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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1943-1951
Simultaneous Antagonism of Interleukin-5, Granulocyte-Macrophage
Colony-Stimulating Factor, and Interleukin-3 Stimulation of Human
Eosinophils by Targetting the Common Cytokine Binding Site of Their
Receptors
By
Q. Sun,
K. Jones,
B. McClure,
B. Cambareri,
B. Zacharakis,
P.O. Iversen,
F. Stomski,
J.M. Woodcock,
C.J. Bagley,
R. D'Andrea, and
A.F. Lopez
From the Hanson Centre for Cancer Research, the Institute of Medical
and Veterinary Science, Adelaide, Australia.
 |
ABSTRACT |
Human interleukin-5 (IL-5), granulocyte-macrophage
colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic
cytokines implicated in allergy in general and in the inflammation of
the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific 
chain and a shared subunit ( c). Although binding of
IL-5, GM-CSF, and IL-3 to their respective receptor chains is the
first step in receptor activation, it is the recruitment of
c that allows high-affinity binding and signal
transduction to proceed. Thus, c is a valid yet untested
target for antiasthma drugs with the added advantage of potentially
allowing antagonism of all 3 eosinophil-acting cytokines with a single
compound. We show here the first development of such an agent in the
form of a monoclonal antibody (MoAb), BION-1, raised against the
isolated membrane proximal domain of c. BION-1 blocked
eosinophil production, survival, and activation stimulated by IL-5 as
well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism
showed that BION-1 prevented the high-affinity binding of
125I-IL-5, 125I-GM-CSF, and
125I-IL-3 to purified human eosinophils and that it bound
to the major cytokine binding site of c. Interestingly,
epitope analysis using several c mutants showed that
BION-1 interacted with residues different from those used by IL-5,
GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed
that BION-1 prevented ligand-induced receptor dimerization and
phosphorylation of c, suggesting that ligand contact
with c is a prerequisite for recruitment of
c, receptor dimerization, and consequent activation.
These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and
generate further agents for the treatment of eosinophil-dependent diseases such as asthma.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN INTERLEUKIN-5 (IL-5), IL-3, and
granulocyte-macrophage colony-stimulating factor (GM-CSF) are cytokines
involved in hematopoiesis and inflammation.1 All 3 cytokines stimulate eosinophil production, function, and
survival2-6 and therefore have the ability to influence
inflammatory diseases, such as asthma, atopic dermatitis, and allergic
rhinitis, in which the eosinophil plays a major effector
role.7 IL-5, being the eosinophil-specific cytokine, has
received most of the initial attention, with IL-5 mRNA and protein
levels noted to be elevated in lung tissue and bronchoalveolar lavage
fluid from symptomatic asthma patients.8-10 Correlations
between IL-5 levels and allergen challenge and disease activity have
also been seen. However, it is becoming apparent that not only IL-5,
but also GM-CSF and IL-3 play a role in eosinophil production and
activation in asthma, because there is evidence of both GM-CSF and IL-3
production at sites of allergic inflammation.11-17 The
concomitant expression of these cytokines probably contributes to the
total number of infiltrating eosinophils as well as to the degree of
eosinophil activation. In addition, each of these cytokines may be
responsible for the different phases and compartmentalization of the
eosinophil infiltrate. Recent kinetic data from patients undergoing
antigen challenge showed that IL-5 levels increased between days 2 and
7 postchallenge, whereas GM-CSF peaked at day 2 and remained elevated
through to day 16. Furthermore, detection of GM-CSF extended beyond the
site of allergen challenge.18
IL-5, GM-CSF, and IL-3 stimulate eosinophils and other cells by binding
to cell surface receptors that comprise a ligand-specific  chain and
a chain that is shared by the 3 receptors
(c).1 Binding to each receptor chain is
the initial step in receptor activation; however, engagement of either
chain alone is not sufficient for activation to occur. Recruitment
of c by each ligand: chain complex follows, a step
that has 2 major functional consequences. Firstly, it allows the
binding of IL-5, GM-CSF, and IL-3 to become essentially irreversible.
