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INTRODUCTION |
Anti-cytokine therapies are aimed at the inhibition of a certain
cytokine that is responsible for the maintenance of a disease. Different strategies have been used to neutralize cytokines in patients. Most effective has been the application of soluble cytokine receptors that consist solely of the ectodomain but lack the
transmembrane and cytoplasmic regions. They bind the respective
cytokine with high affinity and specificity as membrane-bound receptors
do. In the treatment of chronic inflammatory diseases such as
rheumatoid arthritis, the use of dimeric soluble tumor necrosis
factor receptors for the neutralization of tumor necrosis factor
has been a real breakthrough (1).
IL-61 is secreted by several
cell types in response to various inflammatory stimuli. It is the major
mediator of the acute-phase response of the liver and is involved in
the coordination of inflammatory and immune responses at the site of
inflammation (2). In several acute and chronic inflammatory diseases
such as rheumatoid arthritis and inflammatory bowel diseases, in
postmenopausal osteoporosis, but also in certain types of cancer, IL-6
levels are elevated and a causal role for IL-6 in disease progression
has been suggested. In some cases inhibition of IL-6 activity by
receptor antagonists or neutralizing antibodies has beneficial effects
(3, 4).
IL-6 belongs to the family of hematopoietic cytokines (5). It is a
member of the subfamily of IL-6-type cytokines (6) comprising IL-6,
IL-11, ciliary neurotrophic factor, leukemia inhibitory factor,
oncostatin M, cardiotrophin-1, and cardiotrophin-like cytokine. They
all use the hematopoietic cytokine receptor gp130 as a common
signal-transducing receptor subunit (7). As a result of receptor
activation the transcription factor STAT3 becomes tyrosine-phosphorylated and translocates into the nucleus to induce target gene expression (8, 9).
Expression of gp130 is not sufficient for cells to become responsive to
IL-6. They additionally have to express the cytokine specific
-receptor subunit IL-6R
. This
-receptor is not involved in the
initiation of the cytoplasmic signal transduction cascades but is
essential for cytokine binding. Thus, activation of the receptor by
IL-6 requires two steps: (i) low affinity IL-6 binding to IL-6R
and
(ii) subsequent recruitment of the complex of IL-6 and IL-6R
to two
gp130 molecules leading to the formation of a high affinity ternary
complex (10).
Cells lacking IL-6R
can be stimulated with the combination of IL-6
and soluble IL-6R
(sIL-6R
) (10). In such a situation, IL-6 binds
to sIL-6R
in solution and the heterodimer of IL-6/sIL-6R
activates membrane-bound gp130. Soluble gp130 (sgp130) alone acts as a
relatively weak IL-6 antagonist (11). Most interestingly, the
antagonizing activity of sgp130 is substantially increased by the
presence of sIL-6R
(12). Both sIL-6R (13, 14) and sgp130 (11, 12)
are found in high concentrations in human blood (about 50 and 300 ng/ml, respectively). This pair of soluble receptors might act as a
natural IL-6 inhibitor to limit systemic IL-6 responses (12).
Structurally, IL-6 belongs to the family of the
-helix-bundle
cytokines. IL-6R
as well as gp130 belong to the family of class I
cytokine receptors (5). The extracellular regions of IL-6R
and gp130
consist of three (D1-D3) (15) or six domains (D1-D6) (16),
respectively. D2 and D3 of IL-6R
are involved in IL-6 binding (17).
The complex of IL-6 and IL-6R
is bound by D1-D3 of gp130 (18). IL-6
contains three receptor-binding sites. Site I is occupied by D2-D3 of
IL-6R
, and sites II and III bind to D2-D3 and D1 of gp130,
respectively (19-21). Based on the mutagenesis data and the recently
solved structure of D1-D3 of gp130 bound to viral IL-6, which binds
gp130 in the absence of any
-receptor, a reliable model of the
IL-6·IL-6R
·gp130 ternary complex has been proposed (22).
Inhibition of IL-6 activity by the use of soluble receptors is
challenging because of the bipartite nature of the IL-6 receptor. IL-6
alone does not bind to gp130. To be neutralized by sgp130, IL-6 must
first bind to sIL-6R
. A fusion protein of gp130 and sIL-6R
would
therefore guarantee that the agonistic complex of IL-6·sIL-6R
is
immediately neutralized. Only recently, due to the new structural data
on the IL-6·receptor complex (22), a promising rational approach on
how to design an IL-6-antagonist based on a fusion of sgp130 with
sIL-6R
became possible. In this study, we present a highly potent
IL-6 antagonist consisting of the ligand-binding moieties of sgp130 and
sIL-6R
.
