| | The diphtheria toxin/urokinase fusion protein (DTAT) is selectively toxic to CD87 expressing leukemic cellsReceived 4 March 2002; accepted 22 April 2002. Abstract Diphtheria fusion proteins are a novel class of agents for the treatment of chemotherapy resistant acute myelogenous leukemia (AML). We prepared diphtheria toxin/urokinase fusion protein (DTAT) composed of the amino terminal fragment of the urokinase-type plasminogen activator (uPA) fused to the catalytic and translocation domains of diphtheria toxin (DT) and assessed its activity on leukemic cell lines. The number of uPA receptors (uPAR or CD87) was measured using a phycoerythrin conjugated monoclonal antibody to CD87 and flow cytometry. Seven of 23 cell lines (30%) showed CD87 expression (≥5000 receptors/cell). DTAT cytotoxicity (IC50≤30 pM) was observed in all seven of these samples and none of the 16 samples with low or absent CD87 expression. There was a significant correlation between DTAT sensitivity and CD87 density (P=0.0007). These results show that specific CD87 binding is one factor important in the sensitivity of patient’s leukemic blasts to DTAT and demonstrate for the first time that the CD87/uPAR can be used as a target for fusion protein therapy of AML.
Abbreviations:
DTAT, diphtheria toxin/urokinase fusion protein,
DT, diphtheria toxin,
AML, acute myelogenous leukemia,
uPA, urokinase-type plasminogen activator,
uPAR, uPA receptor,
IC50, concentration of fusion protein which inhibits cell proliferation by 50%,
GPI, glycosyl-phosphatidylinositol,
GM-CSF, granulocyte-macrophage colony-stimulating factor,
IL-3, interleukin-3
1. Introduction  Most patients with acute myelogenous leukemia (AML) are older than 60 years and respond poorly to intensified cytotoxic therapy [1]. Both reduced tolerance to the toxicities associated with intensive chemotherapy and chemoresistant blasts contribute to their poor prognosis. Less than 10% of patients will be in remission 3 years after beginning treatment [2]. New treatment strategies are needed for these patients. One new approach is the use of fusion proteins, composed of protein synthesis-inactivating peptide toxins covalently linked to AML-selective peptide ligands. Currently, we are developing fusion proteins consisting of the catalytic and translocation domains of diphtheria toxin (DT388) linked to human granulocyte-macrophage colony-stimulating factor (GM-CSF) and human interleukin-3 (IL-3). These fusion proteins are effective against the leukemic blasts from many but not all patients [3], [4]. We sought an additional ligand for targeting the remaining patients’ blasts. The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycosyl-phosphatidylinositol (GPI) anchored 50kDa membrane glycoprotein with three homologous 90 amino acid residue Ly-6-type domains [5]. The primary function of CD87 is to bind urokinase-type plasminogen activator (uPA) leading to uPA-catalyzed plasminogen activation at the cell surface and controlled breakdown of extracellular matrix during cell migration and invasion [6]. CD87 also binds vitronectin and β2-integrins and thus, plays a role in cell adhesion [7]. CD87 is expressed on the surface of blood neutrophils, eosinophils, monocytes, bone marrow CD34 negative myelomonocytic precursors, and mast cells [8], [9], [10], [11], [12], [13]. The receptor is also expressed on proliferating fibroblasts, fetal thyroid cells, and endothelial cells [14], [15], [16], [17], [18]. The protein is highly overexpressed on a variety of neoplastic cells including megakaryocytic leukemia, bladder transitional carcinomas, glioblastomas and most monocytic leukemias [8], [10], [19], [20], [21], [24]. The region of uPA which binds with high affinity to CD87 is the amino terminal EGF-like fragment, particularly a continuous hydrophobic patch in the Ω-loop region [22]. The uPA-CD87 complex undergoes internalization either mediated by interaction with plasminogen activator inhibitors and the low density lipoprotein receptor-related protein (LRP) [23] or by other poorly characterized