CD64, the high affinity receptor for IgG (FcγRI) 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 β-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.

AML6 is the most common acute leukemia in adults with an incidence of ∼10,000 people/year in the United States (1). AML is characterized by the proliferation of clonal precursor myeloid cells with arrested differentiation (2). The molecular and biological evolution of these malignant clones occurs in a stepwise series of events involving proto-oncogenes, tumor suppressor genes, and interactions with hematopoietic growth factors (3). According to the French-American-British classification system, AML of type M4 and M5 morphology is significantly correlated with expression of the high-affinity receptor for IgG, Fcγ RI (CD64; Ref. 4). CD64 is a 72-kDa cell surface glycoprotein, which is normally expressed on monocytes/macrophages and dendritic cells (5). The biological functions mediated by this receptor include superoxide and cytokine production (tumor necrosis factor α, IL-1, and IL-6), cytotoxicity, endocytosis/phagocytosis, and support of antigen presentation (6, 7). This receptor represents an appropriate target for immunotherapy of hematological malignancies because it is not present on pluripotent stem and CD34+ hematopoietic progenitor cells, thus guaranteeing regeneration of normal CD64-positive immune effector cells (8).

The ultimate goal in the treatment of cancer patients is the elimination of every tumor cell. Patients with AML have a total of 1012 to 1013 malignant cells at the time of diagnosis (9). Per definition, complete remission is achieved after therapy as soon as <5% of malignant cells are detectable in the bone marrow (10). However, these patients still may carry as much as 1010 malignant cells in the blood stream at this moment. These clinically unidentifiable minimal residual cells are the most common cause of relapse (11). Despite advances in polychemotherapy and radiotherapy, only ∼20–30% of patients with AML achieve long-term disease-free survival after first-line therapy (12). Thus, the elimination of minimal residual disease might improve the outcome of patients with AML. Selective approaches, including antibody-based therapies, targeting cytotoxic agents to these cells might offer a promising tool for specific elimination of minimal residual disease (13). To improve the antitumor activity of native antibodies, drugs, isotopes and toxins have been conjugated to mAbs (13).

Recently, a chemically linked anti-CD64 immunotoxin showed rapid binding to and efficient internalization into CD64-positive leukemia cells in vitro and in vivo(14). The authors documented rapid tumor regression of tumor masses ranging from 85 to >90% in a human AML model in NOD/SCID mice. The major obstacle observed in this and other trials were unspecific toxicities, mainly related to the vascular leak syndrome induced by Ricin-A-based chemically linked toxins because of their unspecific binding to endothelial cells (15, 16, 17). In recent studies, our group developed a set of recombinant immunotoxins for treatment of Hodgkin’s lymphoma and neuroblastoma consisting of different anti-CD25, anti-CD30 and anti-GD2 scFv antibody fragments genetically linked to Pseudomonas exotoxin A′ (18, 19, 20). Having established a very efficient expression protocol (21), the recombinant immuntoxins directly isolated from the periplasmic space of Escherichia coli demonstrated specific antitumor activities in vitro and in vivo. On the basis of this expertise we present here the construction, expression and characterization of the anti-CD64 immunotoxin m22(scFv)-ETA′. Furthermore, we demonstrate the specific activity of this novel immunotoxin against human AML cells.

Bacterial Strains, Oligonucleotides, and Plasmids.

E. coli XL1-blue [supE44 hsdR17 recA1 endA1 gyr A46 thi relA1 lacF′(pro AB+lacIqlacZΔM15 Tn10(tetr))] was used for propagation of plasmids and E. coli BL21 Star (DE3) [F-ompT hsdSB(rB-mB-) gal dcm rne131 DE3] as host for synthesis of recombinant immunotoxins. Synthetic oligonucleotides were synthesized by MWG Biotech (Ebersberg, Germany). The bacterial expression vector pBM1.1 is derived from the pET27b plasmid (Novagen, Madison, WI) and is used for NH2-terminal fusion of SfiI/NotI-binding structures to the modified deletion mutant of Pseudomonas aeruginosa Exotoxin A (22). Plasmids were prepared by the alkaline lysis method and purified using plasmid preparation kits from Qiagen (Hilden, Germany). Restriction fragments or PCR products were separated by agarose gel electrophoresis and extracted with QIAquick (Qiagen). All standard cloning procedures were carried out as described by Sambrook et al.(23).

Patient Samples and Cell Lines.

Heparinized peripheral blood samples from an adult patient with AML were obtained after informed consent and with the approval of the clinical research ethics board of the University of Aachen. MNCs were isolated by low-density (<1.007 g/ml) gradient centrifugation using Ficoll-Paque PLUS (Amersham Biosciences, Freiburg, Germany) separation medium. All cell lines, including the CD64-positive AML-derived cell lines HL-60 (provided by Theo. Thepen, Utrecht, the Netherlands) and U937 (DSMZ; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and CD64-negative L540Cy (24) and IIA1.6 (provided by T. Thepen), were cultivated in complete medium (RPMI 1640) supplemented with 10% (v/v) heat-inactivated FCS, 50 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine. All cells were cultured at 37°C in a 5% CO2 air atmosphere.

Construction and Expression of Recombinant m22(scFv)-ETA′.

The m22(scFv) DNA was amplified from m22-bearing plasmid (provided by T. Thepen) by PCR using the oligonucleotide primers m22(scFv)Back [5′-ATG-GCT-CAG-GGT-GCG-GCC-CAG-CCG-GCC-ATG-GCC-CAG-GTG-CAG-CTG-GTG-G-3′; bold letters: SfiI consensus site, in italics: 5′-m22(scFv) region] and m22(scFv)For [5′-GAG-TCA-TTC-TCG-ACT-TGC-GGC-CGC-TTT-GAT-CTC-CAG-CTT-GGT-CC-3′; bold letters: NotI consensus site, in italics: 3′-m22(scFv) region]. After SfiI/NotI-digestion, the 754-bp PCR-fragment was cloned into the bacterial expression vector pBM1.1 (22), digested with the same restriction enzymes. The resulting recombinant construct was verified by DNA sequence analysis.

After transformation into BL21 Star (DE3), m22(scFv)-ETA′ was periplasmically expressed under osmotic stress in the presence of compatible solutes as described by Barth et al.(21). Briefly, recombinant bacteria were harvested 15 h after IPTG induction. The bacterial pellet was resuspended in sonication-buffer [75 mm Tris/HCl (pH 8), 300 mm NaCl, 1 capsule of protease inhibitors/50 ml (Complete, Roche Diagnostics, Mannheim, Germany), 5 mm DTT, 10 mm EDTA, 10% (v/v) glycerol] at 4°C and sonicated 6 times for 30 s at 200 W. m22(scFv)-ETA′ was purified by IMAC using nickel-nitriloacetic chelating Sepharose (Qiagen) and SEC with Bio-Prep SE-100/17 (Bio-Rad, München, Germany) columns according to the manufacturer’s instructions. Recombinant immunotoxin was eluted with PBS (pH 7.4) and 1 m NaCl, analyzed by SDS/PAGE, quantified by densitometry (GS-700 Imaging Densitometer; Bio-Rad) after Coomassie staining in comparison with BSA standards and verified by Bradford assays (Bio-Rad).

SDS-PAGE and Western Blot Analysis.

SDS-PAGE and Western blotting were performed as described previously (18). m22(scFv)-ETA′ was detected by anti-ETA′ mAb TC-1 (Ref. 25; kindly provided by Darrell R. Galloway, Columbus, OH). Bound antibody was stained with an alkaline-phosphatase-conjugated antimouse-IgG mAb (Sigma Chemical Co., Deisenhofen, Germany) and a solution of Tris-HCl (pH 8.0) and 0.2 mg/ml naphtol-AS-Bi-phosphate (Sigma Chemical Co.) supplemented with 1 mg/ml Fast-Red (Serva, Heidelberg, Germany).

CM-ELISA.

The binding activity of the fusion protein m22(scFv)-ETA′ was determined by CM-ELISA using biological active membranes of tumor cells as described by Tur et al.(26, 27). ELISA Maxisorp-Plates (Nalge Nunc International, Roskilde, Denmark) were coated with 100 μl (∼0.9 mg protein/ml) freshly prepared membrane fractions of CD64-positive cell lines HL-60/U-937 and L540Cy as control in 0.02 m bicarbonate buffer (pH 9.6) overnight at 4°C. Plates were washed five times with PBS (pH 7.4) containing 0.2% (v/v) Tween 20 (TPBS) and blocked with 200 μl 2% BSA (w/v) in PBS (PBSA). After overnight incubation at 4°C, plates were washed five times with TPBS and 2–10 μg/ml m22(scFv)-ETA′ diluted with 0.5% BSA (w/v), and 0.05% Tween 20 (v/v) in PBS was added to the plates and incubated at room temperature (23°C) for 1 h. Thereafter, plates were washed, and binding of the recombinant immunotoxin was detected with the anti-ETA′ mAb TC-1 and F(ab′)2 fragments of peroxidase-coupled goat antimouse IgG (Boehringer, Ingelheim, Germany) according to the manufacturer’s recommendations. Bound antibodies were visualized after addition of 100 μl of 2′,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) solution (Roche Molecular Biochemicals) by measuring the extinction at 415 nm with an ELISA Reader (Molecular Devices, Ismaning, Germany).

Binding Specificity.

The binding specificity of m22(scFv)-ETA′ was tested by CM-ELISA following the protocol described above using the parental mAb m22 for competition (Acris, Bad Nauheim, Germany). Briefly, ELISA Maxisorp plates were coated with membrane fractions of CD64-positive HL-60 cells. After blocking, the plates were incubated with a fixed concentration (35 μg/ml) of recombinant immunotoxin m22(scFv)-ETA′. Competition experiments were performed in the presence or absence of different concentrations (100 ng–10 μg/ml) of mAb m22. Binding of m22(scFv)-ETA′ was detected using peroxidase labeled anti-His6 antibodies (Roche Molecular Biochemicals) after addition of 2′,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid).

Flow Cytometric Binding Analyses.