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EXPERIMENTAL PROCEDURES |
Cloning of the Fusion Proteins--
A fragment corresponding to
D2-D3 of IL-6R
(Val110-Lys338) was
amplified by PCR introducing a multiple cloning site (SmaI,
NotI, MluI, NheI) with the sense primer and a
ApaI site, a stop codon instead of Met331, and a
BamHI site with the antisense primer. The product was cut
with SmaI (Roche Diagnostic GmbH, Mannheim, Germany) and
BamHI (MBI Fermentas GmbH, St. Leon-Rot, Germany) and cloned
into pSVL-gp130 (D1-D3) (Met1-Pro326)
digested with the same enzymes. Then three different linkers were added
after digestion of the obtained chimeric construct with MluI
(Promega, Madison, WI) and NheI (MBI Fermentas GmbH). The
first linker (stalk-49) corresponding to the short extracellular membrane proximal part of IL-6R
(Ala323-Val362) was produced by PCR. Its amino
acid sequence is GSAAATRAEN EVSTPMQALT TNKDDDNILF RDSANATSLP VQDSSSVAS.
The two other linkers were constructed with hybridized
oligonucleotides. The 41 amino acids of AGS-41 are GSAAATRGSA
GSGGSATGSG SAAGSGDSVA AGSGGGSGSA S. AGS-33 consists of the sequence
GSAAATRGSA GSGGSATGSG SAAGSGDSVR RAS. A FLAG tag was added to the C
terminus of all fusion proteins using hybridization of an
oligonucleotide pair containing ApaI,
XbaI, and BamHI restriction sites and a stop
codon. The fusion protein constructs were subcloned into the pIB/v5-his
vector (Invitrogen, Groningen, The Netherlands) cut with
BamHI and HindIII (Roche Diagnostic GmbH) to
express the protein in insect cells. The integrity of all constructs
was verified by DNA sequencing.
Expression in Insect Cells--
High 5 (H5) cells cultured in
Sf-900II medium (Invitrogen, Paisley, Scotland) were stably
transfected with the empty pIB/v5-his vector or vectors containing the
fusion protein constructs, using the CellFECTIN method (Invitrogen).
Cell supernatants were harvested every 3 days, cleared by
centrifugation, and stored at
20 °C until use.
Protein Precipitation--
The fusion proteins from cell
supernatants were precipitated overnight at 4 °C with IL-6
covalently linked to CNBr-Sepharose (Amersham Biosciences AB, Uppsala, Sweden).
Western Blotting--
Proteins were separated by SDS-PAGE,
transferred to polyvinylidene difluoride membranes (PALL, Dreieich,
Germany), incubated with the antibodies as indicated in the
figures, and processed for chemiluminescence detection (Amersham
Biosciences AB). Antibodies used for protein detection were as follows:
sIL-6R
(Eurogentec, Philadelphia, PA), Tyr(P) STAT3 (New
England Biolabs, Frankfurt, Germany), and STAT3 (Santa Cruz
Biotechnology Inc., Santa Cruz, CA).
Purification of the Fusion Proteins--
200-500 ml of H5 cell
supernatants containing the respective fusion proteins were applied to
an IL-6-Sepharose column (2 ml) at 4 °C. After rinsing with
phosphate-buffered saline, proteins were eluted with 6 ml of 2 M MgCl2. The eluate was dialyzed against phosphate-buffered saline or cell culture medium, and subsequently the
amounts of fusion proteins were measured by ELISA.
Quantification of Fusion Proteins by ELISA--
An ELISA
procedure was performed as described previously (12), using 0.3 µg/well of FLAG monoclonal antibody (Sigma) for coating and 50 ng/well of biotinylated monoclonal antibody B-T2 (DIACLONE,
Besançon, France) as secondary antibody. The standard curve was
obtained by 2-fold serial dilutions of sgp130-FLAG expressed in COS-7
cells and calibrated by sgp130 ELISA (12).
Ba/F3 Proliferation Assay--
Stably transfected
Ba/F3-gp130-IL6R and Ba/F3-gp130-IL11R cells were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum,
seeded on 96-well plates (20,000 cells/well), and stimulated with IL-6
(0.9 ng/ml) or trx-IL-11 (5 ng/ml) prepared as described previously
(23) in the presence of purified fp stalk-49 (500 ng/ml first
concentration) or purified mock-vector-transfected cell supernatant.
