Schneider H, Chaovapong W, Matthews D J, Karkaria C, Cass R T, Zhan H, Boyle M, Lorenzini T, Elliott S G, Giebel L B
Department of Receptor Biology, Arris Pharmaceutical Corp, South San Francisco, CA 94080, USA.
Blood. 1997 Jan 15;89(2):473-82.
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.
促红细胞生成素（EPO）刺激红系祖细胞的增殖和分化。多项证据表明，EPO激活促红细胞生成素受体（EPO-R）最可能的机制是红细胞前体表面受体的同型二聚化。因此，我们认为应该有可能产生能通过二聚化激活该受体从而模拟EPO作用的EPO-R单克隆抗体（MoAbs）。我们已经鉴定出这样一种针对EPO-R细胞外EPO结合域的激动剂MoAb（MoAb34）。这种二价IgG抗体触发EPO依赖细胞系的增殖并在体外诱导红系前体的分化。相比之下，不能使受体二聚化的单价Fab片段则完全无活性。通过同型二聚化激活受体的机制意味着在高配体浓度下，1:1受体/配体复合物的形成比2:1复合物更受青睐，从而使配体激动剂变成拮抗剂。因此，在细胞增殖和分化试验中，EPO和MoAb34在高浓度时应具有自我拮抗作用。我们的数据确实表明，EPO和MoAb34分别以约20和2 μmol/L的IC50值拮抗配体依赖的细胞增殖。在MoAb34浓度高于1 μmol/L时，红系集落形成（BFUe）受到抑制。此外，我们使用描述抗体介导的受体二聚化的数学模型分析了MoAb34与EPO-R的相互作用。增殖和分化活性的数据与该模型预测的细胞表面受体二聚体形成一致。
