A recombinant trispecific single‐chain Fv derivative directed against CD123 and CD33 mediates effective elimination of acute myeloid leukaemia cells by dual targeting

Markus Kügler,Georg H Fey,Kristin Mentz,Andreas Mackensen,Christoph Stein,Heiko Singer,Christian Kellner,Ingo Schubert,Domenica Saul,Michael Schwenkert,Bernhard Stockmeyer,Fuat Oduncu

doi:10.1111/j.1365-2141.2010.08300.x

Abstract

Two trivalent constructs consisting of single-chain Fv antibody fragments (scFvs) specific for the interleukin-3 receptor alpha chain (CD123), CD33 and the Fcgamma-receptor III (CD16) were designed and characterized for the elimination of acute myeloid leukaemia (AML) cells. The dual targeting single-chain Fv triplebody (sctb) [123 x ds16 x 33] and the mono targeting sctb [123 x ds16 x 123] both specifically bound their respective target antigens and were stable in human serum at 37 degrees C for at least 5 d. Both constructs induced potent antibody-dependent cellular cytotoxicity (ADCC) of two different AML-derived CD33- and CD123 double-positive cell lines in the low picomolar range using isolated mononuclear cells (MNCs) as effector cells. In these experiments the dual targeting molecule produced significantly stronger lysis than the mono targeting agent. In addition, the sctbs showed a high potency in mediating ADCC of primary leukaemia cells isolated from peripheral blood or bone marrow of seven AML patients. Hence, these novel molecules displayed potent anti-leukaemic effects against AML cells in vitro and represent attractive candidates for further preclinical development.

Highlights

Acute myeloid leukaemia (AML) is a haematopoietic malignancy characterized by a block in cell maturation and an accumulation of undifferentiated blasts in the bone marrow and peripheral blood (Lowenberg et al, 2003, Hiddemann et al, 2005)
On double-positive cells, the sctb bound to CD123 and CD33 with a KD-value of 13.5 ± 0.6 nM, ev demonstrating simultaneous binding with both terminal single chain Fv antibody fragment (scFv) and a small avidity effect
No specific lysis was observed when samples were incubated in the absence of NK cells. These results show first, that the sctbs were able to directly activate purified CD16+ NK cells in antibody dependent cellular cytotoxicity (ADCC) reactions; second, that the proteins alone without effector cells had no effect on the target cells; and third, that the protein preparations were free of cytotoxic contaminants

Summary

Introduction

Acute myeloid leukaemia (AML) is a haematopoietic malignancy characterized by a block in cell maturation and an accumulation of undifferentiated blasts in the bone marrow and peripheral blood (Lowenberg et al, 2003, Hiddemann et al, 2005). Autologous or allogeneic bone marrow transplantation can be performed in selected patients (Shipley and Butera 2009). Fo than 60 years and for patients resistant to standard chemotherapy, the immunoconjugate. GO is a CD33-specific antibody chemically coupled to the cytotoxic drug calicheamicin (Sievers et al, 1999, Stasi 2008). CD33, a 67-kDa ee transmembrane cell surface glycoprotein specific for the myeloid lineage, is not expressed on non-haematopoietic tissue but is expressed on a subset of normal haematopoietic stem cells (HSCs) and on AML leukaemia stem cells

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Conclusion