Homodimerization of erythropoietin receptor by a bivalent monoclonal antibody triggers cell proliferation and differentiation of erythroid precursors.
Erythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Several lines of evidence indicate that the most likely mechanism of EPO receptor (EPO-R) activation by EPO is homodimerization of the receptor on the surface of erythrocyte precursors. Therefore, we argued that it should be possible to raise EPO-R monoclonal antibodies (MoAbs) that would activate the receptor by dimerization and thus mimic EPO action. We have identified such an agonist MoAb (MoAb34) directed against the extracellular EPO binding domain of the EPO-R. This bivalent IgG antibody triggers the proliferation of EPO-dependent cell lines and induces differentiation of erythroid precursors in vitro. In contrast, the monovalent Fab fragment, which cannot dimerize the receptor, is completely inactive. The mechanism of receptor activation by homodimerization implies that at high ligand concentrations the formation of 1:1 receptor/ligand complexes is favored over 2:1 complexes, thereby turning the ligand agonist into an antagonist. Thus, EPO and MoAb34 should self-antagonize at high concentrations in both cell proliferation and differentiation assays. Our data indeed demonstrate that EPO and MoAb34 antagonize ligand-dependent cell proliferation with IC50 values of approximately 20 and 2 mumol/L, respectively. Erythroid colony formation (BFUe) is inhibited at MoAb34 concentrations above 1 mumol/L. Furthermore, we analyzed the MoAb34:EPO-R interaction using a mathematic model describing antibody-mediated receptor dimerization. The data for proliferation and differentiation activity were consistent with the receptor dimer formation on the cell surface predicted by the model.
Schneider
H
H
Department of Receptor Biology, Arris Pharmaceutical Corp, South San Francisco, CA 94080, USA.
Chaovapong
W
W
Matthews
D J
DJ
Karkaria
C
C
Cass
R T
RT
Zhan
H
H
Boyle
M
M
Lorenzini
T
T
Elliott
S G
SG
Giebel
L B
LB
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D019281:Dimerization; D004900:Erythroblasts / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D017467:Receptors, Erythropoietin / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects* / Q000276:immunology
D000911:Antibodies, Monoclonal; D017467:Receptors, Erythropoietin