Secondly, it leads to full receptor activation. c is the
major signaling component of these receptors, and its engagement leads
to the activation of JAK-2, STAT-5, and other signaling
molecules,19 culminating in the full plethora of cellular
activities commonly associated with either IL-5, GM-CSF, or IL-3
stimulation, such as eosinophil adherence, priming for degranulation
and cytotoxicity, and prolongation of viability.
To block or antagonize the activity of eosinophil-activating cytokines
in vivo, 3 major approaches are being tried. One of them uses
antibodies to the implicated cytokines. For example, antibodies to
human IL-5 are being used in an animal model of allergen-induced
asthma20,21 and have been shown to have a relatively
long-lasting effect in preventing eosinophil influx into the airways
and bronchial hyperresponsiveness. A second approach relies on IL-5 or
GM-CSF mutants that can bind to their respective chains with
wild-type affinity but that have lost or shown reduced ability to
interact with human c. IL-5 mutants such as E13Q, E13K,
and E13R and the human GM-CSF mutant E21R directly antagonize the
functional activation of eosinophils by IL-5 or GM-CSF,
respectively.22,23 However, at least in the case of E13K,
eosinophil survival is not antagonized, and, in fact, this mutant is
able to support eosinophil survival.24 A third approach
involves the use of soluble receptor chains that can sequester
circulating cytokines.25 However, this carries the risk of
a cytokine:receptor chain complex potentially interacting with
surface-expressed c and triggering receptor activation.
The common theme among these approaches is that they tackle a single
receptor system involving either IL-5, GM-CSF, or IL-3, leaving the
other 2 eosinophil-acting cytokines unaffected. Although the
concomitant administration of IL-5 and GM-CSF antagonists may be
considered, this could be clinically impracticable.
An alternative approach to blocking eosinophil-activating cytokines
involves targeting c. Although c does not
directly bind IL-5, GM-CSF, or IL-3 alone, it does associate with these
cytokines complexed to the appropriate receptor chain. Recent
studies have identified the major binding sites of c for
the IL-5:IL-5R, GM-CSF:GM-CSFR, and IL-3:IL-3R complexes.
Significantly, these sites are used by all 3 complexes and comprise the
predicted B-C loop and F-G loops in the membrane proximal domain of
c.26-28 Thus, targetting c is
not only desirable, but also feasible, with the potential to allow the
simultaneous inhibition of IL-5, GM-CSF, and IL-3 action by a single agent.
We show here the development of the first simultaneous antagonist of
IL-5, GM-CSF, and IL-3 in the form of monoclonal antibody (MoAb) BION-1
directed against the major cytokine binding region of c.
BION-1 blocked the production and activation of human eosinophils stimulated by IL-5, GM-CSF, or IL-3 by inhibiting the high-affinity binding of all 3 cytokines to eosinophils and preventing receptor heterodimerization and c phosphorylation. The results
demonstrate the feasibility of blocking the 3 eosinophilopoietic
cytokines with a single compound and support the notion of
simultaneously inhibiting more than 1 cytokine by targetting the shared
signaling subunit in their receptors.
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MATERIALS AND METHODS |
Domain 4 cDNA.
Domain 4 of c was expressed with an N-terminal
Flag-epitope in the form of an activated c mutant,
 QP.29 An extracellular deletion removes domains 1 to 3 in this construct, but the transmembrane and cytoplasmic
regions are retained. This was cloned into the eukaryotic expression
vector pcDNA3 (Invitrogen, San Diego, CA).
Cytokines, cell lines, and primary cells.
Recombinant human IL-3 and GM-CSF were produced in Escherichia
coli as described.22,30 Recombinant human IL-5 was
purified from E coli by Bresagen (Adelaide, South Australia).
Tumor necrosis factor (TNF) was a gift from Dr J. Gamble (Hanson
Centre for Cancer Research, Adelaide, Australia). COS cells were
transfected with receptor cDNA as described previously.26
CHO c and CHO QP cells stably expressing either
full-length c or QP, respectively, were generated by
electroporation.22 TF1.8 cells were a gift from Dr J. Tavernier (University of Gent, Gent, Belgium). MO7e cells, a human
megakaryoblastic cell line, were from Dr P. Crozier (Auckland, New
Zealand). Human eosinophils were purified from the peripheral blood of
normal or slightly eosinophilic volunteers via sedimentation through
dextran and centrifugation through a discontinuous density gradient of
hypertonic metrizamide, as previously described,31 and were
greater than 95% pure. Human neutrophils and monocytes were purified
from peripheral blood as described previously,2,32 with
greater than 95% purity.
