eberhardt and coworkers used a panel of monoclonal antibodies targeting GM-CSF to characterize their epitope specificities. 47 To identify discontinuous epitopes, they used combinatorial hexapeptide libraries and peptide scans. They could identify the conformation-dependent epitopes from helix A and the C-terminal tail of GM-CSF A18-E21-R23-R24-F119 and R23-E21-N17-W13 being bound by a moderately neutralizing monoclonal antibody called CC5B5.…”