Interleukin-3/erythropoietin fusion proteins: in vitro effects on hematopoietic cells.
Erythropoietin (Epo) acts synergistically with interleukin-3 (IL-3) to induce proliferation and differentiation of erythroid progenitors. This synergy occurs at IL-3 concentrations that have little or no effect alone. To determine whether optimal expansion of erythroid cells results when they are targeted by a molecule with both IL-3 and Epo activities, fusion proteins were generated and analyzed. Expression vectors were constructed in which the coding regions of human IL-3 and Epo cDNAs were joined by either a short (2 to 3 amino acids) or long (23 amino acids) linker sequence and expressed in Chinese hamster ovary (CHO) cells. Analysis of equilibrium binding properties of the IL-3 and Epo moieties revealed that in all fusion proteins each retained the ability to bind receptor. When IL-3 was connected to Epo by a short linker, the binding affinity of the IL-3 moiety was lower. In vitro proliferative activity of each moiety was observed on cell lines responsive to IL-3, Epo or a combination of the two cytokines. Fusion of IL-3 to Epo through its amino terminus was found to result in partial loss of its function. All the fusion proteins were biologically active on human bone marrow. When IL-3 was located at the amino domain of the protein, induction of erythroid colonies was similar to that of a mixture of IL-3 and Epo. These results indicate that biological integrity of both IL-3 and Epo can be maintained when these cytokines are fused, but that enhancement of erythropoiesis over that observed with a mixture of the two cytokines cannot be achieved by their fusion alone. Other requirements such as the coexpression of the IL-3 and Epo receptors and the sharing of a receptor subunit are likely to be needed for an optimal cell response to the fusion growth factors.
R.W. Johnson Pharmaceutical Research Institute, Johnson & Johnson, Raritan, NJ 08869-0602.
N S
NS
Weich
J
J
Tullai
E
E
Guido
M
M
McMahon
L K
LK
Jolliffe
A F
AF
Lopez
M A
MA
Vadas
P A
PA
Lowry
P J
PJ
Quesenberry
J
J
Rosen
D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells; D016466:CHO Cells; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D004247:DNA; D015672:Erythroid Precursor Cells / Q000166:cytology; D004921:Erythropoietin / Q000008:administration & dosage / Q000235:genetics / Q000494:pharmacology*; D006098:Granulocytes / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007377:Interleukin-3 / Q000008:administration & dosage / Q000235:genetics / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*
D007377:Interleukin-3; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D004247:DNA