After 60 h of incubation, metabolically active cells were
quantified using a colorimetric assay based on the cell proliferation
kit II XTT assay (Roche Diagnostic GmbH).
Induction of Acute-phase Protein Synthesis in HepG2
Cells--
1-Anti-chymotrypsin synthesized by HepG2 cells was
measured by immunoprecipitation of radioactively labeled protein as
described previously (12).
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RESULTS |
Rational Design of a Fusion Protein of sgp130 and sIL-6R
as a
Potential IL-6 Inhibitor--
The fusion protein was designed to
contain the minimal regions of IL-6R
and gp130 required for high
affinity IL-6 binding. Moreover, the N terminus of mature gp130 should
not be affected by the fusion, since it is important for ligand binding
(24). Thus, the fusion protein consists of domains D1-D2-D3 of gp130 (including the signal sequence at the N terminus that directs its
secretion) followed by a linker and domains D2 and D3 of IL-6R
(Fig.
1A, upper scheme).
The two receptor fragments have to be connected by the linker in a way
that allows the fusion protein to adopt the conformation required for
efficient neutralization of IL-6. According to the ternary complex
model based on the x-ray structure of viral IL-6 bound to D1-D3 of
gp130 (22), the C terminus of gp130-D3 and the N terminus of
sIL-6R
-D2 are separated by at least 8 nm. This distance can be
bridged by a peptide linker of about 30-40 amino acids (Fig.
1A, lower part). The linker should be of high
conformational flexibility, of low immunogenicity, and resistant to
protease degradation. Three fusion proteins containing different
linkers were constructed. Two of them, AGS-33 and AGS-41, are made of
flexible Ala-, Gly-, and Ser-rich peptides of 33 and 41 amino acids,
respectively. In an extended conformation these linkers span from about
10 (AGS-33) to 12 nm (AGS-41). The third one (fp stalk-49) consists of
a short flexible fragment of the extracellular membrane-proximal part
of IL-6R
(25). Besides its flexibility, this linker is expected to
be of low immunogenicity, since it is derived from the endogenous
IL-6R
. For technical reasons, a FLAG tag epitope was added at the C
termini of all constructs (Fig. 1A, upper
scheme).

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Fig. 1.
Design and characterization of
sgp130/sIL-6R fusion proteins.
A: upper panel, schematic representation
of the fusion protein fp AGS-33. Numbers refer to the amino
acid positions in the fusion protein as indicated. Lower
panel, structural model of IL-6 (yellow) bound to a
fusion protein of sgp130 D1-D3 (green) and sIL-6R D2-D3
(blue). The gp130 and IL-6 part correspond to the solved
structure of viral IL-6 bound to gp130 D1-D3 (22). IL-6R D2-D3 is
adopted from the recently solved structure of sIL-6R (25). The
linker (red) contains 33 amino acids as in fp AGS-33. Domain
D1 of gp130 is involved in dimerization of the depicted ternary
complex, leading to a stable hexameric complex (not shown).
B, 5 ml of supernatants from insect cells stably transfected
with expression vectors encoding the fusion proteins or mock-vector
(control) were incubated with IL-6-Sepharose. Sepharose-bound proteins
were analyzed by immunoblotting using a polyclonal sIL-6R antibody.
C, purification of fp stalk-49 from insect cell supernatant
by IL-6 affinity chromatography. 200 ml of supernatant from insect
cells expressing fp stalk-49 were loaded onto a 2-ml IL-6-Sepharose
column. Bound proteins were eluted with 10 ml of 2 M
MgCl2, and 2 ml fractions were collected. The fusion
protein from 10 ml of supernatant (sn), 10 ml of the
fractions of the flow-through after 100, 150, and 200 ml, and from 1.5 ml of the 2-ml fractions of the eluates were precipitated and analyzed
by immunoblotting using a polyclonal sIL-6R antibody. The indicated
concentrations of fp stalk-49 were determined by ELISA.
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Purification and Characterization of Fusion Proteins Produced in
Insect Cells--
For continuous production of the fusion proteins
stably transfected H5 insect cell lines were generated. The fusion
proteins were precipitated from cell supernatants with IL-6-Sepharose
and analyzed by Western blotting (Fig. 1B). The apparent
molecular masses of the fusion proteins are 83.5 kDa for fp stalk-49,
69.5 kDa for fp AGS-33, and 72 kDa for fp AGS-41. The substantially higher molecular mass of fp stalk-49 is most likely due to an additional N-glycosylation site (Asn-Ala-Thr) introduced
with the linker.