Cell binding activity of m22(scFv)-ETA′ expressed in E. coli BL21 Star (DE3) was evaluated using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, Heidelberg, Germany). Cells were stained with the affinity purified scFv-immunotoxin as described previously (28). Briefly, 10,000 events were collected for each sample, and analyses of intact cells were performed using appropriate scatter gates to exclude cellular debris and aggregates. A total of 5 × 105 cells was incubated for 1 h on ice with 50 μl of the m22(scFv)-ETA′ bacterial protein extract at a concentration of 30–40 μg/ml. The cells were washed with PBS buffer containing 0.2% (w/v) BSA and 0.05% (w/v) sodium azide and then incubated for 30 min with an anti-Pseudomonas ETA mAb (TC-1) diluted 1:2 in PBS buffer. Cells were washed and incubated with FITC-labeled goat-antimouse IgG (Dako Diagnostica, Hamburg, Germany) for 1 h at 4°C. After a final wash, the cells were treated with 2 μl of 6.25 mg/ml PI and subsequently analyzed by fluorescence-activated cell sorting.

Additionally, as positive control, CD64-positive AML cells were directly identified by a m22(scFv) fragment recombinantly fused to eGFP.7

Colorimetric Cell Proliferation Assay.

The cytotoxic effect of m22(scFv)-ETA′ on target cells was determined by measurement of metabolization of XTT to a water soluble orange formazan dye was determined as described previously (28). Briefly, various dilutions of the recombinant immunotoxin were distributed in 100-μl aliquots in 96-well plates. A total of 2 × 104 target cells in 100-μl aliquots of complete medium was added, and the plates were incubated for 48 h at 37°C. Then, the cell cultures were pulsed with 100 μl of fresh culture medium supplemented with XTT/phenazine methosulfate (final concentrations of 0.3 mg and 0.383 ng, respectively) for 4 h. The spectrophotometrical absorbances of the samples were measured at 450 and 650 nm (reference wavelength) with an ELISA reader (Molecular Devices). The concentration required to achieve a 50% reduction of protein synthesis (IC50) relative to untreated control cells was calculated graphically via Microsoft Excel generated diagrams. All measurements were done in triplicate.

Flow Cytometric Assay of Apoptosis.

MNCs from patient-blood samples were isolated by Ficoll-Paque centrifugation. Cell-surface CD64 expression was confirmed by flow cytometry using the m22(scFv)-ETA′ immunotoxin as described above. Additionally, primary leukemic cells were directly stained using the newly developed eGFP-tagged m22(scFv) fusion protein.7 Approximately 5 × 105 MNCs/well were seeded in flat-bottomed 12-well plates in RPMI 1640 supplemented with 10% FCS in triplicate. A total of 100 ng/ml immunotoxin was added into each well, and the cells cultured for 18 h at 37°C and 5% CO2 air atmosphere. Apoptotic cells were detected using an annexin V-FITC apoptosis detection kit I (BD PharMingen, Heidelberg, Germany). Briefly, whole cells were stained simultaneously with FITC-conjugated AnnV and PI in PBS according to the manufacturer’s protocol. Ten thousand cells were analyzed by flow cytometry, and the AnnV-/PI-, AnnV+/PI-, AnnV+/PI+ and AnnV-/PI+ subpopulations were counted. Early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to AnnV-FITC but excluded PI. Thus, the four populations of AnnV-/PI-, AnnV+/PI-, AnnV+/PI+, and AnnV-/PI+ have been found to correspond to living cells, early apoptotic cells, late apoptotic/necrotic cells, and necrotic cells, respectively.

Construction and Expression of m22(scFv)-ETA′.

PCR-amplified m22(scFv) DNA (Fig. 1,A) was directionally cloned into the kanamycin-resistant pBM1.1 expression vector containing an IPTG-inducible lac operator, a pelB signal peptide followed by an enterokinase-cleavable His10 tag, and modified ETA′ (Fig. 1 B). The deleted domain Ia of Pseudomonas Exotoxin responsible for nonspecific cell binding was thus replaced by CD64-specific m22(scFv). Successful cloning was verified by DNA sequence analysis.

After transformation, recombinant E. coli BL21 Star (DE3) clones were cultivated under osmotic stress conditions in the presence of compatible solutes. Recombinant immunotoxin was directed into the periplasmic space and the functional m22(scFv)-ETA′ (∼70 kDa) directly purified by combination of IMAC and SEC to >90% purity. At least 1 mg of purified m22(scFv)-ETA′ protein was routinely prepared from 1 liter of bacterial shaking cultures (Fig. 2,A). Intact recombinant immunotoxin was secreted to the periplasmic compartment, as visualized by immunoblot using TC-1, an ETA′-specific mAb (Fig. 2 B).

Binding Properties of m22(scFv)-ETA′.

