title: Recombinant CD64-specific single chain immunotoxin exhibits specific cytotoxicity against acute myeloid leukemia cells.
abstract: CD64, the high affinity receptor for IgG (FcgammaRI) is expressed on acute myeloid leukemia blast cells and has recently been described as a specific target for immunotherapy. To generate a recombinant immunotoxin, the anti-CD64 single chain fragment (scFv) m22 was cloned into the bacterial expression vector pBM1.1 and fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Genetically modified Escherichia coli BL21 Star (DE3) were grown under osmotic stress conditions in the presence of compatible solutes. After isopropyl beta-D-thiogalactoside induction, the 70-kDa His(10)-tagged m22(scFv)-ETA' was directed into the periplasmic space and purified by a combination of metal-ion affinity and molecular size-chromatography. The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays, using CD64-positive AML cells. Binding specificity of m22(scFv)-ETA' was verified by competition with the parental anti-CD64 monoclonal antibody m22. The recombinant immunotoxin showed significant toxicity toward the CD64-positive cell lines HL-60 and U937 reaching 50% inhibition of cell proliferation at a concentration (IC(50)) of 11.6 ng/ml against HL-60 cells and 12.9 ng/ml against U937 cells. Approximately 41% of primary leukemia cells from a patient with CD64-positive AML were driven into early apoptosis by m22(scFv)-ETA' as measured by flow cytometric analysis. This is the first article documenting the specific cytotoxicity of a novel recombinant immunotoxin with major implications for immunotherapy of CD64-positive diseases.
authors.affiliation: Department of Pharmaceutical Product Development, Fraunhofer IME, Aachen, Germany.
authors.forename: Mehmet K
authors.initials: MK
authors.lastname: Tur
authors.forename: Michael
authors.initials: M
authors.lastname: Huhn
authors.forename: Theo
authors.initials: T
authors.lastname: Thepen
authors.forename: Michael
authors.initials: M
authors.lastname: Stöcker
authors.forename: Regina
authors.initials: R
authors.lastname: Krohn
authors.forename: Simon
authors.initials: S
authors.lastname: Vogel
authors.forename: Edgar
authors.initials: E
authors.lastname: Jost
authors.forename: Rainhardt
authors.initials: R
authors.lastname: Osieka
authors.forename: Jan G
authors.initials: JG
authors.lastname: van de Winkel
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Ricarda
authors.initials: R
authors.lastname: Finnern
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D000328:Adult; D000918:Antibody Specificity; D017209:Apoptosis / Q000187:drug effects; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D005098:Exotoxins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D007951:Leukemia, Myeloid / Q000188:drug therapy* / Q000276:immunology; D017452:Receptors, IgG / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D037521:Virulence Factors / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A