We took advantage of the affinity of the fusion proteins to IL-6 for
their purification and concentration with IL-6-Sepharose. The insect
cell supernatant, the flow-through and the eluate of IL-6 affinity
chromatography were analyzed for the presence of fusion protein by
Western blotting (Fig. 1C). Compared with the supernatant
(sn, left lane), the fusion protein is strongly
enriched in the eluate. No fusion protein is detectable in the
flow-through fractions. The concentrations of fusion protein in the
fractions determined by a newly developed ELISA correlate well with the intensities of the bands in the Western blot (Fig. 1C).
After dialysis, enriched fp stalk-49 was used for the following
studies. Supernatants containing the other fusion proteins and
supernatants of mock-transfected insect cells were treated the same
way. The latter was used as negative control in the bioassays.
Potent IL-6 Antagonistic Activity of the Fusion Proteins--
To
test the IL-6 antagonizing activity of the fusion proteins,
supernatants of stably transfected insect cells were incubated with
IL-6 for 30 min to allow the fusion protein to bind to IL-6. Ba/F3
cells stably transfected with gp130 and IL-6R
(Ba/F3-gp130-IL6R) were stimulated with the IL-6-treated supernatants. After 30 min, cells
were lysed, and STAT3 phosphorylation was analyzed. In the presence of
supernatant from mock-transfected insect cells, stimulation of
Ba/F3-gp130-IL6R cells with 0.5 ng/ml IL-6 is sufficient to induce
prominent tyrosine phosphorylation of STAT3 (Fig.
2A, upper and
lower panels, lanes 1 and 2).
Treatment of Ba/F3-gp130-IL6R cells with IL-6 that was preincubated
with supernatants from cells expressing the fusion proteins did not
result in significant tyrosine phosphorylation of STAT3 (Fig.
2A, upper panel, lanes 3-5). Thus, all three fusion proteins in the supernatants inhibit IL-6 signaling, since no STAT3 phosphorylation is observed in response to IL-6. A
2-fold higher IL-6 concentration (1 ng/ml) is neutralized only incompletely by the supernatant containing fp AGS-33 (Fig.
2A, lower panel, lane 4).

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Fig. 2.
IL-6 antagonistic activity of
sgp130/sIL-6R fusion proteins.
A: upper panel, Ba/F3-gp130-IL6R cells were
incubated with supernatants from insect cells expressing fusion
proteins or from mock-transfected insect cells (control) and
stimulated with 0.5 ng/ml IL-6 (+) for 30 min or left unstimulated ( )
as indicated. Activation of STAT3 was analyzed by immunoblotting of
cellular lysates. Activation of STAT3 was detected using a polyclonal
antibody against tyrosine phosphorylated STAT3. After stripping of the
blot, STAT3 loading was controlled using a polyclonal STAT3 antibody.
Lower panel, Ba/F3-gp130-IL6R cells were incubated with
supernatant from insect cells expressing fp AGS-33 or mock-transfected
insect cells (control) and stimulated with different
concentrations of IL-6 for 30 min as indicated. Cellular lysates were
analyzed for STAT3 phosphorylation as described above. B,
HepG2 cells were incubated with 1 ng/ml IL-6 and 30 ng/ml of
fusion-proteins or an equivalent volume of purified
mock-vector-transfected cell supernatant (control) for
18 h and metabolically pulse-labeled with 35S for
3 h. Secreted 1-anti-chymotrypsin ( 1-ACT) was
immunoprecipitated from cell culture supernatants, separated by
SDS-PAGE, and analyzed by autoradiography.
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IL-6 is the major inducer of acute-phase protein synthesis in
hepatocytes, but also in hepatoma cell lines such as HepG2. IL-6
stimulation (1 ng/ml) leads to a substantially increased
1-anti-chymotrypsin production by HepG2 cells as shown by
immunoprecipitation of metabolically labeled protein (Fig.
2B, lanes 1 and 2). Purified proteins
of control supernatant do not affect
1-anti-chymotrypsin synthesis
(Fig. 2B, lane 3). In the presence of the
concentrated fusion proteins (30 ng/ml),
1-anti-chymotrypsin
synthesis is reduced to the basal level (Fig. 2B,
lanes 4-6). Thus, all three fusion proteins inhibit
IL-6-induced acute-phase protein synthesis.
