University of Minnesota Cancer Center, 1 Departments of Therapeutic Radiology (Section on Experimental Cancer Immunology) and 4 Pediatrics, Division of Bone Marrow Transplantation, Minneapolis, MN 55455, USA, 3 Department of Microbiology, College of Medicine,Dong-A University, Pusan, South Korea and 5 Dana-Farber Cancer Institute, Boston, MA, USA
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Abstract |
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Keywords: bone marrow transplantation/cancer/diphtheria toxin/IL-3/immunotoxin
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Introduction |
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The high affinity IL-3 receptor (IL-3R) is composed of and ß subunits (Hara and Miyajima, 1992
). The binding of IL-3 to its receptor causes rapid internalization of the ligandreceptor complex (Nicola et al., 1988
). Because of the internalization of IL-3, we reasoned that IL-3 could serve as a ligand for delivering a toxic molecule such as diphtheria toxin to the cells bearing the IL-3 receptors. Diphtheria toxin (DT) is a well-studied glycoprotein with a molecular weight of 58 kDa. Diphtheria toxin has potent cytotoxic ability through ADP-ribosylation of elongation factor-2, resulting in inhibition of cellular protein synthesis and cell death. Delivering a single DT molecule into the cytoplasm is sufficient to kill a cell (Yamaizumi et al., 1978
). Native DT contains three domains: the cell binding domain, the translocation domain and the enzymatic cytotoxic domain (Middlebrook et al., 1978
; Lambert et al., 1980
). The cell binding domain of the DT gene can be replaced by a growth factor gene, resulting in a toxin-growth factor hybrid gene, whose protein product is targeted to a specific growth factor receptor (Murphy et al., 1986
; Williams et al., 1987
; Jean and Murphy, 1991
; Lakkis et al., 1991
; Chadwick et al., 1993
; Chan et al., 1995
; Frankel et al., 1997
; Kreitman et al., 1997; Terpstra et al., 1997
).
Because IL-3R is expressed on most myeloid leukemia cells, we evaluated the anti-leukemic potential of murine DT390IL-3. Unlike other models assessing the promise of human agents against human tumors in scid mice, we pursued the study of murine IL-3 in syngeneic mice so that we could assess native toxicity as well as efficacy. Knowing that the autocrine production of IL-3 could contribute to the transformed phenotype of myeloid leukemia cells (Laker et al., 1989; Stocking et al., 1989), we used a model in which the introduction of a BCR/ABL oncogene into an IL-3-dependent myeloid cell line 32D eliminated the requirement for IL-3 for growth and rendered these cells leukemic in syngeneic mice (Daley and Baltimore, 1988
; Daley et al., 1991
; Matulonis et al., 1993
; Salgia et al., 1995
). Because we observed acute toxic effects and lack of uniform activity of DT390IL-3 against uncommitted BM progenitor cells, we studied the potential of this agent as a purging agent for eliminating leukemic cells from BM without destroying the ability of BM cells to engraft and then for its potential for treating leukemia cells in vivo. These studies indicate that DT390IL-3 has anti-leukemia potential, especially for purging BM of lethal myeloid leukemia cells.
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Materials and methods |
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Recombinant DT390anti-IL-3 was synthesized as previously described (Chan et al., 1996). The hybrid gene was constructed by the method of gene splicing by overlap extension (SOE) and ligated into plasmid pET11d (Novagen, Madison, WI). Restriction endonuclease digestion and DNA sequencing analysis (University of Minnesota Microchemical Facility) were used to verify that the hybrid gene had been cloned in-frame. Plasmid was transformed into the Escherichia coli strain BL21(DE3) (Novagen, Madison, WI). Expression was induced and protein was refolded and purified from inclusion bodies. The pellets were washed three times with Triton X-100 buffer and four times with TE buffer by briefly homogenizing with a tissuemizer and incubating for 510 min. Inclusion bodies were collected by centrifugation at 24 000 g for 50 min. Solubilization of the inclusion body pellet was achieved by sonicating in denaturant buffer consisting of 7 M guanidine, 0.1 M Tris, pH 8.0 and 2 mM EDTA. The solution was incubated at room temperature for 16 h in the presence of 65 mM dithioerythritol (DTE). To remove insoluble material, the solution was centrifuged at 40 000 g for 10 min and the supernatant was collected. Renaturation was initiated by a rapid 100-fold dilution of the denatured and reduced protein into chilled refolding buffer consisting of 0.1 M TrisHCl, pH 8.0, 0.5 M L-arginine, 0.9 mM oxidized glutathione (GSSG) and 2 mM EDTA. The samples were incubated at 10°C for 48 h. The refolded protein was diafiltrated and ultrafiltrated against 20 mM TrisHCl, pH 7.8 using a spiral membrane ultrafiltration cartridge on Amicon's CH2 system (Amicon, Beverly, MA). Samples were loaded on a Q-Sepharose (Sigma, St Louis, MO) column and eluted with 0.3 M NaCl in 20 mM TrisHCl, pH 7.8. The protein was diluted fivefold and subsequently applied to a Resource Q column (Pharmacia, Uppsala, Sweden) and eluted with a linear salt gradient from 0 to 0.4 M NaCl in 20 mM TrisHCl, pH 7.8. The main peak from the Resource Q column was purified by size-exclusion chromatography on a TSK 250 column (TosoHass, Philadelphia, PA). DT390 fusion immunotoxins synthesized with GM-CSF (Chan et al., 1996
) and anti-CD3sFv (Vallera et al., 1996
) were used as controls and have been previously reported.
