A RECOMBINANT FUSION TOXIN TARGETED TO THE GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR-RECEPTOR

Human granulocyte-macrophage colony stimulating factor (GMCSF) and its high affinity receptor function to regulate the proliferation and dif ferentiation of myeloid lineage hmatopoietic cells, and may participat e in the pathogenesis of many malignant myeloid diseases. We have used genetic engineering based on the elucidated molecular structures of h uman granulocyte-macrophage colony-stimulating factor and diphtheria t oxin (DT) to produce a recombinant fusion toxin, DT(ct)GMCSF, that tar gets diphtheria toxin to high affinity GMCSF receptors expressed on th e surface of blast cells from a large fraction of patients with acute myeloid leukemia (AML). DT,,GMCSF was specifically immunoreactive with antidiphtheria toxin and anti-GMCSF antiseras, and exhibited the char acteristic catalytic activity of diphtheria toxin, catalyzing the in v itro ADP-ribosylation of purified elongation factor 2. The cytotoxic e ffects of DT(ct)GMCSF 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. DT(ct)GMCSF were specifically cytotoxic to human leukemia cell lines bearing high affinity receptor s for human GMCSF with IC50 Of 10(-9) to 10(-11)M. It was not toxic to mammalian hematopoietic cell lines lacking human GMCSF (hGMCSF) recep tors. In receptor positive cells, cytotoxicity can be specifically blo cked by a large excess of hGMCSF, confirming that its cytotoxicity is mediated through the hGMCSF receptor. Though DT(ct)GMCSF inhibited gra nulocyte-macrophage colony formation by committed myeloid progenitor c ells (CFU-GM), it did not significantly affect erythroid burst formati on by committed erythroid progenitor cells (BFU-E), or mixed granulocy te-erythroid-macrophage-megakaryocyte colony formation by pluripotent multilineage progenitor cells (CFU-GEMM). DT(ct)GMCSF holds promise fo r the treatment of myeloid lineage malignancies, and is a useful reage nt to study hematopoiesis.