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NEOPLASIA
From the Departments of Hematology-Oncology and
Pathology, St Jude Children's Research Hospital, and the University of
Tennessee College of Medicine, Memphis, TN; and Molecular Technologies,
Bayer Biotechnology, Berkeley, CA.
Interleukin 4 (IL-4) suppresses the growth of acute lymphoblastic
leukemia (ALL) cells, but its clinical usefulness is limited by
proinflammatory activity due mainly to the interaction of cytokine with
endothelial cells and fibroblasts. Stroma-supported cultures of
leukemic lymphoblasts were used to test the antileukemic activity of an
IL-4 variant, BAY 36-1677, in which the mutations Arg 121 to Glu and
Thr 13 to Asp ensure high affinity for IL-4R Despite treatment with intensive chemotherapy,
residual disease persists in approximately 20% of children and 65% of
adults with acute lymphoblastic leukemia (ALL).1,2
Recurrent disease is particularly resistant to chemotherapy, as are
some subtypes of ALL, such as those with the t(9;22)(q34;q11) and
MLL gene rearrangements.1,2 For most of these
patients, the current arsenal of antileukemic drugs given at tolerable
doses is clearly insufficient. Bone marrow ablation with high-dose
chemotherapy or total body irradiation, followed by hematopoietic stem
cell engraftment, may be curative in patients resistant to conventional
therapy, but this option is restricted by the availability of suitable
donors. Even when effective, chemotherapeutic regimens for ALL carry
the risk for severe toxicity, including secondary malignancy,
cardiomyopathy, and neuropsychological abnormalities.1,3-7
Thus, there is an urgent need to develop new antileukemic drugs with
better therapeutic indexes.
We and others have observed that interleukin-4 (IL-4), an
immunomodulatory cytokine produced by T cells,8,9 induces
growth arrest and apoptosis in leukemic lymphoblasts in
vitro.10-12 These findings were confirmed in experiments
with human leukemic cells engrafted in immunodeficient
mice.13 The clinical usefulness of IL-4, however, is
limited by the pleiotropic activities of cytokine, which can include
renal, hepatic, neurologic, and gastrointestinal toxicities as well as
capillary leak syndrome.14-19 Although the pathogenesis of
these side effects is unclear, the available evidence suggests that it
may relate in part to IL-4 signal transduction in fibroblasts and
endothelial cells.18
The receptor for IL-4 (IL-4R) consists of a primary binding subunit,
IL-4R Lymphoid-specific IL-4 variant BAY 36-1677
Receptor-binding assay
Cells
Mononuclear cells were separated by density gradient centrifugation (Lymphoprep; Nycomed, Oslo, Norway) and washed 3 times in PBS and once in AIM-V (Gibco, Grand Island, NY), a serum-free, cytokine-free tissue culture medium. Cord blood CD34+ cells were separated with a MACS separation system (Miltenyi Biotec, Bergisch Gladbach, Germany), which, by flow cytometry, consistently afforded a purity of 90% or greater.24 Six B-lineage ALL cell lines Cell cultures Bone marrow stromal cells, depleted of T cells by CD6- and CD8-mediated rabbit complement lysis, were derived from healthy bone marrow donors. Stroma was prepared in 96-well flat-bottomed plates (Costar, Cambridge, MA) and fed with RPMI-1640, 10% FCS, and 10 6 M hydrocortisone (Sigma, St Louis, MO), as previously
described.12,25-30 Human umbilical-cord endothelial cells
(HUVEC), purchased from the American Type Culture Collection (Manassas,
VA), were cultured in Endothelial-Growth Medium (Clonetics, San Diego,
CA) with 10% FCS. Skin fibroblasts were derived from a healthy donor
by punch skin biopsy and then fed with Dulbecco modified Eagle
medium (BioWhittaker) and 10% FCS.