Bioassay
The 32Dp210 cell line is a C3H/HeJ myeloid leukemia expressing the IL-3 receptor (Daley and Baltimore, 1988; Daley et al., 1991
; Matulonis et al., 1993
; Salgia et al., 1995
). 32Dp210 cells (3x105) were plated into individual wells (24-well flat-bottom plate; Costar, Cambridge, MA) in RPMI 1640 plus 10% fetal bovine serum (Hyclone, Logan, Utah) in the presence of varying concentrations of DT390IL-3. Time points were performed in triplicate. For blocking studies, 300 µg/ml polyclonal anti-IL-3 (R&D Systems, Minneapolis, MN) (goat IgG) and IgG was added to the culture. For a control, rat IgG anti-Ly5, not reactive with either DT390IL-3 fusion protein or the cell line, was added. At 24, 48 and 72 h a small sample was removed and stained with trypan blue dye to quantitate the number of cells remaining in the well and their viability.
Fusion toxin administration
C3H/HeJ mice (H2k) mice were purchased from The Jackson Laboratory (Bar Harbor, Maine). Mice were 810 weeks of age. Reported doses are total daily doses administered twice daily (BID) for 46 consecutive days. Fusion toxins were given intraperitoneally (ip) in a 0.2 ml volume in the morning and then again 68 h later as per a previously established schedule (Vallera et al., 1996).
Bone marrow transplantation
Recipients were irradiated with a lethal dose of total body irradiation (8 Gy X-ray at a dose rate of 39.30 rads/min) (Phillips Medical System, Brookfield, WI) 18 h before intravenous injection with a rescue dose of BM. When treated with fusion toxin, BM was incubated overnight at 106 cells/ml in Dulbecco's minimal essential media with 10% FCS (GIBCO BRL, Gaithersberg, MD) and 2-mercaptoethanol in a 10% CO2/90% air atmosphere. Recipient survival was monitored daily.
Assessment of leukocyte, platelet and erythrocyte levels
Peripheral blood (150250 µl) was obtained by retroorbital venipuncture. Leukocyte number, platelet number and morphology were determined by examination of WrightGiemsa stained smears. Hematocrit values were determined by capillary tube volume red blood cell (RBC) to plasma ratios after centrifugation.
Histology
Mice were sacrificed, autopsied and tissues were taken for histopathologic analysis as described (Vallera et al., 1997)2. All samples were imbedded in OCT compound (Miles, Elkhark, IN), snap frozen in liquid nitrogen and stored at 80°C until sectioned. Serial 4 µm sections were cut, thaw mounted onto glass slides and fixed for 5 min in acetone. Slides were stained with hematoxylin and eosin (H&E) for histopathologic assessment.
Blood urea nitrogen, creatinine and alanine transferase (ALT) assays
All three assays were performed on Kodak EKTACHEM clinical chemistry slides on a Kodak ETACHEM 950 by the Fairview University Medical Center (Minneapolis, MN). Mice were sacrificed, individual serum samples collected and analysis was performed blindly on the undiluted samples. Minimum specimen volume was 11 µl for each assay. The blood urea nitrogen assay is read spectrophotometrically at 670 nm. The creatinine assay is read at 670 nm. In the ALT assay, the oxidation of NADH is used to measure ALT activity at 340 nm.
