A Recombinant fusion toxin targeted to the granulocyte-Macrophage colony-Stimulating factor receptor

Abstract

Human granulocyte-macrophage colony stimulating factor (GMCSF) and its high affinity receptor function to regulate the proliferation and differentiation of myeloid lineage hematopoietic cells, and may participate in the pathogenesis of many malignant myeloid diseases. We have used genetic engineering based on the elucidated molecular structures of human granulocyte-macrophage colony-stimulating factor and diphtheria toxin (DT) to pro-duce a recombinant fusion toxin, DTctGMCSF, that targets diphtheria toxin to high affinity GMCSF receptors expressed on the surface of blast cells from a large fraction of patients with acute myeloid leukemia (AML). DTctGMCSF was specifically immunoreactive with anti-diphtheria toxin and anti-GMCSF antiseras, and exhibited the characteristic catalytic activity of diphtheria toxin, catalyzing the in vitro ADP-ribosylation of purified elongation factor 2. The cytotoxic effects of DTctGMCSF were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium (MTT) bromide assay of cell viability and in vivo assays of protein synthesis inhibition. DTctGMCSF were specifically cytotoxic to human leukemia cell lines bearing high affinity receptors for human GMCSF with IC50 of 10−9 to 10−11M. It was not toxic to mammalian hematopoietic cell lines lacking human GMCSF (hGMCSF) receptors. In receptor positive cells, cytotoxicity can be specifically blocked by a large excess of hGMCSF, confirming that its cytotoxicity is mediated through the hGMCSF receptor. Though DTctGMCSF inhibited granulocyte-macrophage colony formation by committed myeloid progenitor cells (CFU-GM), it did not significantly affect erythroid burst formation by committed erythroid progenitor cells (BFU-E), or mixed granulocyte-erythroid-macrophage-megakaryocyte colony formation by pluripotent multilineage progenitor cells (CFU-GEMM). DTctGMCSF holds promise for the treatment of myeloid lineage malignancies, and is a useful reagent to study hematopoiesis.