To prepare test cultures of leukemic cells from patients, we removed
the media from the bone marrow stroma and washed the adherent cells 7 times with AIM-V tissue culture medium.12,25-30 The cells
were then resuspended in AIM-V, and 3 to 4 × 105 cells
were placed on the stromal layer in each well. We then added BAY
36-1677 (Bayer Biotechnology, Berkeley, CA), native IL-4 (Genzyme,
Cambridge, MA), or IL-13 (R&D Systems, Minneapolis, MN) to the test
wells at final concentrations ranging from 1 ng/mL to 100 ng/mL. A
neutralizing antibody to IL-4, an antibody to IL-4R Cell counting Cell numbers and phenotypes were determined by flow cytometry, when cultures were established, and again after 7 days, as described.12,25-30 Briefly, stromal cultures were transferred to Falcon tubes (Becton Dickinson, San Jose, CA). B-lineage ALL cells were incubated with CD19 conjugated to fluorescein isothiocyanate (FITC) and CD3 conjugated to phycoerythrin (PE); in some experiments, cells were stained with CD19 PE and CD10 FITC. Normal CD34+ bone marrow cells were incubated with a combination of CD34 conjugated to peridin chlorophyll protein (PerCP), CD19 PE, and CD13 and CD33, both conjugated to FITC. All monoclonal antibodies and isotype-matched unreactive controls were purchased from Becton Dickinson, with the exception of CD19 PE, CD10 FITC, CD13 FITC, and CD33 FITC, which were purchased from DAKO (Carpinteria, CA). After they were washed twice in PBS with 0.2% BSA and 0.2% sodium azide, the cells were resuspended in 0.5% paraformaldehyde and analyzed with a FACScan flow cytometer and Cell Quest software (Becton Dickinson).At the beginning of each culture, we set "gates" around the area of the light-scatter dot plot that included virtually all leukemic cells. These gates were used to count cells with the predetermined light-scattering properties present after culture with or without IL-4 (variant and native). These cell numbers were corrected for the percentage of cells in each sample expressing a given immunophenotype. Relative cell recovery after drug treatment was calculated by the formula (number of cells recovered with drug/number of cells recovered in parallel culture without drug) × 100. All results are reported as the means of at least duplicate experiments. Colony-forming assays Cord blood mononuclear cells were first suspended in Iscoves modified Dulbecco medium (BioWhittaker) and 2% FCS, and then in complete methylcellulose medium (Methocult GF H4434; StemCell Technologies, Vancouver, BC, Canada) at a 1:10 (vol/vol) ratio, to a final cell concentration of 5 × 103/mL and 2 × 104/mL. The methylcellulose medium contains 30% FCS and the following recombinant human growth factors: erythropoietin (3 U/mL), stem cell factor (50 ng/mL), granulocyte-monocyte colony-stimulating factor (10 ng/mL), and IL-3 (10 ng/mL). Petri dishes (35-mm; Nalge Nunc, Naperville, IL) were filled with 1.1 mL cell suspension. Triplicate cultures with BAY 36-1677 (25 ng/mL) and without the variant IL-4 were placed in an incubator set at 37°C, 5% CO2, and 90% humidity. Colonies (more than 50 cells) were scored after 14 days.Determination of apoptosis DNA content was analyzed as previously described31 using the ModFit software (Becton Dickinson). DNA fragmentation was evaluated after cell permeabilization and staining with fluorescein-12-dUTP.32 To detect apoptosis, we also labeled phosphatidylserine residues exposed on the cell surface with FITC-conjugated annexin-V (Trevigen, Gaithersburg, MD),33 following the manufacturer's instructions. In these experiments, cell membrane permeabilization was demonstrated by labeling cells with 5 µg/mL propidium iodide (Trevigen) for 15 minutes at 20°C.
Antileukemic activity of BAY 36-1677 Most of the ALL cases selected for testing had genetic features that confer a poor prognosis either t(9;22)(q34;q11) (n = 11) or
11q23 abnormalities with MLL gene rearrangements (n = 3). Additionally, in 3 of these patients, the samples were collected at the
first or second relapse (Table 1).
To maintain cell viability of leukemic lymphoblasts in vitro, we seeded
them onto allogeneic bone marrow stromal layers, which suppress
spontaneous apoptosis of ALL cells.27,34 After 7 days of
culture, the number of leukemic lymphoblasts recovered from stromal
layers ranged from 57% to 231% (median, 89.5%) of those originally
seeded. Treatment with BAY 36-1677 (25 ng/mL) reduced the recovery of
leukemic cells by 17% to 95% (median, 85%) compared with
results of parallel cultures not exposed to the cytokine (Table 1).
Only 2 of the cases (nos. 4 and 9) showed absolute resistance to the
cytokine (less than 1% inhibition of cell recovery). The cytotoxic
effect of BAY 36-1677 was dose dependent (Figure 2).
Two of the 6 experimental cell lines studied (KOPN-57bi and OP-1), both
carrying t(9;22)(q34;q11), were also susceptible to the IL-4 variant.