Inflammatory cytokines
Serum IL-1ß and TNF- levels were measured by commercial ELISA kits (R&D Systems, Minneapolis, MN).
Statistical analyses
Groupwise comparisons of continuous data were made by Student's t-test. A computer program for compiling life table and statistical analysis by the Log-Rank test was used to analyze survival data. Probability (P) values 0.05 were considered significant.
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Results |
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To determine the potency on 32Dp210 leukemia, cells were cultured in the presence of various concentrations of DT390IL-3. At 24, 48 and 72 h, cells were stained with trypan blue dye and counted. Figure 1A shows that 0.1 nM DT390IL-3 killed all cells within 72 h, consistent with published studies showing that DT390IL-3 was active in picomolar concentrations (Chan et al., 1996
). In Figure 1B
, the addition of anti-Ly5, an irrelevant control antibody, to DT390IL-3 did not alter the activity of DT390IL-3. However, 20 µM anti-IL3 (Figure 1C
) blocked all killing of leukemia cells at a DT390IL-3 concentration of 0.1 or 1 nM, but not 10 nM. One hundred nM of DT390IL-3 did not kill EL4 thymoma cells, which do not express IL-3R (Figure 1D
). Together, these data indicate that the killing by DT390IL-3 was selective and is attributed to the IL-3 moiety of the hybrid protein.
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To determine whether DT390IL-3 could prove useful as a potent anti-cancer agent when injected in vivo, C3H/HeJ mice (2325 g) were given BID injections for 5 days to establish the MTD. MTD was 0.05 µg/day since all mice survived (Table I). All 10 mice given 0.1 µg/day DT390IL-3 or higher were dead within 5 days.
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To provide some insight into the cause of death of DT390IL-3-treated mice, we first examined the survival curves and weight curves of treated mice. Groups of eight mice given DT390IL-3 in excess of the MTD (0.2 µg/day), died within 24 days with no sign of weight loss indicating an acute toxic death. In a previous report, we observed that the primary cause of death with another DT390 immunotoxin, made by fusing the toxin with a single chain Fv, was renal toxicity (Vallera et al., 1996). Therefore, we measured serum urea nitrogen and creatinine levels to determine whether the kidneys were fatally damaged by treatment. Figure 2
shows that in either C3H/HeJ or C57BL/6 mice, treatment with lethal doses of DT390IL-3 did not significantly elevate the levels of urea nitrogen (Figure 2A
) or creatine (Figure 2B
) above that measured in the untreated controls. Both levels of urea nitrogen and creatinine in DT390IL-3-treated mice were about sixfold less than levels (dashed line) measured with DT390anti-CD3sFv, a fusion toxin made with the same DT390 plus a sFv reactive against the CD3 component of the murine T cell receptor. This agent had profound renal toxicity in a previous study (Vallera et al., 1997
). Histologic examination of kidney tissue sections from treated mice revealed slight damage consisting of a small amount of neutrophilic infiltrate in the glomeruli. Together, these data indicated that acute toxicity induced by DT390IL-3 administration was not renal. Levels of alanine transferase activity were studied in the same manner with results indicating that this acute toxicity was not hepatic in nature.
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Blood analysis revealed a significant reduction in packed red blood cell volume as a measure of hematocrit illustrating a significant drop in red blood cell number (Figure 4A). Although such a drop is not likely to cause death, a precipitous decline in platelets was also observed in DT390IL-3-treated mice (Figure 4B
). Since IL-3R is expressed on endothelial cells, we autopsied treated mice to look for visual evidence of bleeding. Although we did not note dramatic changes in mice given 0.5 µg/day, we observed significant hemorhage in mice given three daily injections of 2.5 µg DT390IL-3. Morphologic examination revealed purpura in tissues from thymus, skin, intestine, Peyer's patches, liver, uterus, fallopian tubes and ovaries in mice given DT390IL-3, but not DT390GM-CSF. Hemorrhage in thymus and uterus is shown in Figure 5
. The presence of heme was observed throughout the liver in mice given DT390IL-3 at the MTD and breaks in several veins indicated that internal bleeding played a role in toxicity. Histology revealed bleeding into the proximal tubules in the kidney. In the small intestine, the intestinal wall was extremely thin with the Peyer's patches ulcerated into the lumen. These data, combined with the knowledge that IL-3R is prominently expressed on platelet progenitors, erythrocyte progenitors and endothelium could be used to argue that lethal effects are primarily related to bleeding.
