Characterization of murine and humanized anti-CD33, gelonin immunotoxins reactive against myeloid leukemias

Abstract

M195 antibodies recognize CD33, an antigen present on acute myeloid leukemia blasts as well as some myeloid progenitor cells, but not on the ultimate hematopoietic progenitor stem cell. Immunotoxins (IT) reactive with human myeloid leukemias were constructed by conjugating gelonin, a single-chain ribosome-inactivating protein, to murine and genetically engineered, humanized M195 antibodies via an N-succinimidyl-3-(2-pyridyl-dithio)-propionate linkage. No losses of gelonin cytotoxic activity or M195 binding activity were observed after conjugation of up to two toxin molecules per antibody. Toxin conjugates displayed specific, potent toxicity for CD33+ cells. The murine and humanized IT were not toxic to CD33- cells and were 600 and 4500 times more potent, respectively, than free gelonin in inhibiting CD33+ HL60 cells. Treatment of HL60 cells with 1 micrograms/ml HuM195-gelonin resulted in more than 1000 times lower colony formation; normal bone marrow mononuclear cell colony-forming units treated with HuM195-IT were reduced by a factor of 10. HL60 leukemia cells could be effectively purged from an excess of normal bone marrow cells. Exposure of target cells to IT for as little as 30 min was as effective as continuous exposure of IT for up to 6 days. However, measures of the efficacy of the immunotoxin were directly related to the length of time of observation after IT exposure and were inversely related to cell concentration. M195-gelonin immunoconjugates are potential candidates for therapeutic use in in vivo or ex vivo bone marrow purging for myeloid leukemias.