mechanisms that may involve the cation-independent mannose 6-phosphate/insulin-like growth factor-II receptor (CIMPR) [24] or the urokinase-type plasminogen activator receptor associated protein (uPARAP) [25]. The uPA and the uPA amino terminal fragment has been fused to saporin and Pseudomonas exotoxin to yield selectively toxic fusion proteins [24], [26]. We chose to fuse the uPA amino terminus to the first 388 amino acid residues of diphtheria toxin encoding the enzymatic and translocation domains [26]. The fusion protein, DTAT, was expressed and partially purified to >95% homogeneity from Escherichia coli inclusion bodies. The goal of the present study was to evaluate the toxicity of the fusion protein towards leukemic cell lines and correlate the blast sensitivity to CD87 expression. We investigated a series of cell lines that exhibited a range of responses to the fusion protein. Our data supports the hypothesis that leukemia cell lines that were responsive to the fusion protein had far greater receptor density than cell lines that were less responsive to the treatment. 2. Material and methods 2.1. Recombinant DTAT DTAT was synthesized and partially purified as previously described [26]. Briefly, DNA encoding amino acid residues 1–390 of diphtheria toxin followed, in-frame, by amino acid residues 1–135 of urokinase was synthesized using the method of gene splicing and subcloned into the pET31d plasmid (Novagen, Madison, WI). Plasmid was then transformed into the E. coli strain BL21 (DE) (Novagen), and protein expression was induced with IPTG. Inclusion bodies were isolated and washed with Triton-X100 containing TE buffer (0.1M Tris pH 8, 2mM EDTA). Inclusion bodies were solubilized in denaturant buffer consisting of 7M guanidine, 0.1M Tris pH 8, and 2mM EDTA. After removal of insoluble material by centrifugation at 40,000×g for 10min, the supernatant was diluted rapidly 100-fold in chilled refolding buffer consisting of 0.1M Tris pH 8, 0.5M l-arginine, and 2mM EDTA. After 48h at 10°C, the protein was diafiltrated and ultrafiltrated against 20mM Tris pH 7.8 using a spiral membrane ultrafiltration cartridge on an Amicon CH2 System (Amicon, Beverly, MA). Samples were then loaded on a Q-Sepharose (Sigma) column and eluted with 1M NaCl in 20mM Tris pH 7.8. Protein was diluted five-fold, applied to a resource Q column (Pharmacia, Uppsala, Sweden), eluted with a linear salt gradient from 0 to 1M NaCl in 20mM Tris pH 7.8, and dialyzed against phosphate buffered saline (PBS). The material was then purified on a TSK 250 size-exclusion chromatography column (TosaHass, Philadephia, PA). The protein was stored in aliquots in PBS at −80°C until used. 2.2. Cell lines HL60, CEM, THP-1, U937, K562, Jurkat, Raji, and RPMI8226 were obtained from the American Type Culture Collection (Rockville, MD). ML2 was a generous gift of Dr. Janet Rowley (University of Chicago, Chicago, IL) [27]. Mono Mac 1, Mono Mac 6, MUTZ-2, CTV-1, OCI-AML-2, KG-1, UT-7, Kasumi-1, OCI-AML-5 and Sig M5 were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). TF/v-SRC was a gift of James McCubrey (East Carolina University, Greenville, NC). PER-377, ML1, and ML17 are cell lines derived in our laboratories [28], [29]. HL60, K562, TF/v-SRC, U937, CEM, Jurkat, Raji, KG-1, Kasumi-1, CTV-1 and ML2 were grown in RPMI 1640 medium with 10% heat-inactivated fetal calf serum supplemented with penicillin/streptomycin and l-glutamine. ML1 and ML17 were grown in Iscove’s modified Dulbecco’s medium with 10% heat-inactivated fetal calf serum supplemented with penicillin/streptomycin and l-glutamine. RPMI8226 was expanded in RPMI 1640 plus 10% heat-inactivated fetal calf serum supplemented with 1gm/l sodium bicarbonate, 4.5gm/l glucose, 10mM Hepes, penicillin/streptomycin and sodium pyruvate. Mono Mac 1 and Mono Mac 6 were grown in RPMI 1640 plus 10% heat-inactivated fetal calf serum supplemented with non-essential amino acids, sodium pyruvate, penicillin/streptomycin, l-glutamine, and 0.9μg/ml insulin. Sig M5 were grown in Iscove’s modified Dulbecco’s