A murine cytokine fusion toxin specifically targeting the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on normal committed bone marrow progenitor cells and GM-CSF-dependent tumor cells.
A fusion protein was synthesized consisting of the murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mGM-CSF hybrid gene was cloned into a vector under the control of an inducible promoter and the protein expressed in Escherichia coli. After induction, a protein was purified from inclusion bodies in accord with the deduced molecular weight of DT390 mGM-CSF. Cell-free studies of the adenosine diphosphate-ribosylating activity of DT390 mGM-CSF showed results that were similar to those of native DT. The DT390 mGM-CSF immunotoxin inhibited FDCP2.1d, a murine myelomonocytic tumor line expressing the GM-CSF receptor with an IC50 (concentration inhibiting 50% activity) of 5 x 10(-11) mol/L. The fusion toxin was specifically cytotoxic and directed by the GM-CSF portion of the molecule because addition of a monoclonal antibody directed against GM-CSF inhibited its ability to kill the cell line. Cell lines that do not express GM-CSF receptor were not inhibited by the fusion toxin. DT390 mGM-CSF was also able to specifically inhibit normal committed bone marrow (BM) progenitor cells as measured in clonal colony-forming unit granulocyte-macrophage assays. Together, these findings indicate that DT390 mGM-CSF may be useful as a novel tool for purging BM of contaminating leukemia cells or in vivo for eliminating residual leukemia cells. Also, it can be used to determine whether committed and/or noncommitted BM progenitor cells express the GM-CSF receptor.
Chan
C H
CH
Department of Therapeutic Radiology, University of Minnesota Hospital and Clinics, Minneapolis 55455, USA.
Blazar
B R
BR
Eide
C R
CR
Kreitman
R J
RJ
Vallera
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D001854:Bone Marrow Cells; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D003114:Colony-Forming Units Assay; D004167:Diphtheria Toxin / Q000235:genetics / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000494:pharmacology*; D006098:Granulocytes / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology
D000970:Antineoplastic Agents; D004167:Diphtheria Toxin; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor