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Variant Diphtheria Toxin–Interleukin-3 Fusion Proteins with Increased Receptor Affinity Have Enhanced Cytotoxicity against Acute Myeloid Leukemia Progenitors

Authors' Affiliations: ¹ Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada and ² Department of Medicine, Scott and White Hospital, Temple, Texas

Requests for reprints: Donna E. Hogge, Terry Fox Laboratory, British Columbia Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3. Phone: 604-675-8138; Fax: 604-877-0712; E-mail: dhogge{at}bccancer.bc.ca.

	Abstract

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A fusion protein linking a truncated form of diphtheria toxin (DT₃₈₈) to human interleukin-3 (DT₃₈₈IL3) kills malignant progenitors from some patients with acute myeloid leukemia (AML) while sparing normal progenitors. This study evaluated two variants of DT₃₈₈IL3 with increased affinity for the IL-3 receptor (IL-3R) for their cytotoxicity to AML progenitors and determined the ability of quantitative reverse transcription-PCR assessment of expression of the IL-3R subunits to predict the effectiveness of wild-type DT₃₈₈IL3 and its variants. Both the IL-3 deletion variant (125-133) and the amino acid substitution variant (K116W) showed enhanced toxicity against AML colony-forming cells (AML-CFC; but not normal CFC) compared with wild-type DT₃₈₈IL3 with the K116W variant achieving >90% AML-CFC kill with 17 of 23 patient samples. This variant was also more effective against AML cells engrafting in nonobese diabetic severe combined immunodeficient mice. There was a significant correlation between the expression of the and, particularly, the common ß subunit of the IL-3R on AML blasts detected by quantitative reverse transcription-PCR and AML-CFC kill. Thus, the combined use of IL-3R expression to select patients most likely to respond to DT₃₈₈IL3 and the improved cytotoxicity of the K116W DT₃₈₈IL3 variant against leukemic progenitors may enhance the clinical usefulness of these fusion proteins.

The human interleukin-3 (IL-3) receptor (IL-3R) is a heterodimeric structure (3). The subunit is essential for ligand binding and confers specificity on the receptor. The common ß (ß_c) subunit, which is shared by the granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-5 receptors among others, is required for high-affinity ligand binding and signal transduction. The IL-3R is expressed on leukemic blasts from the large majority of patients with AML (4–6). The subunit, in particular, is often expressed at much higher levels than are typically seen in normal hematopoietic cells and progenitors and is also detected on subpopulations of AML blasts enriched for malignant progenitors that engraft nonobese diabetic severe combined immunodeficient (NOD/SCID) mice (4, 7–10). Thus, the IL-3R may be an appropriate target for cytotoxic drugs designed to selectively kill AML cells while sparing their normal hematopoietic cell counterparts.

Fusion proteins in which the diphtheria toxin (DT) catalytic and translocation domains are genetically fused to ligands that can selectively target malignant cells are a novel class of molecules that induce cell death by a different mechanism than conventional chemotherapy drugs (11–13). After ligand binding and receptor-mediated endocytosis, the toxin translocates to the cytosol where it ADP-ribosylates elongation factor 2, leading to inactivation of protein synthesis and cell death (14–17). The truncated form of DT₃₈₈ has been linked to a variety of growth factors (18–21). Among the resulting drugs is DT₃₈₈IL3, which, in previous studies, has been shown to kill AML colony-forming cells (AML-CFC), AML long-term culture-initiating cells, and AML cells that engraft in NOD/SCID mice from many patient samples while showing little or no toxicity against analogous normal bone marrow progenitors (5, 22). Although DT₃₈₈IL3 is effective against many AML samples, only partial eradication of malignant progenitors was seen with some leukemias in spite of detectable expression of high affinity IL-3R on target blasts. To attempt to improve on these results, two variants of the DT₃₈₈IL3 molecule have been constructed to enhance IL-3R binding affinity of the toxin. For the K116W variant substitution of a large, bulky hydrophobic tryptophan residue at position 116 in IL-3 was done, which was expected to increase high-affinity interactions with the IL-3R (23, 24). Deletion of COOH-terminal residues 125 to 133 was done in a second construct to enhance the electrostatic interactions between IL-3 and IL-3R. Consistent with these expectations, in initial testing, the K_d values for the wild-type (WT), K116W variant, and the 125-133 variant of DT₃₈₈IL3 were 1,500, 370, and 680 pmol/L, respectively, when tested against the IL-3R-bearing cell line TF/H-ras (25). When tested against a series of IL-3R⁺ AML cell lines, the IC₅₀ for the variant fusion proteins was reduced 6-fold to >10-fold for the K116W variant and 3-fold to 4-fold for the 125-133 variant compared with WT DT₃₈₈IL3 (25). Given these encouraging results, we now report a comparison of the ability of these same variant fusion proteins and WT DT₃₈₈IL3 to kill AML-CFC from 28 newly diagnosed leukemia patients and clonogenic progenitors from three normal bone marrows. In addition, the cytotoxicity of the drugs against NOD/SCID mouse-engrafting AML cells from three AML patients is presented as well as the levels of IL-3R and ß_c subunit RNA in AML blasts as quantitated by real-time reverse transcription-PCR (qRT-PCR). The results show enhanced efficacy for both variants against AML-CFC that express sufficient amounts of the target IL-3R with little change in toxicity detected toward normal CFC. In particular, the K116W variant of DT₃₈₈IL3 showed markedly enhanced cytotoxicity against leukemic progenitors, including those "candidate" leukemic stem cells that engraft in NOD/SCID mice (9, 26).

	Materials and Methods

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AML and normal cells. Peripheral blood samples from newly diagnosed AML patients and normal marrow samples were obtained with the approval of the Clinical Research Ethics Board of the University of British Columbia and after obtaining informed consent from AML patients and processed and cryopreserved as previously described (27). Diagnosis and classification of AML were based on the criteria of the French-American-British group (28). Cytogenetic analysis was done on the bone marrow at initial diagnosis. Clinical characteristics of the 28 patients whose cells were studied are listed in Table 1. Normal bone marrow was obtained from cadaveric donors (North West Tissue Center, Seattle, WA). Mononuclear cells were isolated and cryopreserved as previously described (5).

Cultures of AML and normal bone marrow cells. Peripheral blood cells from AML patients and normal bone marrow cells were incubated at a concentration of 1 x 10⁶ cells/mL with 50 ng/mL granulocyte-CSF with or without WT DT₃₈₈IL3 or its variants at the indicated concentrations. After 24-hour incubation, equal fractions of the cells recovered from cultures with or without toxin were assayed without regard to change in cell numbers. Treated and untreated cells were plated in methylcellulose medium for detection of normal and leukemic CFC or injected i.v. into NOD/SCID mice (7) for detection of engrafting AML cells. Assays for AML-CFC were done by plating cells at 0.2 to 1.0 x 10⁵ cells/mL in methylcellulose medium (StemCell Technologies) supplemented with growth factors as previously described (27). Cultures were scored after 14 days at 37°C for clusters (4-20 cells) and colonies (>20 cells; ref. 27). Normal bone marrow CFCs were detected by plating cells in methylcellulose medium (Methocult H4330; StemCell Technologies) supplemented with growth factors as described (30). Granulopoietic, erythroid, and mixed colonies detected after 16-day incubation at 37°C were scored as described (30).

NOD/SCID mice. NOD/LtSz-scid/scid (NOD/SCID) mice (7) were bred and maintained under sterile conditions in the British Columbia Cancer Research Center Joint Animal Facility according to protocols approved by the Animal Care Committee of the University of British Columbia. Eight- to 10-week-old mice were irradiated with 350 cGy from a ¹³⁷Cs source 24 hours before injection of AML cells. AML cells (2 x 10⁶ to 1 x 10⁷) cultured 24 hours with or without WT DT₃₈₈IL3 or one of its variants were injected into each mouse via the tail vein. After injection of AML cells into mice, a bone marrow aspiration was done every 4 weeks from the femur after anesthesia with inhaled isoflurane to obtain cells for fluorescence-activated cell sorting (FACS) analysis (31). Eight to 16 weeks postinjection, the mice were killed by CO₂ inhalation. Bone marrow was obtained from the four long bones by flushing with -MEM with 50% FCS (26).

Analysis of mice. Cells were prepared for FACS analysis as previously described (26). Half of the cells were incubated 30 minutes on ice with a mouse IgG1 isotype control (Becton Dickinson Immunocytometry Systems, San Jose, CA) and the other half were incubated with fluoresceinated anti-CD45, a human-specific pan-leukocyte marker (prepared in our center from American Type Culture Collection, Rockville, MD, clone HB10508). FACS analysis was done on a Becton Dickinson FACScan or FACSort flow cytometer. The %CD45⁺ cells was determined after excluding 99.9% of cells labeled with the isotype control and nonviable cells. Nonspecific binding of CD45 on mouse bone marrow cells is reliably <0.1% (26). Thus, a difference between the IgG1-negative control and the CD45 expression of the treated mice of >0.1% was regarded as evidence for engraftment of human cells. Values shown for engraftment of AML cells in mouse tissues are the mean values of %CD45⁺ cells obtained for all mice in the cohort that survived to the time of analysis.

Real-time qRT-PCR. Total RNA was extracted from AML cells using the Absolutely RNA Microprep kit (Stratagene, La Jolla, CA). The reverse transcription reaction was done in 20 µL with Superscript II reverse transcriptase (Invitrogen, Burlington, ON, Canada) using random hexamer oligonucleotides (Amersham Pharmacia, Piscataway, NJ). Quantitative PCR was done using 12.5 µL 2 x SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 1 µL of 20 pmol/L specific primers, 1 to 2 µL cDNA, and water to a final volume of 25 µL. Specific forward and reverse primers to produce 100 bp amplicons for optimal amplification in real-time PCR of reverse-transcribed cDNA for the human IL-3R subunit were 5'-GACCTGTACTTGAACGTTGCC and 5'-GAAACGACACCCGATACGTGT, for human IL-3R ß_c subunit were 5'-GCAGCATGTCGGCCTTCACTA and 5'-GTCCCCGAATCCTACAGGGAA, for human IL-3 were 5'-CCAAGCTCCCATGACCCAGACA and 5'-CTGTTAGAGCAGTTAACCCAGC and for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5'-CCCATCACCATCTTCCAGGAG and 5'-CTTCTCCATGGTGGTGAAGACG. qRT-PCR and data analysis were done on an iCycler iQ system, using iCycler iQ Real-time Detection Software (Bio-Rad, Hercules, CA). The relative quantification of human, IL-3R , IL-3R ß_c, and IL-3 compared with a reference gene (GAPDH) was generated on the basis of a mathematical model for relative quantification in qRT-PCR as described (32, 33).

IL-3 ELISA. Serum-free medium (StemCell Technologies) was collected from AML peripheral blood cells that had been cultured at 2 x 10⁶ cells/mL for 72 hours in the absence of growth factors and concentrated 10-fold with Centricon-10 concentrators (Amicon, Beverly, MA). IL-3 concentration was measured using a quantitative sandwich enzyme immunoassay technique with Quantikine kits (R&D Systems, Minneapolis, MN).

Statistical analysis. The mean percentage kill of AML and normal CFC achieved with each dose of WT or variant DT₃₈₈IL3 was compared using a paired, two-tailed Student's t test. Mean CD45⁺ cells detected in the bone marrows of cohorts of mice receiving AML cells treated with the different fusion proteins were also compared using a two-sided Student's t test. P < 0.05 was considered significant. A correlation coefficient between the level of expression of the IL-3R and ß subunits and IL-3 (relative to GAPDH) and the percentage kill of AML-CFC was determined (Excel, Microsoft, Redmond, WA) and the significance of the correlation determined using the Student's t test.

	Results

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Patient characteristics. Table 1 shows the clinical characteristics of the 28 AML patients whose leukemic progenitors were tested in this study. The presenting WBC count was high in the majority (median, 151 x 10⁹/L; range, 24-457 x 10⁹/L) and six presented with cytogenetic abnormalities in their diagnostic bone marrow sample associated with an adverse prognosis as defined by the Medical Research Council (United Kingdom) and/or Southwest Oncology Group criteria (34, 35). Although 15 of these patients obtained an initial complete remission with conventional chemotherapy, six failed to achieve a remission and seven died of early complications. The median survival from the time of diagnosis for the 28 patients was 6.5 months.

Enhanced cytotoxicity of variant DT₃₈₈IL3 molecules against AML but not normal CFC. AML blasts from these 28 newly diagnosed leukemia patients were incubated for 24 hours with concentrations of WT DT₃₈₈IL3 or the K116W or 125-133 variants ranging from 1 to 250 ng/mL. Treated and untreated cells were then plated in AML-CFC assays to assess the relative cytotoxicity of the fusion proteins against these progenitors. Greater than 50% kill of AML-CFC was obtained with one or more of the DT₃₈₈IL3 variants for 23 of these AML samples (Table 2). As shown on Table 3, the mean percentage kill of AML-CFC from these was significantly increased with both the K116W and 125-133 variants compared with WT DT₃₈₈IL3 for each of the drug concentrations tested. The K116W variant also achieved a significantly higher (P < 0.001; two-tailed, paired t test) %AML-CFC kill than the 125-133 variant for three of the four drug concentrations tested. More than 90% kill was achieved with the K116W variant for 17 of these 23 samples with concentrations as low as 1 ng/mL (Table 2). In addition, >95% kill of AML-CFC was achieved with the K116W variant with samples from four of six AML patients that failed to enter remission with induction chemotherapy (samples 6, 7, 8, and 28; Tables 1 and 2).