medium with 20% heat-inactivated fetal calf serum. PER-377 was grown in RPMI 1640 plus 10% heat-inactivated fetal calf serum supplemented with 10μM β-mercaptoethanol, non-essential amino acids, and sodium pyruvate. OCI-AML-M5 and MUTZ-2 cells were grown in α-minimal essential medium plus 20% fetal calf serum supplemented with 10% 5637 cell conditioned medium, l-glutamine and penicillin/streptomycin. OCI-AML-2 was grown in α-minimal essential medium plus 10% fetal calf serum supplemented with penicillin/streptomycin and l-glutamine. UT-7 were grown in the same medium as OCI-AML-2 but were supplemented with 0.5ng/ml GM-CSF. The properties of the cell lines and their sources are described in Table 1.  | Cell line | Description | Marker cytogenetics | Reference | |
| Sig M5 | AML-M5 | +8 | [32] | |
| U937 | AML-M0 | +16, +18, -X, -9, t(1;3)(q21;q27) | [34] | |
| PER-377 | AML-Biphenotypic | t(2;13)(p12;q34) | [29] | |
| ML2 | AML-M4 | t(6;11)(q27;q23) | [27], [51] | |
| Mono Mac 6 | AML-M5 | t(9;11)(p21;q23) | [30] | |
| ML17 | AML-M5 | del11q23b | Unpublished | |
| ML1 | AML-M4 | t(6;11)(q27;q23) | [28], [51] | |
| CTV-1 | AML-M5 | t(1;7)(p34,q34) | [47] | |
| THP-1 | AML-M5 | t(9;11)(p21;q23) | [33] | |
| OCI-AML-2 | AML-M4 | t(1;6)(q13;p12) | [48] | |
| Mono Mac 1 | AML-M5 | t(9;11)(p21;q23) | [31] | |
| Jurkat | T-ALL | -2,-18,del (2)(p21p23), del (18)(p11) | [39] | |
| TF/v-SRC | AML-M6 | t(2;22)(p11;q13), t(10,12) (q21;q15), t(2;14)(q21;p11) | [38] | |
| KG-1 | AML-M6 | t(6;8)(p11;q22) | [49] | |
| RPMI8226 | MM | t(8;22)(q24;q11) | [41] | |
| Raji | B-ALL | +7, +16, -8 | [40] | |
| K562 | CML-blast crisis | t(9;22)(q34;q11) | [35] | |
| HL60 | AML-M2 | -X, -5, +18 | [36] | |
| CEM | T-ALL | t(7;11) | [37] | |
| OCI-AML-5 | AML-M4 | t(1;19)(p13;p13) | [46] | |
| MUTZ-2 | AML-M2 | +8, +10 | [46] | |
| Kasumi-1 | AML-M2 | t(8;21) | [50] | |
| UT-7 | AML-M7 | t(2;4)(p16-23;q27-31) | [46] | | | | |
|
a
Most cell lines possess multiple karyotypic abnormalities. Only single or a few abnormalities listed. AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; T-ALL, T-cell ALL; B-ALL, B-cell ALL; MM, multiple myeloma. FAB classifications noted: M0, undifferentiated AML; M2, differentiated with maturation AML; M4, myelomonocytic AML; M5, monocytic AML; M6, erythroleukemia; M7, megakaryocytic leukemia.
b
Although the karyotype was interpreted as del11q23, molecular analysis demonstrated on MLL-AF5q 31 fusion. |
2.3. CD87 expression Two aliquots of 1 million cells for each of the cell lines were pelleted at 600×g and resuspended in 80 (test) or 100μl (control) PBS/1% bovine serum albumin (BSA). To one of each aliquot of cells (test), 20μl of R-phycoerythrin (PE) conjugated mouse anti-CD87 antibody (BD PharMingen, San Diego, CA) was added. The cells were incubated at 4°C for 30min, washed with PBS/1% BSA and resuspended in 1ml PBS/1% BSA. A volume of 250μl of 10% formalin was added to each tube, and the cells were assayed on an EPICS-XL flow cytometry (Coulter, Hialeah, FL) with filters set for PE fluorescence detection. U937 cells were assayed on every experiment, and QuantiBrite beads (BD Biosciences, Mountain View, CA) were used to determine the ratio of PE fluorescence channel/number of PE molecules. The PE conjugate used had one molecule of PE per antibody. CD87 expression was calculated, assuming single antibody molecules bound per CD87 cell surface antigen (also referred to as ABC or antibody binding capacity), as the product of (median fluorescence channel with antibody minus median fluorescence channel without antibody) × (number of PE molecules/fluorescence channel). All samples were repeated at least once on separate days with inter-assay variability for ABC of ≤50%. The QuantiBrite beads analyzed on an EPICS flow cytometer with 660V FL2 photomultiplier setting yielded peaks at 0.3, 11, 34, and 70 for 570, 13,400, 37,000 and 78,000 phycoerythrin molecules per bead. This corresponds well with the predicted peaks supplied by the manufacturer. 2.4. Cell line sensitivity to DTAT Aliquots of 104 cells were incubated in 100μl medium (same as that used to grow the cells) in Costar 96-well flat-bottomed plates in duplicate. Fifty microlitres of DTAT in medium was added to each column to yield concentrations ranging from 0.1 to 10,000pM, and the cells were incubated at 37°C/5% CO2 for 48h. Then, 1μCi  -thymidine (NEN DuPont, Boston, MA) in 50μl medium was added to each well, and incubation continued for an additional 18h at 37°C/5% CO2. Cells were then harvested using a Skatron Cell Harvestor (Skatron Instruments, Lier, Norway) on to glass fiber mats and counts per minute of incorporated radiolabel were counted using an LKB liquid scintillation counter gated for  . The IC50 was defined as the concentration of toxin which inhibited thymidine incorporation by 50% compared to control wells. The percent maximal  -thymidine incorporation was plotted versus the log of the toxin concentration, and non-linear regression with a variable slope sigmoidal dose response curve was generated along with IC50 using GraphPad Prism software (GraphPad Software, San Diego, CA). Linear regression analysis was used to compare the CD87 density and DTAT IC50 with GraphPad Prism software. All assays were performed at least twice with inter-assay range of ≤30% for IC50. 3. Results Moderate to high level CD87 expression was found on 7 of 23 (30%) leukemia cell lines ranging from 6600 to 25,000 receptors/cell (Table 2). Low level expression (240–3800 receptors/cell) was observed on 10 cell lines (seven AML cell lines and four lymphoid cell lines). The 7/18 (39%) of AML cell lines had moderate to high CD87 expression. Most of the AML cell lines tested were derived from patients with M4 or M5 phenotype. | Cell line | CD87 receptors/cell (mean ± range) | DTAT IC50 (pM, mean ± range) | |
| Sig M5 | 31,000 ± 5,000 | 18 ± 7 | |
| ML17 | 22,000 ± 10,000 | 10 ± 1 | |
| U937 | 19,000 ± 2,000 | 15 ± 3 | |
| PER-377 | 16,000 ± 500 | 30 ± 1 | |
| ML2 | 13,000 ± 500 | 16 ± 2 | |
| Mono Mac 6 | 11,000 | 20 ± 3 | |
| ML1 | 6,600 ± 1,000 | 5 ± 1 | |
| CTV-1 | 2,900 ± 900 | >4000 | |
| THP-1 | 2,500 ± 1,000 | 1000 ± 100 | |
| UT-7 | 1,800 ± 100 | 60 ± 5 | |
| OCI-AML-2 | 920 ± 200 | 210 ± 10 | |
| Mono Mac 1 | 950 ± 400 | 160 ± 15 | |
| Jurkat | 800 ± 50 | 71 ± 3 | |
| TF/v-SRC | 570 | 48 ± 3 | |
| KG-1 | 420 ± 10 | >4000 | |
| RPMI8226 | 420 ± 190 | 200 ± 40 | |
| Raji | 120 ± 100 | >4000 | |
| Kasumi-1 | 80 ± 80 | 410 ± 50 | |
| K562 | 0 | >4000 | |
| HL60 | 0 | 110 ± 1 | |
| CEM | 0 | >4000 | |
| OCI-AML-5 | 0 | >4000 | |
| MUTZ-2 | 0 | >4000 | | | | |
|
a
Receptor density and IC50 determined as described in the text. |
DTAT fusion protein was cytotoxic to CD87 positive leukemia cell lines with seven of 23 cell lines showing IC50 of ≤30pM. All the sensitive cell lines were from CD87 positive AML patients (Fig. 1). In contrast, blasts from patients with either low or absent CD87 expression or with non-myeloid leukemias were insensitive or poorly sensitive to DTAT. 4. Discussion CD87 is expressed in several normal human tissues and many different tumor cells. Since cell surface receptor density is an important determinant of fusion protein toxicity, the relative expression of CD87 on normal and malignant cells may predict normal tissue toxicities and efficacy of CD87-directed fusion protein therapy. Cell surface CD87 has been measured by immunoblots, immunohistochemistry, flow cytometry and radiolabeled uPA binding. Studies of cell surface CD87 have been done for normal hematopoietic cells, for a few normal non-hematopoietic tissues including vascular endothelium, fibroblasts, and thyroid epithelium, and for many tumor cell lines [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. Binding of the  -labeled N-terminal fragment of uPA revealed normal mast cells had high level CD87 expression (200,000/cell), monocytes and neutrophils had moderate level CD87 expression (3000–6000/cell), resting T lymphocytes had 2000 receptors/cell, activated T lymphocytes had 20,000 receptors/cell and B lymphocytes, platelets and red blood cells lacked measurable