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Recombinant CD64-Specific Single Chain Immunotoxin Exhibits Specific Cytotoxicity against Acute Myeloid Leukemia Cells

¹ Department of Pharmaceutical Product Development, Fraunhofer IME, Aachen, Germany;
² Department of Immunology, Immunotherapy Laboratory, University Medical Center Utrecht, Utrecht, the Netherlands;
³ Department of Molecular Biotechnology, Aachen University, Aachen, Germany;
⁴ Medarex, Inc., Annandale, New Jersey; and
⁵ Department of Internal Medicine IV, University Hospital Aachen, Aachen, Germany

CD64, the high affinity receptor for IgG (FcRI) 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 ß-D-thiogalactoside induction, the 70-kDa His₁₀-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 (IC₅₀) 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.

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