View larger version (11K): [in this window] [in a new window]   Fig. 1. IL-3R subunit expression on AML blasts as detected by qRT-PCR. RNA was extracted from leukemic blasts from the 28 patient samples shown on Table 1 and reversed transcribed for qRT-PCR as described in Materials and Methods. Values shown are the levels of expression of the and ßc subunits of the IL-3R relative to the expression of GAPDH arbitrarily set at 1,000. Each data point represents the value for an individual patient sample. Horizontal bars and adjacent numbers, median level of expression for each of the and ßc subunits.

View larger version (15K): [in this window] [in a new window]   Fig. 2. AML-CFC kill correlates with expression of IL-3R subunits on leukemic blasts. The level of expression of the IL-3R subunits is plotted against the percentage kill of AML-CFC achieved for the same patient sample with 50 ng/mL of the K116W variant of DT388IL3. The significance of the correlation coefficient was determined using the Student's t test. A, IL-3R subunit. B, IL-3Rßc subunit.

The K116W variant of DT₃₈₈IL3 has enhanced cytotoxicity against AML progenitors that engraft in NOD/SCID mice. To assess the relative ability of the WT and K116W variant of the DT₃₈₈IL3 molecules to kill AML progenitors that engraft NOD/SCID mice, in a final set of experiments AML blasts from three patient samples were incubated for 24 hours with or without one of the fusion toxins at several concentrations and then injected into cohorts of sublethally irradiated NOD/SCID mice. As shown in Fig. 3, ex vivo treatment with the fusion toxins reduced the level of engraftment of all three AML samples in mice compared with untreated cells. However, complete eradication of leukemic progenitors was not achieved with even the highest concentration of WT DT₃₈₈IL3 tested. The K116W variant was more effective than WT DT₃₈₈IL3 with all three AML samples. In particular, among the mice injected with AML cells previously treated with 50 or 250 ng/mL of the K116W variant, 6 of 13 and 7 of 8 mice, respectively, had no AML cells detectable in their bone marrow 16 weeks after injection of the treated AML cells (Fig. 3) or at the prior bone marrow examinations on weeks 4, 8, and 12. As shown in the table below Fig. 3, the increased cytotoxicity of the K116W variant was also seen against AML-CFC from the same patient samples.

View larger version (32K): [in this window] [in a new window]   Fig. 3. The K116W variant of DT388IL3 shows enhanced cytotoxicity against NOD/SCID leukemia-initiating cells. AML cells from three patients listed on Table 1 were incubated for 24 hours with or without WT DT388IL3 or the K116W variant at various concentrations. Cohorts of five or six sublethally irradiated NOD/SCID mice were then injected i.v. with the same dose of treated or untreated cells. Data are shown for the last point of analysis 12 to 16 weeks after injection of the mice with leukemia cells. Columns, mean percentage of human AML cells in mouse bone marrow among the cohort of mice receiving the same patient cells and the same treatment; bars, SE for each mouse cohort. *, cohorts of mice where the mean percentage of human AML cells in mouse marrow was <0.1% (i.e., below the limits of detection in the FACS assay).

	Discussion

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The results show that both variants of DT₃₈₈IL3 are more cytotoxic against AML progenitors than the WT molecule. In addition, the K116W variant was more effective in killing AML-CFC than the 125-133 variant. The AML samples studied were obtained from patients with a generally poor prognosis as illustrated by their relatively high presenting WBC count, the frequency of cytogenetic abnormalities associated with an adverse prognosis and their poor overall survival in spite of conventional cytarabine and anthracycline-based induction and consolidation chemotherapy. Nevertheless, the K116W variant of DT₃₈₈IL3 achieved >90% kill of AML-CFC from most patient samples (Table 2) and almost completely eradicated leukemic cells capable of engrafting in NOD/SCID mice from three samples (Fig. 3). In contrast, WT DT₃₈₈IL3 was able to kill 90% of AML-CFC from only 7 of 28 samples and eradication of NOD/SCID mouse engrafting progenitors was also much less effective. Thus, the K116W variant of DT₃₈₈IL3 seems to be more effective than either the WT or 125-133 variant against AML progenitors detected either in vitro or in vivo. On the other hand, this increased toxicity is less evident against normal bone marrow CFC, many of which survived treatment with even the highest doses of these drugs (maximum normal CFC kills with 250 ng/mL of the K116W variant were 54%, 60%, and 48% for each of the three marrows tested).