Coupling of the m22(scFv) coding regions to the truncated ETA′ coding sequences did not affect the binding activity of the VH/VL antibody format. Purified recombinant anti-CD64 immunotoxin always bound to AML cell membrane fractions but not to CD64-negative L540Cy membranes and corresponding intact cells as measured by CM-ELISA (Fig. 3,A) and flow cytometry (Fig. 3,B), respectively. CD64 specificity was documented by competitive CM-ELISA experiments: binding of m22(scFv)-ETA′ against CD64-positive HL-60 membrane fractions was inhibited by ∼70% upon addition of 10 μg/ml mAb m22 (Fig. 3 C).

In Vitro Cytotoxic Activity.

To characterize the cytotoxic activity of the recombinant anti-CD64 immunotoxin in vitro, we evaluated the proliferation of different target cells after incubation with different amounts of m22(scFv)-ETA′. Growth inhibition of AML-derived cell lines HL-60 and U937 were documented by a XTT-based colorimetric assay. Toxic effects were observed against CD64-positive cells with a calculated median IC50 of 11.6 ng/ml on HL-60 cells (Fig. 4,A) and 12.9 ng/ml on U937 cells, respectively (Fig. 4 B). The CD64-negative Hodgkin-derived cell line L540Cy and the murine T-cell leukemia-derived cell line IIA1.6 were not affected by recombinant immunotoxin concentrations of up to 10 μg/ml.

Analysis of Apoptosis on Primary AML Cells.

The effects of m22(scFv)-ETA′ on the induction of apoptosis in a freshly prepared population of acute myeloma cells from a patient were examined by flow cytometry. Immunophenotyping revealed ∼90% leukemic cells. The expression of CD64 on the primary cells was verified directly with eGFP-tagged m22(scFv) and in a sandwich approach with m22(scFv)-ETA′ (Fig. 5,A). Two color flow cytometric analysis using AnnV-FITC and PI (Fig. 5,B) discriminated four populations, viable (Fig. 5,B, bottom left quadrant), early apoptotic (Fig. 5,B, bottom right quadrant), late apoptotic/necrotic (Fig. 5,B, top right quadrant), and necrotic cells (Fig. 5 B, top left quadrant). Primary patient-derived CD64-negative leukemic cells treated with m22(scFv)-ETA′ for 18 h remained mostly viable (∼90%). Primary patient-derived CD64-positive AML cells treated with the recombinant immunotoxin after incubation for 18 h showed viable (∼44%), early apoptotic (∼41%), and late apoptotic/necrotic cell populations (∼15%).

In this study, we report the construction of the first recombinant anti-CD64 immunotoxin targeting CD64-positive AML cells (29). The overall expression of CD64 on AML cells is ∼58% (14). To realize the construction of the immunotoxin, we fused the anti-CD64 scFv m22 to a truncated Pseudomonas exotoxin A (ETA′). The major findings to emerge from our study are: (a) functional m22(scFv)-ETA′ was directly isolated from the periplasmic space of E. coli cultured under osmotic stress conditions in the presence of compatible solutes and additionally purified by a combination of immobilized metal affinity and molecular size chromatography; (b) m22(scFv)-ETA′ bound to CD64-positive cells as documented by CM-ELISA and flow cytometry; (c) CD64-specific binding activity was shown by competition CM-ELISA using increasing concentrations of parental monoclonal antibody m22; and (d) the recombinant immunotoxin exhibited specific cytotoxic activity toward CD64 receptor-expressing AML-derived cell lines HL-60 and U937 and destroyed CD64-positive patient-derived primary AML cells.

Targeting malignant cells selectively via cell-surface receptors is inherently different from surgery, radiation, and chemotherapy and is often considered a new modality for cancer therapy. Recently, AML cells were targeted using both anti-CD33 and anti-GM-CSF immunotoxins. The Food and Drugs Administration recently approved the anti-CD33 immunotoxin Gemtuzumab ozogamicin (Mylotarg) for the treatment of relapsed AML in the United States (30). However, hepatotoxicity, including severe hepatic veno-occlusive disease, has been reported in association with the use of Mylotarg, which may result from targeted delivery of the toxin moiety calicheamicin to CD33-expressing cells found in hepatic sinusoids (31). The fusion toxin DT388-GM-CSF combining DT with GM-CSF was evaluated in a Phase I dose-escalation trial in patients with relapsed AML (32). DT388-GM-CSF induced complete and partial remissions in chemotherapy-resistant AML-patients but produced liver injury characterized by transient transaminasemia and severe liver dysfunction, which was speculated to be a result of cytokine release from GM-CSF receptor-positive liver Kupffer cells. Thus, additional immunotoxins for AML-targeting alternative cell surface receptors and using different cytotoxic components might be beneficial for patients. Furthermore, very recently no liver damage was observed in carcinoma xenograft-bearing mice after repeated application of the recombinant Pseudomonas exotoxin A-based immunotoxin 4D5MOCB-ETA targeting the epithelial cell adhesion molecule in a dose range up to 500 μg · kg-1(33).

Additional problems identified in clinical trials with chemically coupled immunotoxins are (a) the development of neutralizing antibodies against both the murine IgG and the toxic moiety resulting in a limited number of application in ∼40–60% of the patients (15, 16), and (b) the unspecific cytotoxicity related to unspecific binding of Ricin-A-based toxins to endothelial cells because of their (x)D(y)-motif (17). These problems might, at least in part, be circumvented by using recombinant DNA technology to construct smaller and less immunogenic immunotoxins with reduced unspecific toxicities. Recently, it had been reported in first clinical trials that recombinant scFv- or IL-immunotoxin carrying truncated ETA variants show reduced antibody responses in patients (34, 35).

