Progenipoietins: biological characterization of a family of dual agonists of fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor

Abstract

The progenipoietins, a class of engineered proteins containing both fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor receptor agonist activities, were functionally characterized in vitro and in vivo. Four representative progenipoietins were evaluated for receptor binding, receptor-dependent cell proliferation, colony-forming unit activity, and their effects on hematopoiesis in the C57BL/6 mouse.The progenipoietins bound to fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor with affinities within twofold to threefold of the native ligands, and each progenipoietin bound simultaneously to both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. The progenipoietins exhibited different levels of activity in receptor-dependent cell proliferation assays. The fetal liver tyrosine kinase-3-dependent cell proliferation activity of three of four progenipoietins was decreased sixfold to 33-fold relative to native fetal liver tyrosine kinase-3 ligand, while granulocyte colony-stimulating factor receptor-dependent activity of the progenipoietins was within twofold to threefold of native granulocyte colony-stimulating factor. At nonsaturating concentrations, the progenipoietins stimulated colony formation to a greater extent than the equimolar combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor. Treatment of mice with the progenipoietins yielded dramatic increases in peripheral blood and splenic white blood cells, polymorphonuclear leukocytes, and dendritic cells. These preclinical results demonstrate that the progenipoietins are potent hematopoietic growth factors that stimulate cells in a receptor-dependent manner. When administered in vivo, the progenipoietins effectively promote the generation of multiple cell lineages. Thus, in both in vitro and in vivo settings, the progenipoietins as single molecules exhibit the synergistic activity of the combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor.