Modulation of the Binding Affinity of Myelopoietins for the Interleukin-3 Receptor by the Granulocyte Colony-Stimulating Factor Receptor Agonist William F. Hood,* Yiqing G. Feng, Roger J. Schilling, Yatin Gokarn, Cindy Jarvis, Edward E. Remsen, Jeng-Jong J. Shieh, William Joy, Barbara K. Klein, Joseph O. Polazzi, Joseph K. Welply, John P. McKearn, and Joseph B. Monahan Searle Research and DeVelopment, Pharmacia Corporation, 700 Chesterfield Parkway North, St. Louis, Missouri 63198 ReceiVed March 23, 2001; ReVised Manuscript ReceiVed July 23, 2001 ABSTRACT: Myelopoietins (MPOs) are a family of recombinant chimeric proteins that are both interleukin-3 (IL-3) receptor and granulocyte colony-stimulating factor (G-CSF) receptor agonists. In this study, MPO molecules containing one of three different IL-3 receptor agonists linked with a common G-CSF receptor agonist have been examined for their IL-3 receptor binding characteristics. Binding to the R-subunit of the IL-3 receptor revealed that the affinity of the MPO molecules was 1.7-3.4-fold less potent than those of their individual cognate IL-3 receptor agonists. The affinity decrease was reflected in the MPO chimeras having approximately 2-fold slower dissociation rates and 2.7-5.5-fold slower association rates than the corresponding specific IL-3 receptor agonists alone. The affinity of binding of the MPO molecules to the heteromultimeric R IL-3 receptor expressed on TF-1 cells was either 3-, 10-, or 42-fold less potent than that of the individual cognate IL-3 receptor agonist. Biophysical data from nuclear magnetic resonance, near-UV circular dichroism, dynamic light scattering, analytical ultracentrifugation, and size exclusion chromatography experiments determined that there were significant tertiary structural differences between the MPO molecules. These structural differences suggested that the IL-3 and G-CSF receptor agonist domains within the MPO chimera may perturb one another to varying degrees. Thus, the differential modulation of affinity observed in IL-3 receptor binding may be a direct result of the magnitude of these interdomain interactions. Interleukin-3 (IL-3) 1 and granulocyte colony-stimulating factor (G-CSF) are cytokines which stimulate the prolifera- tion and differentiation of hematopoietic cells. IL-3, or multicolony stimulating factor, promotes the differentiation of multipotent progenitor cells into the granulocyte, mono- cyte, erythroid, and megakaryocyte lineages (1, 2). G-CSF, on the other hand, specifically stimulates the proliferation, differentiation, and maturation of myeloid progenitor cells of the neutrophil lineage (3). When used in combination, IL-3 and G-CSF have been shown to have an advantage over using either of them alone in the treatment of neutropenia and thrombocytopenia as a result of cancer chemotherapy, bone marrow transplant, or congenital defects (4-7). My- elopoietin (MPO) proteins, which have been engineered to contain both IL-3 and G-CSF receptor agonist activities, demonstrate synergistic hematopoietic activities that exceed those observed by a combination of G-CSF and IL-3 (8). MPO proteins were shown to be superior to the two single molecules in both cellular (colony-forming unit activity in CD34 + cells derived from human bone marrow) and in vivo (mobilization of peripheral blood progenitor cells in non- human primates) assays (9, 10). Furthermore, clinical trials have indicated that the MPO molecules are well-tolerated in humans and are effective in mobilizing neutrophil and multilineage precursors in patients undergoing chemotherapy (11-13). Single molecules possessing dual agonist activity have been described previously. Those molecules have variable activity compared with the individual agonists (14-19). For example, a chimera of IL-3, bombyxin, and human insulin- like growth factor II was reported to be 25-30 times more active than IL-3 in a proliferation assay of the human proerythroleukemic cell line TF-1 (14). PIXY321, in which IL-3 and GM-CSF were combined (15), exhibited enhanced receptor affinity and in vitro proliferative activity compared to either cytokines alone or in combination. In contrast, when IL-3 and erythropoietin were fused (16), no enhancement of erythropoiesis over that observed with the combination of the individual cytokines could be shown. These prior studies demonstrate that the biological activity and receptor binding activity of the dual receptor agonist proteins depend on the complementarity of the biological profiles of the cytokines, as well as the linker and the orientation, i.e., the sequential order, of the cytokines that are engineered into the chimeras. * To whom correspondence should be addressed. Phone: (636) 737- 6564. Fax: (636) 737-6599. E-mail: william.f.hood@pharmacia.com. 1 Abbreviations: MPO, myelopoietin; IL-3, interleukin-3; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte/macro- phage colony-stimulating factor; IL-5, interleukin-5; CD, circular dichroism; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; RAMFc, rabbit anti-mouse Fc; HEPES, N-(2-hydroxyethyl)- piperazine-N′-2-ethanesulfonic acid; HBS, HEPES-buffered saline; PBS, phosphate-buffered saline; NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum coherence; SEC, size exclusion chroma- tography; DLS, dynamic light scattering; SE, sedimentation equilibrium; SV, sedimentation velocity. 13598 Biochemistry 2001, 40, 13598-13606 10.1021/bi010590t CCC: $20.00 © 2001 American Chemical Society Published on Web 10/17/2001