The most important prerequisite for effective immunotoxin therapy is internalization of target antigen after binding of the immunotoxin to allow its translocation into the cytosol and cell killing. This internalization behavior was recently proven using a chemically linked anti-CD64 immunotoxin (36); it both developed specific functional activity against CD64 expressing cells in vitro and in a transgenic mice mouse model expressing human CD64.

Although CD64 is a normal marker during the myeloid lineage differentiation pathway, this surface receptor is an ideal target for selective immunotherapy because it is not expressed on CD34-positive hematopoietic stem cells. Self-renewing of these cells is a prerequisite for long-term multilineage reconstitution of hematopoiesis after immunotherapy eliminating human immune effector cells (8). Additionally, it has been shown that only activated CD64-positive cells are killed, whereas CD64-expressing nonactivated cells are not affected (14, 36).

The periplasmically expressed, nonglycosylated recombinant scFv-immunotoxin constructed in this study exhibited specific cytotoxic activity in vitro in the same concentration range (ng/ml) as reported for other ETA-based fusion proteins (28, 37, 38, 39).

In summary, we have shown that CD64-positive AML-derived tumor cell lines and primary patient-derived AML cells can be specifically eliminated by a novel recombinant anti-CD64 immunotoxin in vitro. Having demonstrated the functional activity of m22(scFv)-ETA′, this selective immunotherapeutic compound might also be used to eliminate deregulated, tissue-infiltrating CD64-positive monocytes/macrophages in patients with local inflammatory diseases (36).

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.

Requests for reprints: Stefan Barth. Fraunhofer IME, Pharmaceutical Product Development, Worringerweg 1, 52074 Aacher, Germany, Phone: 49-241-9632132; fax: 49-241-871062; E-mail: barth@ime.fraunhofer.de

6

The abbreviations used are: AML, acute myeloid leukemia; IL, interleukin; mAb, monoclonal antibody; MNC, mononuclear cell; IPTG, isopropyl-1-thio-β-d-galactopyranoside; IMAC, immobilized metal-ion affinity chromatography; SEC, size exclusion chromatography; CM-ELISA, cell membrane ELISA; PI, propidium iodide; 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt; AnnV, Annexin V; GM-CSF, granulocyte macrophage colony-stimulating factor; DT, diphtheria toxin.

7

M. Stöcker, M. K. Tur, A. Klimka, T. Klockenbring, M. Huhn, R. Fischer, and S. Barth, Development of a novel system for functional secretion of eGFP-based fusion proteins, manuscript in preparation.

Fig. 1.

Cloning of m22(scFv)-ETA′. A, PCR amplification of m22(scFv) [Lane M, 100-bp ladder; Lanes 1–4, m22(scFv) (790 bp); Lane C, negative control]. B, schematic structure of the m22(scFv)-ETA′ coding region in the pET27b E. coli expression vector. The expression module is composed of the IPTG-inducible T7-lac operator, the signal peptide of the pectate lyase of Erwinia carotovora (pelB), a synthetic and enterokinase-cleavable His10 cluster (His-Tag), the murine anti-CD64 scFv m22 and the ETA′ coding region.

Fig. 1.

Cloning of m22(scFv)-ETA′. A, PCR amplification of m22(scFv) [Lane M, 100-bp ladder; Lanes 1–4, m22(scFv) (790 bp); Lane C, negative control]. B, schematic structure of the m22(scFv)-ETA′ coding region in the pET27b E. coli expression vector. The expression module is composed of the IPTG-inducible T7-lac operator, the signal peptide of the pectate lyase of Erwinia carotovora (pelB), a synthetic and enterokinase-cleavable His10 cluster (His-Tag), the murine anti-CD64 scFv m22 and the ETA′ coding region.

Close modal
Fig. 2.

Bacterial expression and purification of m22(scFv)-ETA′. A, m22(scFv)-ETA′ protein after IMAC and SEC as documented after 10% (w/v) SDS-PAGE and Coomassie staining; Lane 1, m22(scFv)-ETA′ (67 kDa); Lane M, prestained Marker (Bio-Rad). B, immunoblot stained with anti-ETA′ mAb TC-1 (lane identification corresponds to A).

Fig. 2.

Bacterial expression and purification of m22(scFv)-ETA′. A, m22(scFv)-ETA′ protein after IMAC and SEC as documented after 10% (w/v) SDS-PAGE and Coomassie staining; Lane 1, m22(scFv)-ETA′ (67 kDa); Lane M, prestained Marker (Bio-Rad). B, immunoblot stained with anti-ETA′ mAb TC-1 (lane identification corresponds to A).

Close modal
Fig. 3.

