Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization.
Recombinant human erythropoietin (rhuEpo)-specific mouse monoclonal antibodies (MoAbs) have been produced and characterized. All antibodies were specifically reactive with rhuEpo in enzyme-linked immunosorbent assay (ELISA). Epitope exclusion studies showed three distinct epitope regions, A, B, and C, recognized by neutralizing MoAbs. An additional epitope region D was recognized by non-neutralizing MoAbs. Antibodies defining an epitope region competed with each other for binding sites, but did not compete with antibodies defining a different epitope region. Group B antibodies were able to compete for the receptor binding site on rhuEpo with a soluble human Epo-receptor-lg fusion protein. No single peptide sequences were found to specifically interact either with group B MoAbs or with the rhuEpo-receptor. Therefore, it is suggested that epitope region B and the receptor binding site share binding determinants that are primarily composed of conformational epitopes. Because group A and group C antibodies did not compete with the receptor for binding to the receptor binding site of the rhuEpo molecule, it is suggested that neutralization via epitope regions A and C is mediated through binding inhibition caused by conformational changes, transmuting the binding site(s) for the receptor. Conversely, binding to the receptor seems to induce conformational changes in the hormone molecule, eliminating epitopes for group A and C antibodies.
Fibi
M R
MR
Department of Preclinical Development of Therapeutics, BEHRINGWERKE AG, Marburg, Germany.
Aslan
M
M
Hintz-Obertreis
P
P
Pauly
J U
JU
Gerken
M
M
Lüben
G
G
Lauffer
L
L
Siebold
B
B
Stüber
W
W
Nau
G
G
D000818:Animals; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D004921:Erythropoietin / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D009500:Neutralization Tests; D017467:Receptors, Erythropoietin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004921:Erythropoietin