Specificity of the IL-6-inhibiting sgp130/sIL-6R
Fusion Proteins--
To demonstrate the specificity of the
inhibitory fusion proteins, we compared the proliferation of Ba/F3
cells stably transfected with gp130 and IL-6R
or gp130 and IL-11R
(Ba/F3-gp130-IL11R) in response to 0.9 ng/ml IL-6 or 5 ng/ml IL-11,
required for 50 or 80% of maximal cell proliferation, respectively.
Trx-IL-11 is a fusion protein of thioredoxin and IL-11 exhibiting IL-11 activity indistinguishable from IL-11 wild type (23). The proliferation of Ba/F3-gp130-IL6R cells treated with a constant amount of IL-6 is
inhibited by the fusion protein fp stalk-49 in a
concentration-dependent manner. Purified control
supernatant had no significant effect (Fig.
3A, left diagram).
At a fusion protein concentration of 250-500 ng/ml, cell proliferation
is completely abrogated. Proliferation of Ba/F3-gp130-IL11R cells in
response to trx-IL-11, however, is not significantly disturbed by fp
stalk-49 (Fig. 3A, right diagram). Thus fp
stalk-49 specifically inhibits IL-6, but not IL-11 responses. Similar
observations were made for the remaining fusion proteins (data not
shown).

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Fig. 3.
Specificity and efficiency of the
IL-6-antagonizing sgp130/sIL-6R fusion
proteins. A, Ba/F3-gp130-IL6R (left panel)
or Ba/F3-gp130-IL11R cells (right panel) were incubated with
a constant amount of IL-6 (0.9 ng/ml) or trx-IL-11 (5 ng/ml),
respectively, and serial 2-fold dilutions of inhibitors starting with a
concentration of 500 ng/ml. Equivalent volumes of purified
mock-vector-transfected cell supernatant were used as a control. After
60 h of incubation, viable cells were quantified using the
colorimetric XTT assay (Roche Diagnostic GmbH). Dashed lines
and dotted lines correspond to proliferation in the absence
of cytokine or inhibitor, respectively, derived from the standard
curves (not shown). Mean values from three independent experiments are
shown with S.D. values. B, Ba/F3-gp130-IL6R cells were
stimulated for 20 min with 0.5 ng/ml IL-6, which was preincubated for
15 min at 4 °C with 1.5 ng/ml fp stalk-49 or the equivalent volume
of purified mock-vector-transfected cell supernatant
(control) or with increasing amounts of soluble receptors
(sR): 1 ng/ml sIL-6R and 2 ng/ml sgp130 (lane
4), 10 ng/ml sIL-6R and 20 ng/ml sgp130 (lane 5);
and 100 ng/ml sIL-6R and 200 ng/ml sgp130 (lane 6).
Activation of STAT3 was analyzed by immunoblotting of cellular lysates.
STAT3 tyrosine phosphorylation was detected as described in legend to
Fig. 2A. sIL-6R and sgp130 were prepared as described previously
(12, 33).
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Inhibitory Activity of the Fusion Proteins Compared with Separately
Expressed sgp130 and sIL-6R
--
Next, we proved that the
appropriate fusion of the ligand-binding domains of gp130 and IL-6R
leads to a more potent inhibitor than sgp130 and sIL-6R
separately
expressed in insect cells. The inhibition of STAT3 phosphorylation in
Ba/F3-gp130-IL6R cells induced by 0.5 ng/ml IL-6 achieved with 1.5 ng/ml fp stalk-49 was compared with inhibition by the combination of
sgp130 and sIL-6R
(Fig. 3B). The
approximately equimolar ratio of IL-6 and fp stalk-49 is sufficient for
almost full suppression of IL-6 induced STAT3 activation (lanes
1-3). In contrast to this extremely high antagonistic potency of
the fusion protein, a molar ratio of 1:100 of IL-6 and the combination
of sIL-6R
and sgp130 is required to achieve inhibitory activity
(lanes 4-6). We conclude that the fusion proteins are of
~100-fold increased inhibitory activity compared with the separate
soluble receptor proteins.
