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

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.

Clinical-scale production of granulocyte progenitor and post-progenitor cells using daniplestim, leridistim, Progenipoietin, Promegapoietin and autologous plasma.

BACKGROUND: Supplementation of PBPC autografts with ex vivo expanded PBMC may significantly reduce or eliminate the period of neutropenia associated with high-dose chemotherapy. METHODS: Unmanipulated growth-factor mobilized PBMC were expanded in media containing daniplestim, leridistim, Promegapoietin, and Progenipoietin (DLPP) and 2% autologous plasma at 4 x 10(5) PBMC/mL, first in 25 cm(2) T-flasks, with sampling on Days 7, 10, 13 and 15, and then in 1264 cm(2) Nunclon Cell Factories, with sampling on Days 7 and 13. RESULTS: In T25-flasks, maximal CFU-GM expansion ([38.2 +/- 9.5]-fold) occurred on Day 10, whereas maximal total cell expansion ([6.7 +/- 1.1]-fold) occurred on Day 15. Production of CD15(+)CD11b(-) and CD15(+)CD11b(+) granulocytic post-progenitors (3.0 +/- 0.4 x 10(6) and 3.7 +/- 0.9 x 10(6), respectively) was also maximal at Day 15. Compared with the previously studied combination of Flt3L, PIXY321, G-CSF, GM-CSF and Epo, the DLPP cocktail performed similarly, with the exception of yielding larger GM colonies at Day 10 and fewer granulocyte post-progenitors on Day 15. In Cell Factories, CFU-GM were expanded (31.6 +/- 14.5)-fold, while total nonadherent cells were expanded (2.6 +/- 0.5)-fold. The two stack Cell Factory cultures seeded with 1.0 x 10(8) unselected PBMC produced approximately 3.3 x 10(6) CFU-GM and 1.3 x 10(8) myeloid post-progenitors. DISCUSSION: Whereas expansion of cell numbers, CFU-GM and granulocytic post-progenitors in Cell Factories mirrored that achieved in T25-flasks, future preclinical studies with the DLPP cytokine combination may be performed in small volumes, with subsequent translation to the larger volume Cell Factories. Sufficient expansion can be achieved using the DLPP cytokine combination in the Cell Factories to provide the numbers of progenitors required for clinical trials.