Reactivity of murine cytokine fusion toxin, diphtheria toxin390-murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor cells.
Myeloid leukemias can express interleukin-3 receptors (IL-3R). Therefore, as an antileukemia drug, a fusion immunotoxin was synthesized consisting of the murine IL-3 (mIL-3) 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-mIL-3 hybrid gene was cloned into a vector under the control of an inducible promote. The fusion protein was expressed in Escherichia coli and then purified from inclusion bodies. The fusion toxin was potent because it inhibited FDC-P1, an IL-3R-expressing murine myelomonocytic tumor line (IC50 = 0.025 nmol/L or 1.5 ng/mL). Kinetics were rapid and cell-free studies showed that DT390-mIL-3 was as toxic as native DT. DT390-mIL-3 was selective because anti-mIL-3 monoclonal antibody, but not irrelevant antibody, inhibited its ability to kill. Cell lines not expressing IL-3R were not inhibited by the fusion protein. Because the use of DT390-mIL-3 as an antileukemia agent could be restricted by its reactivity with committed and/or primitive progenitor cells, bone marrow (BM) progenitor assays were performed. DT390-mIL-3 selectively inhibited committed BM progenitor cells as measured by in vitro colony-forming unit-granulocyte-macrophage and in vivo colony-forming unit-spleen colony assays. To determine if this fusion protein was reactive against BM progenitor cells required to rescue lethally irradiated recipients, adoptive transfer experiments were performed. Eight million DT390-mIL-3-treated C57BL/6 Ly5.2 BM cells, but not 4 million, were able to rescue lethally irradiated congenic C57BL/6 Ly5.1 recipients, suggesting that progenitor cells might be heterogenous in their expression of IL-3R. This idea was supported in competitive repopulation experiments in which DT390-mIL-3 treated C57BL/6 Ly5.2 BM cells were mixed with nontreated C57BL/6 Ly5.1 BM cells and used to reconstitute C57BL/6 Ly5.1 mice. A significant reduction, but not elimination, of Ly5.2-expressing cells 95 days post-BM transplantation and secondary transfer experiments indicated that IL-3R is not uniformly expressed on all primitive progenitor cells. The fact that some early progenitor cells survived DT390-mIL-3 treatment indicates that this fusion toxin may be useful in the treatment of myeloid leukemias that express the IL-3R.
Chan
C H
CH
Department of Therapeutic Radiology, University of Minnesota Hospital and Clinics, Minneapolis, USA.
Blazar
B R
BR
Greenfield
L
L
Kreitman
R J
RJ
Vallera
D A
DA
D000818:Animals; D001483:Base Sequence; D001853:Bone Marrow / Q000187:drug effects*; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004167:Diphtheria Toxin / Q000008:administration & dosage*; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D000922:Immunotoxins / Q000737:chemistry / Q000494:pharmacology*; D007377:Interleukin-3 / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011828:Radiation Chimera; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D017931:DNA Primers; D004167:Diphtheria Toxin; D000922:Immunotoxins; D007377:Interleukin-3; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins