An Immunodominant Epitope in a Functional Domain Near the N-Terminus of Human Granulocyte-Macrophage Colony-Stimulating Factor Identified by Cross-Reaction of Synthetic Peptides with Neutralizing Anti-Protein and Anti-Peptide Antibodies

Abstract

We produced polyclonal and monoclonal antibodies (MAbs) against recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and performed studies of epitope mapping by ELISA, using five synthetic peptides corresponding to sequences along this molecule. Additionally, anti-peptide MAbs were generated. The antibody ability to inhibit rhGM-CSF activity was determined using as bioassay the MO7e cell line, which is dependent on hGM-CSF for growth in vitro. An immunodominant epitope able to induce the highest neutralization antibody titers was identified near the N terminus of hGM-CSF. A synthetic peptide 14-24, homologous to a sequence including part of the first alpha-helix of the molecule, was recognized by neutralizing anti-protein antibodies. Similarly, MAbs anti- 14-24 cross-reacted with rhGM-CSF and specifically blocked its function. Replacement of Val16 or Asn17 with alanine greatly reduced the antibody-binding capacity to peptide 14-24, whereas substitution of Gln20 or Glu21 was less critical. Monoclonal antibodies generated against residues 30-41 (corresponding to an intrahelical loop) and 79-91 (homologous to a sequence including part of the third alpha-helix) or its analog [Ala88](79-91)beta Ala-Cys, were conformation dependent and nonneutralizing: they failed to react or bound poorly to rhGM-CSF in ELISA, but readily recognized the homologous sequence in the denatured protein, by Western blotting.

Identification of an Antigenic Domain Near the C Terminus of Human Granulocyte-Macrophage Colony-stimulating Factor and Its Spatial Localization

“…Only one of the two sera (labeled 813) showed an appreciable specific affinity for the synthetic peptide-(102-121) in direct ELISA. Instead, both sera contained Abs able to bind the synthetic peptide-(14 -24), analogous of the hGM-CSF N-terminal region, confirming our previous results (26). Of these two sera, neither contained Abs specific for the peptide-(55-65), analogous to the region including the second ␣-helix of the hGM-CSF.…”

“…Previous results showed immunogenicity of the synthetic peptide-(14 -24), analogous to the region 14 -24 of the hGM-CSF N terminus (26). Anti-rhGM-CSF antisera produced in our laboratory did not contain antibodies against the region comprising the second of the four ␣-helices of hGM-CSF.…”

“…Consequently, the destruction of the ␣-helix conformation in the analogous region of the murine GM-CSF (with the insertion of a proline) does not inhibit the biological activity of recombinant protein (58). Other experiments suggested that a region near the fourth ␣-helix has a partial reactivity with monoclonal antibodies produced against the whole recombinant hGM-CSF molecule (26,52,59).…”

“…2A), which may be involved with the interaction of the polypeptide with specific neutralizing Abs of hyperimmune sera (26). Here we concentrated on the region around the ␣-helix D, characterized by a hydrophilic N terminus and a hydrophobic C terminus (Fig.…”

“…Previous studies (22)(23)(24)(25)(26)(27)(28) using synthetic peptides and neutralizing antibodies, substitution, and deletion mutants and interspecies chimeric polypeptides have indicated several areas whose integrity is fundamental to maintaining the normal function of the growth factor.…”