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Research Articles: Therapeutics, Targets, and Development

Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes

¹ Helmholtz-Institute for Biomedical Engineering, Department of Experimental Medicine and Immunotherapy, ² Institute for Molecular Biotechnology, and ³ University Hospital, Medical Clinic IV, RWTH Aachen University; ⁴ Fraunhofer IME, Department of Pharmaceutical Product Development, Aachen, Germany

Requests for reprints: Stefan Barth, Fraunhofer IME, Department of Pharmaceutical Product Development, Forckenbeckstr. 6, 52074 Aachen, Germany. E-mail: stefan.barth{at}ime.fraunhofer.de

Abstract

Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (FcRI), the high-affinity receptor for IgG, which is normally expressed at high levels only on activated cells of the myeloid lineage. CD64 is therefore a prime target for the specific delivery of cytotoxic agents. A promising toxin candidate is granzyme B, a human serine protease originating from cytotoxic granules of CD8⁺ T lymphocytes and natural killer cells. After evaluating the sensitivity of the AML-related cell line U937 toward cytosolic granzyme B, we genetically fused granzyme B to H22, a humanized single-chain antibody fragment (scFv) specific for CD64, to obtain Gb-H22(scFv), a fusion protein lacking the immunogenic properties of nonhuman immunofusions. Gb-H22(scFv) was successfully expressed in human 293T cells, secreted, and purified from cell culture supernatants. The purified protein bound specifically to CD64⁺ U937 cells. Despite linkage to the binding domain, the proteolytic activity of functional Gb-H22(scFv) was identical to that of free granzyme B. Target cell-specific cytotoxicity was observed with a half-maximal inhibitory concentration (IC₅₀) between 1.7 and 17 nmol/L. In addition, the induction of apoptosis in U937 cells was confirmed by Annexin A5 staining and the detection of activated caspase-3 in the cytosol. Finally, apoptosis was observed in primary CD64⁺ AML cells, whereas CD64^– AML cells were unaffected. This is the first report of a completely human granzyme B-based immunotoxin directed against CD64, with activity against an AML-related cell line and primary AML cells. [Mol Cancer Ther 2008;7(9):2924–32]

Grant support: German Cancer Aid Association grant 106521 and MAVO-IMHOTEP of the Fraunhofer-Gesellschaft (S. Barth).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/22/08; revised 6/13/08; accepted 6/22/08.