Generation of anti-c MoAbs.
BALB/c mice were immunized intraperitonally with 1 × 107 COS cells transfected with c or QP
expression constructs. The immunizations were repeated 4 times at
2-week intervals. Four weeks after the final immunization, a mouse was
boosted with 2 × 106 COS transfectants intravenously.
Three days later, splenocytes were harvested and fused with NS-1
myeloma cells as previously described.33 Hybridoma
supernatants were screened on CHO c or CHO QP cells
by flow cytometry, with untransfected CHO cells as a control. All
antibodies were from single hybridoma clones as selected by a limiting
dilution method. Using this procedure, several anti-c
MoAbs were generated; 8B8, 8E4,34 and 1C135 are
nonfunctional MoAbs raised against full-length c and
have been used as control anti-c MoAbs throughout these
studies. MoAbs were purified from ascites fluid or hybridoma
supernatant by a protein A sepharose column. The isotypes of MoAbs were
tested with a Mouse MoAb Isotyping Kit (Boehringer Mannheim, Mannheim, Germany). Fab fragments were generated using a Fab
Preparation kit (Pierce, Rockford, IL) following the supplied protocol.
Immunofluorescence.
Freshly purified neutrophils, eosinophils, monocytes, or CHO and COS
cell transfectants (5 × 105) were incubated with 50 µL of hybridoma supernatant or 0.25 µg of purified MoAb for 45 to
60 minutes at 4°C. Cells were washed twice and then incubated with
fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse Ig
(Silenus, Hawthorn, Victoria, Australia) for another 30 to 45 minutes.
Cell were then washed and fixed before analyzing their fluorescence
intensity on an EPICS-Profile II Flow Cytometer (Coulter Electronics,
Hialeah, FL).
Ligand binding assay.
IL-3 and GM-CSF were radio-iodinated using the iodine monochloride
method.36 125I-IL-5 was purchased from Dupont
NEN (North Sydney, New South Wales, Australia). MoAb BION-1 was
radio-iodinated using the chloramine T method.37 Binding
assays were performed as previously described.38 Briefly, 1 to 2 × 106 cells were preincubated with either BION-1
whole IgG, Fab fragments, or control MoAbs or with a range of
concentrations of IL-3 or TNF for 1 hour at 4°C. Radio-labeled
ligand was then added and incubated for a further 2 hours before the
cells were separated from free label by centrifugation through fetal
calf serum (FCS). Counts associated with the resulting cell pellets
were determined by counting on a  -counter (Cobra Auto Gamma; Packard
Instruments Co, Meridien, CT). Nonspecific binding was determined for
each ligand by determining binding in the presence of a 200-fold excess of unlabeled ligand.
c mutants and MoAb epitope mapping.
Alanine substitutions in the B-C and F-G loops of domain 4 of the
c have been described previously.26,27 The
cDNAs for wild-type c and each of the c
mutants in the B-C and F-G loops were introduced into COS cells by
electroporation,26 and binding assays were performed on the
transfected cells 48 hours posttransfection. The anti-c
MoAb, BION-1, and 1C1 were labeled using the chloramine T
method,37 and binding assays were performed essentially as described previously.27
TF-1.8 cell proliferation assay.
TF-1.8 cells were grown in the presence of 2 ng/mL of GM-CSF. The cells
were starved for 24 hours before setting up proliferation assays as
described previously.33 From dose-response curves, the
half-maximal proliferation dosage of IL-5 (0.3 ng/mL), GM-CSF (0.03 ng/mL), IL-3 (0.1 ng/mL), or erythropoietin (EPO; 5 ng/mL) was chosen to perform proliferation experiments in the presence of a
range of concentrations of MoAbs. The 3H-Thymidine
incorporation of each sample was determined by liquid scintillation.
Colony assay.