Binding properties of the recombinant anti-CD64 immunotoxin m22(scFv)-ETA′. A, binding of m22(scFv)-ETA′ to CD64-negative cell membranes L540Cy and CD64-positive AML-derived cell membranes HL-60 documented by CM-ELISA. B, binding of m22(scFv)-ETA′ to antigen-positive cells by flow cytometry. Cells were stained with purified immunotoxin (transparent curves) or with PBS as negative control (gray curves). a, L540Cy cells stained with CD64-specific immunotoxin. b, HL-60 cells stained with CD64-specific immunotoxin. C, documentation of specific binding activity of m22(scFv)-ETA′ using a CM-ELISA with different dilutions of mAb m22 for competition. Binding of m22(scFv)-ETA′ was detected with peroxidase-conjugated anti-His mAb. Presented are data from three independent experiments.

Fig. 3.

Binding properties of the recombinant anti-CD64 immunotoxin m22(scFv)-ETA′. A, binding of m22(scFv)-ETA′ to CD64-negative cell membranes L540Cy and CD64-positive AML-derived cell membranes HL-60 documented by CM-ELISA. B, binding of m22(scFv)-ETA′ to antigen-positive cells by flow cytometry. Cells were stained with purified immunotoxin (transparent curves) or with PBS as negative control (gray curves). a, L540Cy cells stained with CD64-specific immunotoxin. b, HL-60 cells stained with CD64-specific immunotoxin. C, documentation of specific binding activity of m22(scFv)-ETA′ using a CM-ELISA with different dilutions of mAb m22 for competition. Binding of m22(scFv)-ETA′ was detected with peroxidase-conjugated anti-His mAb. Presented are data from three independent experiments.

Close modal
Fig. 4.

Growth inhibition of AML-derived cell lines after incubation with m22(scFv)-ETA′ as documented by cell-viability assays. A, HL-60 (CD64+) or L540Cy (CD64) and (B) U937 (CD64+) or IIA1.6 (CD64) were treated with various dilutions of recombinant anti-CD64 immunotoxin. HL-60, U937 (———) or L540Cy, IIA1.6 (— — —) were treated with m22(scFv)-ETA′, and their ability to metabolize the XTT to a water-soluble formazan salt (formed by mitochondrial dehydrogenase activity) was measured as absorbance at 450 and 650 nm. Measurements were performed in triplicate. Results are presented as percentage of untreated control cells.

Fig. 4.

Growth inhibition of AML-derived cell lines after incubation with m22(scFv)-ETA′ as documented by cell-viability assays. A, HL-60 (CD64+) or L540Cy (CD64) and (B) U937 (CD64+) or IIA1.6 (CD64) were treated with various dilutions of recombinant anti-CD64 immunotoxin. HL-60, U937 (———) or L540Cy, IIA1.6 (— — —) were treated with m22(scFv)-ETA′, and their ability to metabolize the XTT to a water-soluble formazan salt (formed by mitochondrial dehydrogenase activity) was measured as absorbance at 450 and 650 nm. Measurements were performed in triplicate. Results are presented as percentage of untreated control cells.

Close modal
Fig. 5.

Flow cytometric analysis of induced apoptosis on fresh patient-derived AML cells by the recombinant anti-CD64 immunotoxin m22(scFv)-ETA′. A, binding of m22(scFv)-ETA′ (———) and eGFP-tagged m22(scFv) (····) on primary cells. Cells were stained with AnnV-FITC and PI simultaneously. B, treatment of both primary patient-derived CD64-negative, as well as CD64-positive AML cells with 100 ng/ml m22(scFv)-ETA′. Numbers in the lower and higher right quadrant of each plot represent the percentage of cells in early apoptosis (AnnV+/PI-) and late apoptosis/necrosis (AnnV+/PI+), respectively. The data are representative for three separate experiments performed with these samples.

Fig. 5.

Flow cytometric analysis of induced apoptosis on fresh patient-derived AML cells by the recombinant anti-CD64 immunotoxin m22(scFv)-ETA′. A, binding of m22(scFv)-ETA′ (———) and eGFP-tagged m22(scFv) (····) on primary cells. Cells were stained with AnnV-FITC and PI simultaneously. B, treatment of both primary patient-derived CD64-negative, as well as CD64-positive AML cells with 100 ng/ml m22(scFv)-ETA′. Numbers in the lower and higher right quadrant of each plot represent the percentage of cells in early apoptosis (AnnV+/PI-) and late apoptosis/necrosis (AnnV+/PI+), respectively. The data are representative for three separate experiments performed with these samples.

Close modal

We thank Nicole Redding for excellent technical assistance.

