Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor α-chain and functions as a specific IL-3 receptor antagonist

Abstract

The human interleukin-3 receptor (IL-3R) is expressed on myeloid, lymphoid, and vascular endothelial cells, where it transduces IL-3-dependent signals leading to cell activation. Although IL-3R activation may play a role in hematopoiesis and immunity, its aberrant expression or excessive stimulation may contribute to pathologic conditions such as leukemia, lymphoma, and allergic reactions. We describe here the generation and characterization of a monoclonal antibody (MoAb), 7G3, which specifically binds to the IL-3R α-chain and completely abolishes its function. MoAb 7G3 immunoprecipitated and recognized in Western blots the IL-3R α-chain expressed by transfected cells and bound to primary cells expressing IL- 3Rα. MoAb 7G3 bound the IL-3R α-chain with a k(d) of 900 pmol/L and inhibited 125I-IL-3 binding to high- and low-affinity receptors in a dose- dependent manner. Conversely, IL-3 but not granulocyte-macrophage colony- stimulating factor (GM-CSF) inhibited 125I-7G3 binding to high- and low- affinity IL-3Rs, indicating that MoAb 7G3 and IL-3 bind to common or adjacent sites. In keeping with the inhibition of IL-3 binding, MoAb 7G3 antagonized IL-3 biologic activities, namely stimulation of TF-1 cell proliferation, basophil histamine release, and IL-6 and IL-8 secretion from human endothelial cells. Two other anti-IL-3R α-chain MoAbs failed to inhibit IL- 3 binding or function. Epitope mapping experiments using truncated IL-3R α- chain mutants and IL-3Rα/GM-CSFRα chimeras revealed that 31 amino acids in the N-terminus of IL-3Rα were required for MoAb 7G3 binding. MoAb 7G3 may be of clinical significance for antagonizing IL-3 in pathologic conditions such as some myeloid leukemias, follicular B-cell lymphoma, and allergy. Furthermore, these results implicate the N-terminal domain of IL-3Rα in IL- 3 binding. Since this domain is unique to the IL-3/GM-CSF/IL-5 receptor subfamily, it may represent a novel and common binding feature in these receptors.