After 7 days of culture, the respective reductions in cell recovery
were 94.1% ± 2.3% (mean ± SD; n = 8) and 56.1% ± 9.8%
(n = 6). The cytotoxic effects of BAY 36-1677 were less pronounced
but readily apparent with the 380 cell line (29.0% ± 15.0%;
n = 6), whereas among the remaining 3 cell lines (RS4;11, REH, and
NALM6) cell killing was negligible. The growth-suppressive effects of
BAY 36-1677 on KOPN-57bi, OP-1, and 380 were completely abrogated by an
IL-4-neutralizing antibody (data not shown). All the leukemic cell
lines tested (except REH) reacted with an antibody against the IL-4R To determine the relative affinity of BAY 36-1677 and native IL-4 for
IL-4R
To assess the potency of BAY 36-1677 relative to that of its parent
cytokine, we compared the cytotoxicity of BAY 36-1677 and native IL-4
(both at 25 ng/mL) against leukemic cells from 11 patients with ALL and
2 cell lines (KOPN57bi and OP-1). As shown in Figure
4, BAY 36-1677 was cytotoxic in 10 of 11 ALL cases; neither BAY 36-1677 nor IL-4 had any detectable toxicity on
cells from case 9. Remarkably, IL-4 lacked detectable cytotoxicity in 4 of the 10 cases susceptible to BAY 36-1677, 2 of whom were highly sensitive to BAY 36-1677 (nos. 10 and 11). IL-4 had markedly lower activity in 2 others compared to BAY 36-1677 (47% vs 69% cell kill
for no. 7 and 44% vs 95% cell kill for no. 8). Both BAY 36-1677 and
IL-4 exhibited similar cytotoxicity on the cell lines KOPN57bi and
OP-1. We also tested whether IL-13, which signals through the
IL-4R
Apoptotic cell death induced by BAY 36-1677 BAY 36-1677 consistently induced changes in the light-scattering properties of leukemic cells. These effects, which resembled those ascribed to antileukemic drugs that act by triggering apoptosis,32 consisted of decreased forward light scatter, indicative of a reduction in cell size, accompanied by increased orthogonal light scatter, indicative of augmented intracellular granularity (not shown). Microscopic changes in cell morphology, such as nuclear fragmentation in cells with apparently intact surface membranes, were also typical of apoptosis.32 Other reliable signs of apoptosis apparent after treatment with BAY 36-1677 included exposure of phosphatidyl serine residues on cell membranes by annexin V binding, massive DNA fragmentation demonstrated by incorporation of dUTP conjugated to FITC, and hypodiploidy detected by DNA content analysis (Figure 5).32,33
Effects of BAY 36-1677 on the growth of normal hematopoietic cells To test the effects of BAY 36-1677 on normal hematopoietic cells, we cultured CD34+ cells purified from cord blood on bone marrow stromal layers. Under these culture conditions, the cells expand and differentiate into myeloid cells at discrete stages of maturation.24 In 3 experiments, the total numbers of viable cells recovered after 7 days of culture in control conditions were 377%, 565%, and 654% of those originally seeded. Cell recovery in cultures containing BAY 36-1677 (25 ng/mL) was 103.3% ± 15.3% of control values. Although it did not substantially affect the total number of hematopoietic cells recovered, BAY 36-1677 appeared to have contrasting effects on different subpopulations of myeloid differentiation (Figure 6). Recovery of the most mature myeloid cells (strong expression of CD13 and CD33 but weak expression or absence of CD34) was reduced in cultures with BAY 36-1677 (52.1% ± 17.1%; n = 3), whereas recovery of the more immature cells (strong expression of CD34) was enhanced (158.4% ± 19.5%; n = 3) in control cultures.
The lack of suppressive effects on normal in vitro hematopoiesis was
corroborated by results of colony-forming assays (Table 2). BAY 36-1677 (25 ng/mL) did not
suppress the formation of erythroid, myeloid, or mixed colonies derived
from cord blood mononuclear cells. By contrast, in one experiment,
erythroid colony formation was slightly but significantly enhanced by
the variant IL-4 (Table 2).
To directly compare the effects of BAY 36-1677 on normal and leukemic
cells, we prepared mixtures of the cell line KOPN57bi and normal
CD34+ cells (Figure 7). With
mixtures containing 20% leukemic cells and 80% normal cells, the
proportion of leukemic cells increased to 73% after 7 days of culture.
The addition of BAY 36-1677 (25 ng/mL) markedly changed the outcome of
the cultures: the percentage of leukemic cells was reduced to 3%. In 2 experiments with a lower starting proportion of leukemic cells (5%),
this value increased to 32% and 20% after 7 days of culture in the
absence of BAY 36-1677, but it dropped to 1% and 0.8% when the
cytokine was present. These experiments were repeated with
slower-growing primary ALL cells (no. 5, Table 1). Results were
similar. In experiments with a starting population of 35% leukemic
cells, these represented 40% of cells after 7 days of culture without
BAY 36-1677 and 5% in cultures with the variant IL-4. With a lower
starting proportion of leukemic cells (15%), this value increased to
25% without BAY 36-1677 and decreased to 2% with BAY 36-1677.