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To determine the ability of DT390IL-3 to destroy leukemia cells in vivo, groups of five mice were given a lethal dose of 1x103 32Dp210 leukemia cells i.p. followed by an i.p. course of DT390IL-3 at the MTD (0.05 µg/day), a dose in excess of the MTD (0.1 µg/day) or no treatment. Not surprisingly, all animals that received a dose in excess of the MTD (0.1 µg/day) died by day 21. In the group receiving the MTD (0.05 µg/day) one mouse died early on day 3. Autopsy of this animal revealed no evidence of splenomegaly, which is a hallmark of this leukemia, so it is likely that the mouse died of toxicity. The remaining four mice survived to day 82 when an additional mouse died of leukemia. All but one of the untreated controls developed leukemia and died by day 20. At day 68 the last mouse in the group died. Treatment at the MTD resulted in a significant (P < 0.025) anti-leukemia effect compared with the untreated controls. In a separate experiment, mice (five/group) were given i.v. leukemia cells and DT390IL-3 was administered i.v. There was no anti-leukemia effect and all mice in both groups were dead by day 27. These data suggest that although it is possible to achieve localized anti-leukemia effects by administering DT390IL-3 i.p. against i.p. cancer cells, it is more difficult to obtain leukemia responses against tumor cells that have been injected i.v. and are systemically distributed.
Leukemia purging studies in mice
Because of its acute toxicity in vivo and because IL-3R is not expressed uniformly on early BM progenitors (Chan et al., 1996), we investigated whether DT390IL-3 might be useful for selectively killing leukemia cells in autologous BM grafts contaminated with leukemia. To determine if exposure to DT390IL-3 affected the tumorgenicity of IL-3R+ 32Dp210 cells prior to their injection into mice, leukemia cells were preincubated with 108 M immunotoxin for about 12 h overnight and then injected i.v. into normal C3H/HeJ recipients. As shown in Figure 6A
, all control mice receiving 1x103, 1x104, 1x105 or 1x106 untreated cells died by day 22. In contrast, experimental mice receiving 1x103, 1x104 or 1x105 treated cells were alive on day 120 (P < 0.00001) (Figure 6B
). Protection was selective since mice given DT390GM-CSF-treated 32Dp210 cells died.
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Discussion |
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A number of questions are raised regarding the use of IL-3 as a targeting ligand. On the one hand, many human AML cells have been shown to be responsive to exogenous IL-3 in vitro (Delwel et al., 1988; Miyauchi et al., 1988
; Park et al., 1989
) and some AML samples have been shown to produce IL-3 (Hermann and Vallenya, 1990
). On the other hand, IL-3R is widely expressed on hematopoietic cells, their progenitors and the vasculature (Korpelainen et al., 1996
). Regarding expression on hematopoietic cells, IL-3R is present on cells of erythroid, myeloid and megakaryocytic lineage. It is also present on some T and B cells (Guba et al., 1989
; Tadmori et al., 1989
) raising the issue as to whether IL-3R on these vital tissues will limit therapeutic application.
IL-3R expression is complex and thus dose limiting toxicities may be difficult to predict (Hara and Miyajima, 1992). The receptor exists as a heterodimeric
- and ß-chain. One form of the ß-chain is uniquely inactive with the IL-3
-chain, while a second form of the ß-chain is shared with the
-chain of IL-3, GM-CSF and IL-5 (Colatta et al., 1993
). Although some tissues (e.g. connective tissue) express either
- or ß-chains, the expression of both results in the highest affinity receptorligand interaction and signal transduction. Expression of the receptor is also inducible on certain tissues and regulated by inflammatory cytokines such as IFN-
and TNF-
.