Ethics approval was obtained to collect bone marrow cells from healthy
adult donors. The mononuclear cells were isolated after dextran
sedimentation and density gradient centrifugation. The cells
(50,000/mL) were cultured in semisolid methylcellulose medium for 14 days in humidified conditions supplied with 5% CO2.
Colonies of more than 40 cells were scored after staining as previously described.39
Eosinophil survival assays.
The maximal dose of IL-5 required to support eosinophil survival after
36 hours was determined. Eosinophils were then cultured with 1 nmol/L
of IL-5, GM-CSF, or IL-3 plus anti-c MoAbs for 36 hours.
The viability of eosinophils was quantitated by propidium iodide
staining and flow cytometry analysis as described.40
CD69 expression.
Eosinophils were treated with a range of concentrations of cytokines
for 3 hours either in the presence or absence of anti-c MoAbs. CD69 expression was measured by immunofluorescence using an
anti-CD69 monoclonal MoAb coupled to phycoerythrin (PE;
Becton Dickinson Immunocytochemistry Systems, San Jose, CA).
Coimmunoprecipitation of and chains and the c phosphorylation assays.
MO7e cells were surface labeled with 125I using the
lactoperoxidase method, as described previously.41 The
labeled cells were preincubated with MoAbs BION-1, control
anti-c MoAb 1C1 (0.5 mg/mL), or medium alone for 1 minutes before being stimulated or not with IL-3 (6 nmol/L) for 5 minutes. Cells were lysed in lysis buffer consisting of 137 mmol/L
NaCl, 10 mmol/L Tris-HCl (pH 7.4), 10% glycerol, and 1% Nonindet P-40
with protease and phosphatase inhibitors (10 µg/mL leupeptin, 2 mmol/L phenylmethlysulphonyl fluoride, 10 µg/mL aprotonin,and 2 mmol/L sodium vanadate) for 30 minutes at 4°C, followed by
centrifugation of the lysate at 10,000g for 15 minutes to
remove cellular debris. The lysate was precleared with
mouse-Ig-coupled Sepharose beads for 18 hours at 4°C and incubated
with anti-IL-3R, anti-c MoAb beads for 2 hours at
4°C. The beads were washed 6 times with lysis buffer and
immunoprecipited proteins were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The gel was transferred onto nitrocellulose and the immunoprecipited proteins were detected by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The blot was then probed with an
antiphosphotyrosine MoAb, 3-365-10 (Boehringer Mannheim, Frankfurt,
Germany), as described previously.35
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RESULTS |
Development of MoAb BION-1.
We have previously shown by site-directed mutagenesis that domain 4 of
c and, more specifically, the putative B-C and F-G loops
are involved in high-affinity binding and receptor activation by IL-5,
GM-CSF, and IL-3.26-28 To ascertain the feasibility of targetting this region of c for developing molecules
that can simultaneously block IL-5, GM-CSF, and IL-3 stimulation of
eosinophils, we have attempted to raise functional MoAbs against this
domain. However, previous attempts using cells expressing the
full-length c as the immunogen failed to produce any
MoAb to domain 4 of c, and immunization with chemically
synthesized peptides encompassing either the predicted B-C and F-G
loops or the whole domain 4 led to MoAb that recognized the immunogen
but failed to bind to native c. We have now succeeded in
obtaining a blocking MoAb by immunizing mice with COS cells
overexpressing a cDNA encoding only the extracellular domain 4 of
c (QP). This MoAb, termed BION-1, immunoprecipitated full-length c as well as QP from transfected COS
cells and recognized native c in purified eosinophils,
neutrophils, and monocytes as judged by flow cytometry
(Fig 1).
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| Fig 1.
Flow cytometry analysis of the staining of MoAb BION-1
(solid line) and an isotype-matched IgG1 control MoAb
(dotted line) to (A) COS cells transiently transfected with
c, (B) CHO cells constitutively expressing
c, (C) TF-1.8 cells, (D) neutrophils, (E) eosinophils,
and (F) monocytes.
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BION-1 inhibits the high-affinity binding of IL-5, GM-CSF, and IL-3
to human eosinophils.