1
Jemal A., Murray T., Samuels A., Ghafoor A., Ward E., Thun M. J. Cancer statistics, 2003.
CA - Cancer J. Clin.
,
53
:
5
-26,  
2003
.
2
Jordan C. T. Unique molecular and cellular features of acute myelogenous leukemia stem cells.
Leukemia (Baltimore)
,
16
:
559
-562,  
2002
.
3
Baer M. R., Bloomfield C. D. The clinical significance of biological characteristics of the cells in acute myeloid leukemia.
Annu. Rev. Med.
,
42
:
381
-389,  
1991
.
4
Ball E. D., McDermott J., Griffin J. D., Davey F. R., Davis R., Bloomfield C. D. Expression of the three myeloid cell-associated immunoglobulin G Fc receptors defined by murine monoclonal antibodies on normal bone marrow and acute leukemia cells.
Blood
,
73
:
1951
-1956,  
1989
.
5
Fossati G., Bucknall R. C., Edwards S. W. Fcγ receptors in autoimmune diseases.
Eur. J. Clin. Investig.
,
31
:
821
-831,  
2001
.
6
Booth J. W., Kim M. K., Jankowski A., Schreiber A. D., Grinstein S. Contrasting requirements for ubiquitylation during Fc receptor-mediated endocytosis and phagocytosis.
EMBO J.
,
21
:
251
-258,  
2002
.
7
van de Winkel J. G., Capel P. J. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications.
Immunol. Today
,
14
:
215
-221,  
1993
.
8
Menendez P., del Canizo M. C., Orfao A. Immunophenotypic characteristics of PB-mobilized CD34+ hematopoietic progenitor cells.
J. Biol. Regul. Homeost. Agents
,
15
:
53
-61,  
2001
.
9
Lilleyman J. S., Blanchette V. S., Hann I. M. Pathology of AML John S. Lillegman Ian M. Hann Victor S. Blanchette eds. .
Pediatric Hematology
,
369
-385, Churchill Livingstone New York  
1999
.
10
Cheson B. D., Cassileth P. A., Head D. R., Schiffer C. A., Bennett J. M., Bloomfield C. D., Brunning R., Gale R. P., Grever M. R., Keating M. J., et al Report of the National Cancer Institute-sponsored workshop on definitions of diagnosis and response in acute myeloid leukemia.
J. Clin. Oncol.
,
8
:
813
-819,  
1990
.
11
Liu Yin J. A. Minimal residual disease in acute myeloid leukaemia.
Best Pract. Res. Clin. Haematol.
,
15
:
119
-135,  
2002
.
12
Cassileth P. A., Harrington D. P., Appelbaum F. R., Lazarus H. M., Rowe J. M., Paietta E., Willman C., Hurd D. D., Bennett J. M., Blume K. G., Head D. R., Wiernik P. H. Chemotherapy compared with autologous or allogeneic bone marrow transplantation in the management of acute myeloid leukemia in first remission.
N. Engl. J. Med.
,
339
:
1649
-1656,  
1998
.
13
Carter P. Improving the efficacy of antibody-based cancer therapies.
Nat. Rev. Cancer
,
1
:
118
-129,  
2001
.
14
Zhong R. K., van de Winkel J. G., Thepen T., Schultz L. D., Ball E. D. Cytotoxicity of anti-CD64-ricin a chain immunotoxin against human acute myeloid leukemia cells in vitro and in SCID mice.
J. Hematother. Stem Cell Res.
,
10
:
95
-105,  
2001
.
15
Grossbard M. L., Gribben J. G., Freedman A. S., Lambert J. M., Kinsella J., Rabinowe S. N., Eliseo L., Taylor J. A., Blattler W. A., Epstein C. L., et al Adjuvant immunotoxin therapy with anti-B4-blocked ricin after autologous bone marrow transplantation for patients with B-cell non-Hodgkin’s lymphoma.
Blood
,
81
:
2263
-2271,  
1993
.
16
Vitetta E. S., Thorpe P. E., Uhr J. W. Immunotoxins: magic bullets or misguided missiles?.
Immunol. Today
,
14
:
252
-259,  
1993
.
17
Baluna R., Rizo J., Gordon B. E., Ghetie V., Vitetta E. S. Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome.
Proc. Natl. Acad. Sci. USA
,
96
:
3957
-3962,  
1999
.
18
Barth S., Huhn M., Wels W., Diehl V., Engert A. Construction and in vitro evaluation of RFT5(scFv)-ETA′, a new recombinant single-chain immunotoxin with specific cytotoxicity toward CD25+ Hodgkin-derived cell lines.
Int. J. Mol. Med.
,
1
:
249
-256,  
1998
.
19
Barth S., Huhn M., Matthey B., Tawadros S., Schnell R., Schinkothe T., Diehl V., Engert A. Ki-4(scFv)-ETA′, a new recombinant anti-CD30 immunotoxin with highly specific cytotoxic activity against disseminated Hodgkin tumors in SCID mice.
Blood
,
95
:
3909
-3914,  
2000
.
20
Tur M. K., Sasse S., Stocker M., Djabelkhir K., Huhn M., Matthey B., Gottstein C., Pfitzner T., Engert A., Barth S. An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines.
Int. J. Mol. Med.
,
8
:
579
-584,  
2001
.
21
Barth S., Huhn M., Matthey B., Klimka A., Galinski E. A., Engert A. Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions.
