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

OGGERO EBERHARDT, M.; FRANK, R.; KRATJE, R. B.; ETCHEVERRIGARAY, M.

Santa Fe. Pcia. Santa Fe. Argentina

Congreso; 3rd Mercosur Congress on Process Systems Engineering – 1st Mercosur Congress on Chemical Engineering (ENPROMER 2001); 2001

Facultad de Ingeniería Química (UNL); CERIDE (CONICET); INTEC (UNL-CONICET); INGAR (CONICET)

Human Granulocyte Macrophage Colony Stimulating Factor (hGM CSF) is a member of the four helix bundle family of cytokines that regulates the proliferation and differentiation of hematopoietic progenitor cells. It has potential clinical utility enhancing the rate of hematopoietic recovery after cancer chemoterapy. The GM-CSF biological activity is exerted by a high affinity cell surface receptor (R). It consist of a ligand specific α subunit that confers low affinity binding and a β subunit which can also associate with interleukin 3 and 5. Here, we describe two receptor binding sites using four monoclonal antibodies (MAbs) against E.coli derived GM CSF (MAbs 1B8, CC5B5, 7E10 and CC1H7). Biological activity inhibition assays of GM CSF demostrated that MAbs 1B8, CC5B5 and 7E10 bind cytokine domains which are responsible for the interaction with GM-CSFR. The antibody combining sites were mapped using sets of overlapping peptides and hexapeptide libraries prepared by SPOT synthesis technique. We identified the conformational dependent epitopes A18E21R23R24F119 and R23E21N17W13 bound by CC5B5 and the non linear epitope P118F119W13E14 bound by MAb 1B8. The epitopes recognized by these two MAbs are very closely located on the native protein surface. The peptide L61YKQGKLRGSLTK72 was recognized by MAb 7E10 and the peptide sequence A1PAR4, representing the N terminal sequence of the protein, was bound by the non neutralizing MAb CC1H7. The antibodies 1B8 and CC5B5, whose binding domain include aminoacids from helix A and from the carboxy terminal tail of the molecule, showed lower neutralizing ability that antibody 7E10 whose binding domain is composed by the last residues of helix B and the initial 9 residues of the loop that join helix B with helix C. Further experiments, based on neutralization of biological activity using CHO (Chinesse Hamster Ovary) cells derived GM CSF, confirmed the identification of two GM CSFR binding sites involved in the cytokine biological activity.