Effects of BAY 36-1677 on endothelial cells and fibroblasts In contrast to its toxic effects on leukemic cells, BAY 36-1677 did not affect the growth or the appearance of umbilical cord-derived endothelial cells, even when added to the cultures at 100 ng/mL. Native IL-4, on the other hand, markedly suppressed cell growth and altered cell appearance (Figure 8), as previously described.36 A similar effect was produced by IL-13 (not shown).36 This suggests that, in endothelial cells, IL-4R /IL-13R receptors likely mediate the inhibitory signals
of IL-4.
Both native IL-4 and IL-13 stimulated fibroblast proliferation, whereas
BAY 36-1677 (100 ng/mL) lacked any discernible effect on fibroblast
proliferation or morphology (Figure 8). These results are consistent
with the ability of IL-4 and IL-13 to induce signal through the
IL-4R
One of the important objectives of modern cancer research is to
develop molecules capable of suppressing cancer cell growth while
sparing normal cells. In this study, we tested the cytotoxicity of a
molecularly engineered IL-4 variant, BAY 36-1677, against cells from
patients with ALL, the most common form of cancer in children. The
cytokine variant induced apoptosis in leukemic cells from patients with
high-risk leukemia but did not suppress the growth of normal
hematopoietic cells. Consequently, BAY 36-1677 conferred a growth
advantage to normal hematopoietic cells over leukemic lymphoblasts in
cultures designed to test the growth potential of each cell type. By
contrast, in control cultures lacking BAY 36-1677, the leukemic cells
prevailed, recapitulating the fate of patients with residual aggressive
leukemia. BAY 36-1677 was considerably more cytotoxic than native IL-4.
We speculate that this is owing to its higher affinity for the IL-4R Early findings of IL-4-mediated killing of leukemic
lymphoblasts12 raised the possibility that this cytokine
could be a useful addition to ALL treatment protocols. When
administered in tolerable doses to patients with solid tumors to boost
tumor immune surveillance, IL-4 reached serum levels that would be
expected to trigger apoptosis in leukemic cells,14,16
supporting a therapeutic role for this agent in patients with ALL.
There were, however, several side effects that could prevent the use of
IL-4 at higher, potentially more effective, doses.14-19
Capillary leak syndrome, presumably caused by IL-4 signaling to
endothelial cells, was among the most severe toxicities
observed.14,16 IL-4 also induces the release of
proinflammatory cytokines from fibroblasts,38 which could
contribute to multi-organ toxicity. The amino acid substitutions
introduced in the IL-4 molecule to generate BAY 36-1677 were
specifically chosen to eliminate activation of the IL-4R IL-4 is toxic not only for leukemic immature B cells but also for
their normal human and murine counterparts.12,39 The toxicity toward murine immature B cells was previously thought to be
the indirect outcome of IL-4-induced secretion of unknown factors from
stroma.39 Although leukemic cells from patients were
maintained on bone marrow stroma in the current study, we doubt that
they were killed through indirect actions of the cytokine. First, BAY
36-1677 does not activate the IL-4R Our findings demonstrate that molecular manipulation can be used to increase the specificity and potentially improve the therapeutic index of anticancer compounds. Corticosteroids, microtubule poisons, topoisomerase inhibitors, and DNA synthesis inhibitors are the most commonly used classes of antileukemic drugs.1 With the possible exception of corticosteroids, none of these agents are selective for leukemic cells, and their toxicity extends to diverse types of normal hematopoietic cells and to nonhematopoietic cells. The results reported here suggest that BAY 36-1677 might be a useful addition to contemporary treatment regimens for ALL. With its strong selectivity toward leukemic cells, BAY 36-1677 could be safely introduced in patients with minimal residual disease after remission induction therapy to suppress leukemia cell growth and to aid the expansion of normal hematopoietic cells.
We thank the Roczniak Lab (Bayer Biotechnology) for technical contributions to the development of the IL-4 binding assay, and we thank Elaine Coustan-Smith for flow cytometric analysis of apoptosis.
Submitted July 6, 2000; accepted October 3, 2000.
Supported by National Cancer Institute grants RO1-CA58297 and P30-CA21765 and by the American Lebanese Syrian Associated Charities.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dario Campana, Department of Hematology-Oncology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794; e-mail: dario.campana{at}stjude.org.
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© 2001 by The American Society of Hematology.
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Top
Abstract
Introduction
receptors
expressed by lymphoid cells, without activation of the IL-4R
gt11 library
(Stratagene Cloning Systems, La Jolla, CA), and it was fused to DNA
corresponding to the peptide sequence
Ser-Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly at the C-terminus generating
sIL-4R
) and BAY 36-1677 (
) for
IL-4R
) or native IL-4 (
)
(both at 25 ng/mL). The results of duplicate cultures with either
cytokine were compared to control cultures without cytokines.
