REACTIVITY OF MURINE CYTOKINE FUSION TOXIN, DIPHTHERIA TOXIN(390)-MURINE INTERLEUKIN-3 (DT390-MIL-3), WITH BONE-MARROW PROGENITOR CELLS

Myeloid leukemias can express interleukin-3 receptors (IL-3R), Therefo re, as an antileukemia drug, a fusion immunotoxin was synthesized cons isting 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 D T390-mIL-3 hybrid gene was cloned into a vector under the control of a n inducible promoter. The fusion protein was expressed in Escherichia coli and then purified from inclusion bodies. The fusion toxin was pot ent because it inhibited FDC-P1, an IL-3R-expressing murine myelomonoc ytic tumor line (IC50 = 0.025 nmol/L or 1.5 ng/mL), Kinetics were rapi d 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 line s not expressing IL-3R were not inhibited by the fusion protein. Becau se the use of DT390-mIL-3 as an antileukemia agent could be restricted by its reactivity with committed and/or primitive progenitor cells, b one marrow (BM) progenitor assays were performed. DT390-mIL-3 selectiv ely inhibited committed BM progenitor cells as measured by in vitro co lony-forming unit-granulocyte-macrophage and in vivo colony-forming un it-spleen colony assays. To determine if this fusion protein was react ive against BM progenitor cells required to rescue lethally irradiated recipients, adoptive transfer experiments were performed, Eight milli on DT390-mIL-8-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 expre ssion of IL-3R. This idea was supported in competitive repopulation ex periments in which DT390-mIL-8-treated C57BL/6 Ly5.2 BM cells were mix ed with nontreated C57BL/6 Ly5.1 BM cells and used to reconstitute C57 BL/6 Ly5.1 mice, A significant reduction, but not elimination, of Ly5. 2-expressing cells 95 days post-BM transplantation and secondary trans fer 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 b e useful in the treatment of myeloid leukemias that express the IL-3R. (C) 1996 by The American Society of Hematology.