AML-CFC from 5 of the 28 AML samples showed <50% kill even when treated with the highest concentrations of DT₃₈₈IL3 and its variants. Among the 23 samples where greater AML-CFC kill was obtained, the concentration of fusion toxin required to achieve maximum kill varied from 1 to 250 ng/mL. Previous data using radiolabeled ligand to quantitate high- and low-affinity IL-3 or GM-CSF binding sites on leukemic cells had shown that such binding was necessary for both DT₃₈₈IL3 and DT₃₈₈GM-CSF cytotoxicity (6, 38, 39). However, this measurement did not explain all the variability in cell kill observed with these agents. In addition, the technique used to show binding requires large cell numbers and would not be practical as a rapid screening technique for clinical purposes. Thus, the possibility that IL-3R subunit expression as detected by qRT-PCR might predict the sensitivity of AML progenitors to DT₃₈₈IL3 was investigated. As expected from previously published data using flow cytometry (4, 8) to detect IL-3R expression, the IL-3R subunit was expressed at much higher levels than the ß_c subunit on AML blasts. The relative abundance of the mRNA for both subunits varied by >10-fold among AML samples. A significant correlation between AML-CFC kill and the expression of both subunits was seen (Fig. 2). However, this correlation was stronger for the ß_c subunit than for the subunit. This finding and the generally lower level of ß_c subunit expression on the target cells suggests that IL-3R ß_c expression may be a limiting factor in permitting high-affinity binding and internalization of DT₃₈₈IL3 and subsequent killing of leukemic progenitors. The current data also suggest that it may be possible to determine a threshold level of IL-3Rß_c expression above which effective cytotoxicity of DT₃₈₈IL3 or its variants can be expected (Fig. 2). On the other hand, interference with binding of DT₃₈₈IL3 to the target IL-3R by endogenous production of IL-3 from AML blasts does not seem to explain the occasional lack of effectiveness of this drug because only a minority of blast samples express IL-3 and the killing of AML-CFC was very similar from samples where IL-3 was or was not detected. Other possible explanations for the ability of some leukemic progenitors to escape the toxicity of DT₃₈₈IL3 and its variants include lower expression of IL-3R on the subpopulation of AML cells with progenitor activity compared with the general population of blasts, lack of correlation between IL-3R mRNA and protein expression, ineffective receptor internalization, and intrinsic resistance of some AML cells to apoptotic mechanisms induced by the fusion proteins (29). These possibilities are currently being investigated. Previous experiments have tested the possibility that reduced growth factor receptor expression among some AML progenitors might allow them to escape DT-fusion protein treatments and regenerate leukemia in NOD/SCID mice. However, no difference was detected between the number of growth factor receptors detected on AML cells recovered from mice that had received DT-growth factor fusion protein therapy and the same AML cells before injection into mice (21).

Interestingly, other investigators have shown a correlation between the degree of AML blast cell cytotoxicity seen with DT₃₈₈IL3 and the K116W variant and the level IL-3R subunit expression as detected by flow cytometry (40). Thus, not surprisingly, it seems that the effectiveness of DT₃₈₈IL3 and its variants requires substantial expression of the receptor on target cells. It is also encouraging that much of the variability in responsiveness that was previously observed among AML samples treated with WT DT₃₈₈IL3 seems to have been eliminated with the use of the variant K116W fusion protein even when the drug is tested against AML progenitors that engraft in NOD/SCID mice, which had previously proved to be relatively resistant to WT DT₃₈₈IL3 compared with AML-CFC (5, 37).

New therapeutic options are needed in AML where the standard treatment offered to most patients has not changed substantially for three decades and the outcome has remained poor for the majority of these individuals (1, 2). This is particularly true for elderly AML patients and those whose disease is refractory to initial chemotherapy or relapses after an initial remission. Drugs such as DT₃₈₈IL3 with novel mechanisms of action that bypass the usual pathways of chemotherapy resistance offer a promising new therapeutic approach. The current data suggest that the K116W variant of DT₃₈₈IL3 is worthy of further preclinical development. They also suggest that quantitation of IL-3R subunit expression on AML blasts and progenitors may allow selection of patients who are most likely to benefit from such new agents.

	Footnotes

 
Grant support: Canadian Institute for Health Research (D.E. Hogge), NIH grants R01CA76178 and R01CA90263 (A.E. Frankel), and Leukemia and Lymphoma Society grant 6006-05 (A.E. Frankel).

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 9/21/05; revised 11/29/05; accepted 12/ 9/05.

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