Given that domain 4 of c is crucial for the
high-affinity binding of IL-5, GM-CSF, and IL-3, we examined whether
BION-1 was able to affect this binding. Initially, we found that
BION-1, either as a whole IgG or as a Fab fragment, inhibited, in a
dose-dependent manner, the binding of 125I-IL-5,
125I-GM-CSF, and 125I-IL-3 to the human
erythroleukemic cell line TF-1.8 (data not shown). Significantly,
BION-1 blocked the binding of IL-5, GM-CSF, and IL-3 to primary
purified human eosinophils (Fig 2). In
contrast, other anti-c MoAb or IgG1 MoAb
controls failed to do so. For each cytokine, we used the lowest
practicable concentration of radioligand, thereby allowing binding to
high-affinity sites with minimal binding to low-affinity sites.
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| Fig 2.
BION-1 blocks high-affinity binding of IL-5, GM-CSF, and
IL-3 to human eosinophils. Human eosinophils (1.8 × 106)
were preincubated either alone ( ) or in the presence of 1 µmol/L
BION-1 MoAb ( ) or an isotype IgG1-matched control
antibody ( ) before the addition of radiolabeled cytokine.
Nonspecific binding was in each case less than 1% of total counts
added. Each point is the mean of duplicate determinations and error
bars represent 1 standard deviation.
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To demonstrate that the inhibitory effect of BION-1 was solely due to
the blocking of high-affinity receptors, saturation binding studies
were performed on TF-1 cells and peripheral blood mononuclear cells
(PBMNC) in the presence or absence of BION-1 (Fig 3). Binding was performed with GM-CSF
over a wide range of concentrations, ie, 10 pmol/L to 10 nmol/L.
Scatchard transformation of the binding data obtained shows that BION-1
but not anti-c antibody 8E4 completely blocked GM-CSF
binding to high-affinity binding sites but not to low-affinity sites
(Fig 3). These results, together with those in Fig 2, suggest that
BION-1 recognizes the same binding site on c used by
IL-5, GM-CSF, and IL-3 or binds to an epitope in close proximity to it.
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| Fig 3.
BION-1 blocks high-affinity binding of GM-CSF to TF-1 and
PBMNC. Scatchard transformation of saturation binding studies performed
on (A) TF-1 cells or (B) PBMNC preincubated either alone ( ) or in
the presence of 1 µmol/L BION-1 MoAb ( ) or 1 µmol/L of a control
anti-c MoAb, 8E4 () before the addition of
radiolabeled GM-CSF. Binding assays were performed with
125I-labeled GM-CSF over a concentration range of 10 pmol/L
to 10 nmol/L. The dashed line indicates the best fit for BION-1
noninhibitable binding and the solid line indicates the high-affinity
GM-CSF binding in the absence of BION-1.
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Epitope mapping of BION-1.
In an attempt to define the region in c recognized by
BION-1, we used several mutants of c and examined
whether substitutions of individual aminoacids in the predicted B-C
loop or F-G loop impaired BION-1 binding. To perform these experiments,
we transfected COS cells with wild-type and mutant c and
measured the affinity of 125I-BION-1 and
125I-1C1 (as binding control). The results showed that
c mutants carrying either substitutions M363A/R364A,
E366A, or R418A were not recognized by BION-1
(Table 1), indicating that these residues form part of the BION-1 epitope.
To address this issue further, we performed a reciprocal experiment in
which increasing concentrations of IL-3 were used to compete for
125I-BION-1 binding. As can be seen in
Fig 4, the addition of IL-3 to TF1.8 cells
expressing endogenous IL-3 receptor and c chains prevented, in a dose-dependent manner, the binding of
125I-BION-1 to c. In contrast, TNF did
not prevent the binding of 125I-BION-1 to
c. These results emphasize the close proximity of the
epitopes on c that the cytokines and BION-1 bind.
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| Fig 4.
The binding of BION-1 to TF1.8 cells is inhibited by IL-3
but not by TNF. TF1.8 cells (2 × 106) were incubated
with a range of concentrations of IL-3 () or TNF () in the
presence of 125I-labeled MoAb BION-1 (1 nmol/L). Each point
is the mean of duplicate determinations and error bars represent 1 standard deviation.
|
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BION-1 specifically inhibits eosinophil colony formation and
activation induced by IL-5, GM-CSF, and IL-3.