In this report, the toxicity of DT390IL-3 was different than that observed for other fusion toxins which have been reported and which were assembled with the identical DT390 toxin. For example, the similarly sized DTIL-2 (Kirkman et al., 1989) and DTIL-4 (Lakkis et al., 1991
) fusion toxins demonstrated significantly elevated serum enzyme levels and abnormal renal chemistry and renal histopathology at doses of 1042 µg/day. A recent study of a DTsFv fusion toxin showed similar renal effects (Vallera et al., 1997
). In all of these studies, toxicity was generally attributed to the rapid clearance and concentration of these small 60 kDa agents by the kidney. In contrast, DT390IL-3 induced no significant damage to kidney or liver, but was still about 100-fold more toxic to mice than the DT390anti-CD3sFv. Our data indicate that the toxic effects are directed against the blood, a finding that can be explained by the expression of IL-3R on hematopoietic cells, hematopoietic progenitors and vascular endothelial cells. Direct damage could result from the constitutive expression of the
/ß-chain receptor complex on vascular endothelial cells (Korpelainen et al., 1993
, 1995
). If this were the case, the fusion toxin could compromise blood vessel integrity resulting in the bleeding and hemorrhage that was observed in thymus, skin, intestine, Peyer's patches, liver, uterus, fallopian tubes and ovaries. An alternative explanation is that the reduction in packed red cell volumes caused bleeding. This could be due to the expression of IL-3R on erythroid cells and erythroid progenitors such as burst-forming-unit erythroid (BFU-E) (Bot et al., 1988
; Migliaccio et al., 1988
; Umermura et al., 1989
) and day 8 CFU-S (Van Zant, 1984
; Inaba et al., 1987
). The risk of bleeding would be high since DT390IL-3 administration entirely eliminated platelets. Studies show that IL-3R is expressed on megakaryocytes and/or their precursors (Bruno et al., 1989
; Hoffman et al., 1990
; Mazur et al., 1990
). The absence of elements that promote clotting would promote bleeding, thereby placing mice at even greater risk. Experiments designed to determine if platelet transfusions can reverse DT390IL-3-induced bleeding are warranted. Because it is known that IL-3R is inducible by inflammatory cytokines such as TNF-
(Korpelainen et al., 1993
, 1996
) and neutrophil levels were elevated following DT390IL-3 treatment, we measured the serum levels of TNF-
and IL-1-ß after DT390IL-3 administration. These cytokines were not involved in some indirect mechanism of action.
Despite the MTD of 0.05 µg/day, our toxicity data did not exclude the use of DT390IL-3 for in vivo therapy. Intraperitoneal administration of the toxin was able to save mice that had been given i.p. injection of the lethal IL-3R+ 32Dp210 leukemia. However, protection was local since i.v. administration was ineffective at the doses tested. These findings indicate that the agent may prove useful when administered locally. Chen et al. (1997) has reported that immunotoxin retroviruses can be used to infect lymphoid cells and these can be used to deliver immunotoxin locally to the tumor. DT390IL-3 could be highly selective for this purpose and may have additional advantages in its activity against the tumor vasculature.
Another important finding was the favorable therapeutic window for leukemia cell elimination observed in our autologous BMT model. Autologous BMT is an important alternative therapy for drug refractory myeloid leukemias and published studies have indicated that purging potentially contaminated BM prior to transplantation contributes to a favorable transplant outcome (Gorin et al., 1990). Immunotoxins have been used in this capacity (Kersey et al., 1988
). Most clinical purging regimens result in at least a 2 log elimination of leukemic cells. Since in the same experiment, control mice given 1x103 untreated cells died and mice given 1x105-treated cells lived, treatment killed at least 2 logs of leukemia cells. Since in separate experiments, even 10 untreated 32Dp210 cells were sufficient to kill mice, it is possible that as much as 4 logs of cells were killed. The therapeutic window can be explained by our earlier competitive repopulation studies with DT390IL-3 in mice indicating that the IL-3R is mostly not expressed on the earliest BM progenitor cells and 32Dp210 cells are highly susceptible to the rapidly internalized fusion toxin (Chan et al., 1996
). DT390IL-3 has been shown to have a more pronounced effect against IL-3R+ committed BM progenitors. The destruction of these cells could place transplanted animals at added risk for engraftment failure. However, our data indicate that this problem might be addressed by simply increasing the dose of donor BM. Further studies are warranted.
In conclusion, since IL-3R is expressed on a high frequency of myeloid leukemias, DT390IL-3 may prove a useful anti-leukemia agent when given in vivo or used for ex vivo purging of BM. Although in vivo administration may be limited by its toxicities, only nanomolar quantities are required to kill leukemia cells, indicating that further studies are warranted.
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Acknowledgments |
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Notes |
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References |
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Received February 22, 1999; revised April 11, 1999; accepted April 16, 1999.