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Blood, 1 October 2005, Vol. 106, No. 7, pp. 2527-2529. Prepublished online as a Blood First Edition Paper on May 31, 2005; DOI 10.1182/blood-2005-02-0540.
NEOPLASIA Diphtheria toxin fused to variant human interleukin-3 induces cytotoxicity of blasts from patients with acute myeloid leukemia according to the level of interleukin-3 receptor expressionFrom the Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità; Hematology, Department of Cellular Biotechnologies and Hematology, University "La Sapienza," and the Department of Biopathology, University of Tor Vergata, Rome, Italy; and the Department of Medicine, Wake Forest University School of Medicine, Winston-Salem, NC.
Leukemic blasts from patients with acute myeloid leukemia (AML) frequently express high levels of the interleukin-3 receptor  chain (IL-3R). In the present study, we have explored the sensitivity of primary leukemic blasts obtained from 34 patients with AML to a diphtheria toxin (DT) composed of the catalytic and translocation domains of DT (DT388) fused to IL-3 (DT388IL-3) and to DT388 fused to a variant IL-3 with increased binding affinity (DT388IL-3[K116W]). On a molar basis, DT388IL-3[K116W] was significantly more active than DT388IL-3 in mediating leukemic cell killing. The rate of cell killing induced by the 2 DT/IL-3 fusion proteins was significantly correlated with the level of IL-3R/IL-3R expressed on leukemic blasts. These observations support a potential use of DT388IL-3[K116W] in the treatment of refractory AMLs and provide a simple biochemical parameter for the selection of eligible patients. (Blood. 2005;106:2527-2529)
Acute myeloid leukemia (AML) encompasses a group of heterogeneous disorders characterized by the expansion of malignant clones of hematopoietic cells blocked at various stages of differentiation. Several lines of evidence suggest that multiple molecular mechanisms are involved in the pathogenesis of AMLs.1 For example, chromosome translocations resulting in fusion genes that generate new proteins have been shown to play a major role in the genesis of leukemias.2 However, for the development of an overt leukemia condition, additional mutations and phenotypic abnormalities are required. Among them, a key role is played by mutations or alterations in the level of expression of membrane receptors involved in the control of the proliferation of hemopoietic cells, such as fms-related tyrosine kinase 3 (flt3), cellular-receptor tyrosine kinase (c-kit), and interleukin-3 receptor (IL-3R).
Flt3 is mutated and/or overexpressed in 30% to 40% of AMLs,3 whereas IL-3R is overexpressed in about 45% of AMLs.4 Furthermore, the analysis of an AML subpopulation enriched in leukemic stem cells showed that these cells express elevated levels of the IL-3R A genetically engineered fusion toxin composed of the first 388 amino acid residues of diphtheria toxin (DT) with a His-Met (H-M) linker is fused to human IL-3.7 This IL-3 DT was found to be toxic toward leukemic blasts8 and in vivo studies in Cynomolgus monkeys have shown that it is relatively well tolerated up to 100 µg/kg.9,10 These results contrast with the limited tolerance to the granulocyte macrophage-colony-stimulating factor (GMCSF) DT fusion protein (DT388GMCSF), which produced liver injury above 4 µg/kg to 7 µg/kg in monkeys and patients with AML.11,12 Preclinical studies with rodent-cell-directed DT fusion proteins (DT388mGMCSF and DT388mIL-3) showed Kupffer-cell-mediated liver injury for the GMCSF DT fusion protein but not the IL-3 DT fusion protein, consistent with the presence of GMCSFR but not IL-3R on the target cells.13 Based on these findings, a phase 1 clinical trial has recently been initiated with DT388IL-3 for the treatment of patients with AML. To date, 6 patients with AML have been treated with DT388IL-3 without evidence of severe toxicities or liver damage.14
Finally, with the aim of improving antileukemic activity, diphtheria toxin has been fused to IL-3 variants. Based on mutagenesis studies by several laboratories, substitution of a large, bulky hydrophobic residue (tryptophan) at IL-3 position 116 enhanced the hydrophobic interaction with IL-3R Using primary blasts from patients with AML in vitro, we compared in the present study the antileukemic cytotoxic activity of DT388IL-3 and that of DT388IL-3[K116W]. We found that the variant IL-3 fusion protein exhibited a more potent antileukemic activity than the wild-type (WT) IL-3 fusion protein.