To ascertain whether the inhibition of IL-5, GM-CSF, and IL-3 binding
by BION-1 was translated into inhibition of IL-5, GM-CSF, and IL-3
stimulation, we initially used the factor-dependent TF-1.8 cell line
that proliferates in response to either IL-5, GM-CSF, IL-3, or EPO. We
found that BION-1 inhibited the stimulation of TF-1.8 cell
proliferation by IL-5, GM-CSF, and IL-3 in a dose-dependent manner,
with an ED50 of approximately 0.2 to 2 mmol/L, whereas a
control anti-c MoAb , 8E4, had little or no effect
(Table 2). The Fab fragment of BION-1
behaved similarly with virtually identical ED50 values to
the BION-1 antibody (data not shown). Additionally, the stimulating
ability of EPO was not inhibited by BION-1, showing the specificity of
BION-1 for the IL-5/GM-CSF/IL-3 receptors system (data not shown).
Because eosinophils are believed to be the major effector cells in
asthma and they respond to IL-5, GM-CSF, and IL-3, we examined BION-1
for its ability to block eosinophil production and survival in response
to these 3 cytokines. We found that BION-1, but not another
anti-c MoAb, 8E4, inhibited the ability of IL-5, GM-CSF, and IL-3 to stimulate the formation of eosinophil colonies from human
bone marrow cells (Table 3). In addition,
BION-1 inhibited the prosurvival activity of IL-5, GM-CSF, and IL-3 on
purified peripheral blood human eosinophils
(Fig 5). Because these cytokines are
essential for maintaining eosinophil viability, blocking of c by MoAb BION-1 promoted eosinophil cell death to
levels similar to those observed in the absence of cytokines (Fig 5).
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| Fig 5.
BION-1 blocks IL-5-, GM-CSF-, and IL-3-induced
survival of eosinophils. (A) The percentage of viability of eosinophils
after 36 hours in the presence of a range of concentrations of IL-5
(), GM-CSF (), IL-3 ( ), or MoAb BION-1 ( ). (B) The
percentage of viability of eosinophils after 36 hours in the presence
of IL-5, GM-CSF, and IL-3 (1 nmol/L) and different concentrations of
MoAb BION-1 (open symbols) and a control anti-c MoAb,
8E4 (solid symbols). Each point is the mean of triplicate
determinations and error bars represent 1 standard deviation.
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Eosinophil functional activity can be activated by IL-5, GM-CSF, and
IL-3 as well as by TNF, with the latter using p55 and p75 of the
TNF receptor but not c. A sign of eosinophil
activation is the upregulation of the CD69 surface antigen, a marker of
eosinophil activation in asthma42 and in vitro
(Fig 6A). We found that BION-1 inhibited
the upregulation of eosinophil CD69 induced by IL-5, GM-CSF, and IL-3
(Fig 6B), whereas another anti-c MoAb failed to do so.
The blocking effect of BION-1 was found to be specific for the IL-5,
GM-CSF, and IL-3 receptors, because the stimulating activity of TNF
was not inhibited (Fig 6B).
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| Fig 6.
MoAb BION-1 inhibits IL-5-, GM-CSF-, and
IL-3-stimulated but not TNF-stimulated CD69 upregulation on human
eosinophils. (A) CD69 upregulation in the presence of different
concentrations of IL-5 (), GM-CSF (), IL-3 (), and TNF
( ). (B) CD69 upregulation stimulated by 0.3 nmol/L of IL-5, GM-CSF,
IL-3, or TNF in the presence of different concentrations of MoAb
BION-1 (open symbols) or control anti-c MoAb, 8E4 (solid
symbols). Results are expressed as the percentage increase or change in
CD69 expression relative to unstimulated cells. Each point is the mean
value of 3 replicates and error bars represent 1 standard deviation.
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BION-1 specifically inhibits IL-3 receptor dimerization and
activation.
