Cytotoxicity of a recombinant diphtheria toxin-granulocyte colony-stimulating factor fusion protein on human leukemic...

Granulocyte colony-stimulating factor (G-CSF) is a potent stimulator of the growth of normal and malignant hematopoietic cells and synergizes with other factors such as interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The action of G-CSF is mediated through a specific membrane receptor, however it is not clear if all of the effects of G-CSF are direct or indirect. As a step towards addressing this problem, a recombinant diphtheria toxin (DT)-related human G-CSF fusion protein has been constructed and purified from E. coli. The 70,000 dalton chimeric protein has immunologic determinants characteristic of both DT and G-CSF. At high concentrations, DAB486-G-CSF is cytotoxic towards G-CSF-dependent OCI/AML1 cells, but not factor independent OCI/AML3 cells; colony formation by G-CSF-responsive leukemic blasts from a patient with acute myeloblastic leukemia (AML) was also inhibited. The G-CSF fusion toxin displayed ADP-ribosyltransferase activity in a cell-free system. Genetic conjugation of G-CSF to an enzymatically inactive DT mutant, CRM197, resulted in a 200-fold reduction in the ability of G-CSF to stimulate normal bone marrow colony formation. These results suggest that fusion of G-CSF to DT sequences interferes with some of the activity but not the specificity of the ligand binding domain of the molecule. Nevertheless, DAB486-G-CSF may be included with the increasing number of other toxin-hormone fusion proteins whose toxicity is directed towards specific receptor-bearing cells, and may represent a novel approach towards the study and treatment of leukemia.