Cells Fresh leukemic blasts from 34 patients with AML, obtained after informed consent, were isolated from either bone marrow or peripheral blood by Ficoll-Hypaque density gradient centrifugation and were immediately processed. All patients were consecutively diagnosed at the Division of Hematology, Department of Cellular Biotechnologies and Hematology of the University "La Sapienza," Rome, Italy. Leukemias were classified by morphologic criteria according to the French-American-British (FAB) classification and samples contained more than 70% leukemic blasts. Approval for these studies was obtained from the institutional review board of the Istituto Superiore di Sanità, Rome, Italy. Informed consent was obtained in accordance with the Declaration of Helsinki.
Preparation of DT388IL-3 The preparation and purification of DT388IL-3 and DT388IL-3[K116W] has been previously reported in detail.7,8 Hematopoietic growth factor receptor expression
Phycoerythrin (PE)-labeled anti-IL-3R In vitro incubation of leukemic blasts with IL-3 diphtheria fusion proteins and study of apoptosis Leukemic cells were grown in vitro in freshly prepared Iscoves-modified minimal essential medium (IMDM; Gibco, New York, NY) containing 10% fetal calf serum (Gibco) at a final concentration of 5 x 105 cells/mL either in the absence (control) or in the presence of either DT388IL-3 or DT388IL-3[K116W] at various concentrations (10-11 M-10-7 M). At various days of culture, one cell aliquot was removed and assayed by FACS analysis for the presence of apoptotic cells using the annexin V binding assay.
Newly diagnosed patients with AML were investigated for their in vitro sensitivity to the DT fused to the wild-type IL-3 (DT388IL-3) and to the DT fused to variant IL-3 (DT388IL-3[K116W]). Leukemic blasts from 2 M0, 4 M1, 8 M2, 2 M3, 10 M4, and 8 M5 were incubated in vitro for 24 to 48 hours either in the absence (control) or in the presence of increasing concentrations (from 1 x 10-11 to 1 x 10-7 M) of either DT388IL-3 or DT388IL-3[K116W] and the percentage of apoptotic cells was measured by the annexin V-propidium iodide (PI) labeling. A representative experiment reported in Figure 1A clearly indicated that maximal cytotoxicity was observed using the 1 x 10-7 M concentration of the 2 toxins and that DT388IL-3[K116W] was more active than DT388IL-3 in killing AML blasts. We therefore tested the effects of the 2 fusion proteins at 1 x 10-7 M on AML cells. The results of this analysis showed that after 24 hours of incubation, DT388IL-3[K116W] was more active than DT388IL-3 in inducing apoptosis of leukemic blasts (percentage of apoptotic cells equal to 52.4 ± 4.5, versus 27.1 ± 3.5; P < .001; Figure 1B).
It is of interest to note that the rate of cell killing induced by these diphtheria IL-3 fusion proteins varied among the different AML cases tested. In order to understand the origin of this heterogeneity, we investigated whether the rate of blast-cell killing induced by the IL-3 diphtheria fusion proteins was dependent on the levels of IL-3R expression on leukemic cells. For this purpose, the expression of IL-3R
AML cases expressing very low or low levels of IL-3R
Based on these observations, we conclude that the levels of IL-3R
The results of the present study clearly indicate that DT388IL-3[K116W] exerts a potent in vitro antileukemic activity. The cytotoxic activity of this IL-3/diphtheria toxin is particularly pronounced in samples from patients with AML expressing high IL-3R In summary, our data suggest that the variant DT388IL-3[K116W] is a promising new agent for patients with chemotherapy-refractory AML. Because of the persistently poor prognosis of a large proportion of patients with AML, the further preclinical development of this agent appears warranted.
Submitted February 9, 2005; accepted May 16, 2005.
Prepublished online as Blood First Edition Paper, May 31, 2005; DOI 10.1182/blood-2005-02-0540.
Supported by grants from the Italian Stem Cell Research Program, Ministry of Health, Rome, Italy, and by 1% Progetto Finalizzato 4AC/F, Italian Ministry of Health (U.T.) and by grants from the National Institutes of Health (R01CA76 178 and R01CA90 263) and the Leukemia and Lymphoma Society (A.E.F.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ugo Testa, Department of Hematology, Oncology and Molecular Medicine, Viale Regina Elena 299, 00161 Rome, Italy; e-mail: u.testa{at}iss.it.
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Abstract
Introduction
indicates DT388IL-3; 
, DT388IL-3[K116W]. (B) Percentage of apoptotic cells observed on the leukemic blasts of 25 patients with AML grown for 24 hours in the presence of either DT388IL-3 or DT388IL-3[K116W]. 
indicates DT388IL-3; 