We have previously shown that IL-3, GM-CSF, and IL-5 induce
dimerization of the respective chains with c, a
phenomenon that leads to receptor activation as measured by
phosphorylation of c on tyrosine
residues.35,43 We used this system to show that
preincubation of Mo7e cells with BION-1 blocks receptor dimerization and tyrosine phosphorylation of c, whereas
anti-c MoAb 1C1 was unable to prevent receptor
dimerization and activation (Fig 7). This
result suggests that, although IL-5, GM-CSF, and IL-3 direct binding to
c is not detectable, a direct interaction is obligatory for receptor dimerization and subsequent cellular activation.
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| Fig 7.
Inhibition of IL-3-induced and chain
dimerization and phosphorylation by MoAb BION-1. (A)
Immunoprecipitations using anti-IL-3R MoAb, 9F5, or
anti-c MoAb, 8E4, from 125I-surface-labeled
MO7e cells preincubated with MoAbs BION-1, control
anti-c MoAb 1C1, or medium alone ( ), before being
stimulated (+) or not () with IL-3. The radiolabeled proteins were
spearated by SDS-PAGE and visualized by PhosphorImaging. The position
and molecular weight of marker proteins are shown. (B)
Immunoprecipitated proteins were probed using antiphosphotyrosine MoAb
3-365-10.
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| |
DISCUSSION |
We show here that targetting the common subunit of the human IL-5,
IL-3, and GM-CSF receptors allows for the simultaneous blocking of the
binding and stimulating activities of all 3 eosinophilopoietic cytokines. The MoAb BION-1 and its Fab fragment thus represent initial
reagents with which to inhibit eosinophil production, survival, and
activation in vitro and in vivo. Furthermore, the ability of BION-1 to
directly bind to the cytokine-interacting region of c
offers a new approach to identifying small molecule antagonists of
c.
IL-5, GM-CSF, and IL-3 are the only cytokines so far discovered that
stimulate the production of eosinophils from the bone marrow. In
addition, these cytokines stimulate the adhesion capacity of
eosinophils, their cytotoxic activity, and their survival. Because of
these properties, IL-5, GM-CSF, and IL-3 have been implicated in
chronic inflammatory conditions mediated by eosinophils, of which
allergic inflammation such as asthma is one of the best studied. In
bronchial asthma, eosinophils have long been recognized to play a
central role, as judged by their accumulation in the bronchoalveolar
lavage fluid and the presence in the sputum of asthmatics of eosinophil
cationic proteins believed to contribute to local tissue
damage.7 Of the different eosinophil regulators, IL-5 has
been a major focus of attention, largely due to its specificity for the
eosinophil lineage. Elevated IL-5 mRNA and protein levels have been
observed in biopsies of bronchial mucosa in asthma.8,9 Furthermore, recruitment of eosinophils is associated with elevated IL-5 in the airways of asthma patients.10 Given the
involvement of IL-5 in allergic inflammation, new therapeutic
approaches have involved the use of anti-IL-5
antibodies20,21 and the development of specific IL-5
antagonists.23,24
BION-1 may offer an alternative approach by blocking IL-5 through its
interaction with c, with the added advantage of
also blocking GM-CSF and IL-3. It is becoming clear that not
only IL-5, but also other eosinophilopoietic (GM-CSF and
IL-3)11,14,15,44-46 as well as noneosinophilopoietic
cytokines (IL-4)47-49 are elevated in asthma. Furthermore,
the production of IL-5, GM-CSF, and IL-3 by CD4 cells is believed to
prolong eosinophil survival in vivo.12,13 In the case of
GM-CSF, for example, transgene expression allows the development of
allergic airway inflammation.50,51 The extent to which
these cytokines contribute to the lung inflammation is not clear. Their
concomitant production may accentuate local eosinophilia or,
alternatively, induce temporal and site-specific phases of eosinophil
recruitment and activation.18 In either case, the simultaneous antagonism of the 3 eosinophilopoietic cytokines may more
profoundly downregulate eosinophil-dependent inflammation.