Appl. Environ. Microbiol.
,
66
:
1572
-1579,  
2000
.
22
Matthey B., Engert A., Klimka A., Diehl V., Barth S. A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins.
Gene (Amst.)
,
229
:
145
-153,  
1999
.
23
Sambrook J., Fritsch E. F., Maniatis T. .
Molecular Cloning: A Laboratory Manual
, Ed. 2 Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY  
1989
.
24
Kapp U., Wolf J., von Kalle C., Tawadros S., Rottgen A., Engert A., Fonatsch C., Stein H., Diehl V. Preliminary report: growth of Hodgkin’s lymphoma derived cells in immune compromised mice.
Ann. Oncol.
,
3 (Suppl. 4)
:
21
-23,  
1992
.
25
Galloway D. R., Hedstrom R. C., Pavlovskis O. R. Production and characterization of monoclonal antibodies to exotoxin A from Pseudomonas aeruginosa.
Infect. Immun.
,
44
:
262
-267,  
1984
.
26
Tur M. K., Rothe A., Huhn M., Goerres U., Klimka A., Stocker M., Engert A., Fischer R., Finnern R., Barth S. A novel approach for immunization, screening and characterization of selected scFv libraries using membrane fractions of tumor cells.
Int. J. Mol. Med.
,
11
:
523
-527,  
2003
.
27
Tur M. K., Huhn M., Sasse S., Engert A., Barth S. Selection of scFv phages on intact cells under low pH conditions leads to a significant loss of insert-free phages.
Biotechniques
,
30
:
412
-413,  
2001
.
28
Huhn M., Sasse S., Tur M. K., Matthey B., Schinkothe T., Rybak S. M., Barth S., Engert A. Human angiogenin fused to human CD30 ligand (Ang-CD30L) exhibits specific cytotoxicity against CD30-positive lymphoma.
Cancer Res.
,
61
:
8737
-8742,  
2001
.
29
Krasinskas A. M., Wasik M. A., Kamoun M., Schretzenmair R., Moore J., Salhany K. E. The usefulness of CD64, other monocyte-associated antigens, and CD45 gating in the subclassification of acute myeloid leukemias with monocytic differentiation.
Am. J. Clin. Pathol.
,
110
:
797
-805,  
1998
.
30
Bross P. F., Beitz J., Chen G., Chen X. H., Duffy E., Kieffer L., Roy S., Sridhara R., Rahman A., Williams G., Pazdur R. Approval summary: Gemtuzumab ozogamicin in relapsed acute myeloid leukemia.
Clin. Cancer Res.
,
7
:
1490
-1496,  
2001
.
31
Rajvanshi P., Shulman H. M., Sievers E. L., McDonald G. B. Hepatic sinusoidal obstruction after Gemtuzumab ozogamicin (Mylotarg) therapy.
Blood
,
99
:
2310
-2314,  
2002
.
32
Frankel A. E., Powell B. L., Hall P. D., Case L. D., Kreitman R. J. Phase I trial of a novel diphtheria toxin/granulocyte macrophage colony-stimulating factor fusion protein (DT388GMCSF) for refractory or relapsed acute myeloid leukemia.
Clin. Cancer Res.
,
8
:
1004
-1013,  
2002
.
33
Di Paolo C., Willuda J., Kubetzko S., Lauffer I., Tschudi D., Waibel R., Pluckthun A., Stahel R. A., Zangemeister-Wittke U. A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecule-specific single-chain antibody fragment has potent and selective antitumor activity.
Clin. Cancer Res.
,
9
:
2837
-2848,  
2003
.
34
Kreitman R. J., Wilson W. H., Robbins D., Margulies I., Stetler-Stevenson M., Waldmann T. A., Pastan I. Responses in refractory hairy cell leukemia to a recombinant immunotoxin.
Blood
,
94
:
3340
-3348,  
1999
.
35
Kawakami K., Kawakami M., Puri R. K. Overexpressed cell surface interleukin-4 receptor molecules can be successfully targeted for antitumor cytotoxin therapy.
Crit. Rev. Immunol.
,
21
:
299
-310,  
2001
.
36
Thepen T., van Vuuren A. J., Kiekens R. C., Damen C. A., Vooijs W. C., van de Winkel J. G. Resolution of cutaneous inflammation after local elimination of macrophages.
Nat. Biotechnol.
,
18
:
48
-51,  
2000
.
37
Klimka A., Barth S., Matthey B., Roovers R. C., Lemke H., Hansen H., Arends J. W., Diehl V., Hoogenboom H. R., Engert A. An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFv)-ETA′) is a potent immunotoxin against a Hodgkin-derived cell line.
Br. J. Cancer
,
80
:
1214
-1222,  
1999
.
38
Barth S., Huhn M., Matthey B., Schnell R., Tawadros S., Schinkothe T., Lorenzen J., Diehl V., Engert A. Recombinant anti-CD25 immunotoxin RFT5(SCFV)-ETA′ demonstrates successful elimination of disseminated human Hodgkin lymphoma in SCID mice.
Int. J. Cancer
,
86
:
718
-724,  
2000
.
39
Peipp M., Kupers H., Saul D., Schlierf B., Greil J., Zunino S. J., Gramatzki M., Fey G. H. A recombinant CD7-specific single-chain immunotoxin is a potent inducer of apoptosis in acute leukemic T cells.
Cancer Res.
,
62
:
2848
-2855,  
2002
.