receptors [8], [9], [11], [12]. Flow cytometry with or without quantitation by fluorescent beads showed absence of CD87 from CD34 positive myeloid stem cells, and 3000–5000 receptors/cell on normal granulocytes, monocytes, eosinophils and committed myeloid progenitors [10], [13]. CD87 density has been measured quantitatively on non-hematopoietic tissues by  -labeled uPA binding and qualitatively by flow cytometry, immunoblots, cell extract enzyme immunoassays and immunohistochemistry. By radiolabeled uPA binding, there were 10,000 CD87 molecules per human microvascular endothelial cell [14], 100,000 receptors/human umbilical cord endothelial cell [15] and 100,000/cell for fetal lung fibroblasts [16], [17]. Immunoblots revealed CD87 on thyroid cells [18]. Malignant cell lines of megakaryocytic leukemia [19], bladder transitional carcinoma [20] and glioblastoma origin [21], [24] showed high numbers of CD87 (20,000–100,000 receptors/cell). Overexpression (>5000–10,000/cell) has also been reported for leukemic blasts of patients with monocytic leukemia, leukemia cutis, hepatosplenomegaly, involvement of the central nervous system, and/or complex chromosomal abnormalities or abnormalities of chromosomes 11q or 5 [8], [10]. Our results extend the above findings. Among the 23 leukemia cell lines tested, seven (30%) showed >5000 antibody binding sites or CD87 molecules/cell. Among the 18 AML cell lines tested, seven (39%) showed ≥5000 antibody binding sites or CD87 molecules/cell. Most of these cell lines were derived from patients with acute monocytic or myelomonocytic leukemias. A second requirement for fusion protein cytotoxicity is internalization of the fusion protein–receptor complex. Diphtheria toxin and diphtheria fusion proteins must be transported to the endosomal compartment where they are acidified and processed by furin-like proteases [42]. The translocation domain amphipathic helices insert in the endosomal membrane facilitating the escape of the catalytic A fragment to the cytosol where it refolds and inactivates multiple elongation factor 2 molecules leading to arrest of protein synthesis and cell death. The uPA and uPA conjugate endocytosis occurs by one of several mechanisms. Binding of plasminogen activator–inhibitor (PAI) to uPA and CD87 recruits the LRP. The uPA–CD87–PAI–LRP complex is internalized [43]. While the N-terminus of uPA lacks the binding motif for PAI, residues in peptide toxins are able to directly recruit LRP, the CIMPR, or other unknown receptors [23], [24], [25], [26]. Thus, the sensitivity of leukemic cell lines to DTAT may depend upon the density of CD91 (LRP) or other unknown receptors. All seven of the CD87 over-expressing leukemic cell lines were sensitive to DTAT suggesting leukemic blasts contain the necessary co-receptors for internalization. DTAT potency on leukemic blasts compares favorably with the other fusion proteins targeted to CD87 [24], [44], [45]. The Pseudomonas exotoxin fusion had an IC50 on U937 cells indistinquishable from DTAT [24]. The saporin fusion had an IC50 of 60pM on the same cells [43], [44]. Thus, CD87 appears to be an excellent target for delivery of a number of peptide toxins to leukemic blasts. In contrast to the correlation between receptor density and DTAT fusion protein sensitivity of leukemic blasts, no such relationship was found for non-hematopoietic tumors and ATF-PE38KDEL sensitivity [24]. The lack of correlation for these tumors may be due to the use of a different toxin moiety or the variable expression on these heterogeneous tumors of the required co-receptors for internalization such as LRP, CIMPR, or uPARAP. The finding of potent cytotoxicity of DTAT to AML blasts suggests that this may be a useful therapeutic for treatment of chemoresistant CD87 positive AML patients. DTAT may produce injury to CD87 positive, proliferating vascular endothelium in patients with consequent vascular leak [14], [15]. However, the effect of DTAT on resting endothelium in vivo is unknown. 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