The use of BION-1 or similar molecules that can simultaneously inhibit
IL-5, GM-CSF, and IL-3 would be advantageous not only in asthma, but
also in other eosinophil-dependent inflammatory diseases such as
chronic hyperplastic sinusitis52 and nasal polyposis,16 in which all 3 cytokines have been found to be elevated. Furthermore, BION-1 and functionally analogous molecules may
also be antagonists of choice in certain leukemias that produce and
respond to GM-CSF and IL-3. A novel model of chronic myeloid leukemia
has recently implicated bcr/abl in the production of excess GM-CSF and
IL-3 and the development of disease in mice.53 The degree
of c inhibition required would have to be carefully ascertained in each case, because the complete absence of
c can lead, at least in mice, to lung
disease.54
BION-1 was able to greatly decrease eosinophil colony formation by
IL-5, GM-CSF, and IL-3. This illustrates the importance of
c for eosinophil production and is consistent with
c gene knockout experiments showing a major reduction in
eosinophil numbers in the peripheral blood and bone marrow of these
mice.54 In addition, BION-1 profoundly inhibited eosinophil
viability down to levels observed in the absence of cytokines, a
property similar to that seen by some glucocorticoids such as
fluticasone 17-propionate.55 This ability of BION-1 to
inhibit IL-5-, IL-3-, and GM-CSF-mediated eosinophil viability
suggests its use as an adjuvant of steroid therapy, its use to reduce
the dosage of concomitant steroid therapy, and its use in
steroid-resistant asthma.
The membrane proximal domain 4 of c was chosen as the
immunogen to generate BION-1, because this domain contains the major sites supporting cytokine high-affinity binding and receptor
activation.26-28 We and others have previously shown that
the B-C and F-G loops in domain 4 of c support
high-affinity binding to IL-5, IL-3, and GM-CSF and are important for
receptor activation. Consistent with this and with the inhibition of
BION-1 and IL-3 for each other's binding, we found that residues M363
R364, E366 in the predicted B-C loop, and R418 in the predicted F-G
loop form part of the BION-1 epitope. Interestingly, these residues do
not appear to be directly involved in mediating high-affinity binding;
however, they may be close in the 3-dimensional structure to Y365,
H367, I368, and Y421, the mutation of which has a profound effect on cytokine binding and activation.26-28 Thus, these data
suggest a more complex cytokine binding site than that found by
mutagenesis and identify new target amino acids in c for
the development of novel antagonists. Furthermore, these data, together
with the ability of BION-1 to prevent IL-3-mediated dimerization of
the IL-3 receptor chain and c, support the concept
that receptor dimerization and subsquent activation require ligand
contacting both receptor subunits. However, we cannot rule out that,
given the size of BION-1 as a Fab molecule of 50,000 MW, inhibition of
receptor dimerization is the result of steric hindrance.
The development of BION-1 may have 2 immediate uses. Firstly, it may be
used as a therapeutic in its own right after humanization and affinity
maturation to improve its ED50. The use of MoAbs in
clinical medicine is rapidly gaining momentum to the point that they
now encompass 27% of biotechnology therapeutics in development and
several anticytokine antibodies such as anti-TNF56 are already showing their efficacy in clinical trials. In relation to IL-5,
animal models show that anti-IL-5 antibodies are proving to be
efficacious in asthma.31 Secondly, BION-1 may be used as
the basis for a novel screening assay for antagonists of
c. The observation that IL-3 could reciprocally inhibit
the binding of 125I-labeled BION-1 to c (Fig
4) suggests that compounds with the same specificity may be obtained.
The major advance lies in the fact that, unlike IL-5, GM-CSF, and IL-3,
which require prior binding to each subunit before interacting with
c, BION-1 can directly bind to c, thereby
facilitating a cell-free solid-phase assay on which natural or
engineered products may be screened for inhibition of binding of
labeled BION-1 to immobilized c or domain 4 of
c. The approach described here may also be extended to
other receptors systems, such as the common subunit of the IL-4 and
IL-13, which mediates the allergen-induced asthma triggered by IL-4 and
IL-13.57,58
| |
FOOTNOTES |
Submitted December 23, 1998; accepted May 12, 1999.
Supported by the NH&MRC of Australia.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to A.F. Lopez, MD, PhD, The Hanson Centre for
Cancer Research, IMVS, Frome Road, Adelaide, SA 5000, Australia.
| |
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ABSTRACT
INTRODUCTION