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DISCUSSION |
In this study we present a highly potent IL-6 inhibitor based on
the ligand-binding domains of the IL-6 receptor subunits IL-6R
and
gp130. Many of the existing IL-6 receptor antagonists are IL-6 mutants
of binding sites to gp130 (sites II and III). They block IL-6R
by
binding via the intact site I but do not recruit gp130 (26,
27). Since the interaction of IL-6 with IL-6R
is of low affinity, so
called superantagonists were created by mutating the site I of these
antagonists to strengthen
-receptor binding. Although the
superantagonists perform better, they still have to be applied in a
large excess to IL-6 (27). Furthermore, due to the many mutations the
proteins are highly immunogenic (28). Neutralizing IL-6 or IL-6R
antibodies have also been used as IL-6 inhibitors. They were tested in
clinical trials for the treatment of rheumatoid arthritis (3) or
AIDS-associated Kaposi's sarcoma (29) but turned out to be of rather
low efficiency. Very recently, potent low molecular mass IL-6 receptor
antagonists were described for the first time (30, 31). These
antagonists have to be applied in the micromolar range to
inhibit picomolar amounts of IL-6.
A new generation of cytokine antagonists is based on soluble receptor
fragments that bind the ligand with high affinity and specificity. In
the case of IL-6, two receptor subunits are required for high affinity
binding, IL-6R
and gp130. Moreover, the complex of IL-6 and
sIL-6R
acts agonistically on cells expressing gp130 (10).
Conversely, sIL-6R
supports neutralization of IL-6 by sgp130 due to
formation of a soluble high affinity ternary complex (12). The new IL-6
receptor antagonist presented in this study stems from the idea that
appropriate fusion of the ligand-binding domains of IL-6R
and gp130
should result in a superior antagonist that neutralizes IL-6 with
highest affinity and specificity. In the present study three different
linkers were used to connect the ligand-binding domains of gp130 and
IL-6R
. It turned out that the fusion proteins exhibit similar
inhibitory activities, indicating that the estimation of the required
linker length has been correct and appropriate peptide linkers were chosen.
All three fusion proteins bind IL-6 as shown by precipitation with
IL-6-Sepharose. The fusion protein present in the insect cell
supernatant is sufficient to completely antagonize the activity of 0.5 ng/ml (25 pM) IL-6 in the short term STAT3 phosphorylation assay using transfected Ba/F3 cells (Fig. 2A). Since the
concentrations of the fusion proteins in the insect cell supernatants
are in the range of 1-2 ng/ml (15-30 pM), this points to
an inhibitory activity at a molar ratio between agonist and antagonist
of 1:1.
In a long term assay such as induction of acute-phase protein synthesis
in HepG2 cells, the activity of 1 ng/ml (50 pM) IL-6 was
totally blocked by the addition of inhibitory fusion protein at a
nearly 10-fold molar excess (450 pM). In the Ba/F3
proliferation assay with fp stalk-49, we determined an IC50
of 6 ng/ml (90 pM) for the inhibition of 0.9 ng/ml IL-6 (45 pM). Thus, in long term assays and therefore also for
studies of the inhibitory activity of the fusion proteins in
vivo, an about 10-fold molar excess of fusion protein over IL-6
should be applied.
Besides their inhibitory activity the specificity of the fusion
proteins is an important feature to assess their potential value for
anti-cytokine therapies. IL-11 most closely resembles IL-6 because it
also signals via gp130 homodimers but binds to a different
-receptor, namely IL-11R
. In the Ba/F3-proliferation assay,
amounts of fusion proteins that significantly inhibit IL-6 activity had
no effect on IL-11 induced proliferation (data shown only for fp
stalk-49). Therefore, at the concentrations used in our assays each of
the three fusion proteins is a potent and specific inhibitor of IL-6 activity.
The superior activity of the fused ligand-binding domains of gp130 and
IL-6R
compared with the separate soluble receptors sgp130 and
sIL-6R
is probably the most important issue left to be proven to
confirm the value of our concept. IL-6-induced STAT3 phosphorylation in
Ba/F3 cells is inhibited by the presence of equimolar amounts of fusion
protein. To achieve a similar inhibition an at least 100-fold molar
excess of sgp130 and sIL-6R
has to be applied. This intriguing
result clearly demonstrates the extraordinary high inhibitory activity
of the fusion protein. What is the explanation for this finding? In the
above assay, a low amount of IL-6 that is in the range of
pathophysiological IL-6 concentrations (500 pg/ml) was applied. When
the separate soluble receptors were used, IL-6 first binds to the
sIL-6R
. This interaction is of low affinity, and therefore the
complex of IL-6 and sIL-6R
might dissociate before it encounters
sgp130. In the fusion protein, the initial complex of IL-6 bound to
domains D2 and D3 of IL-6R
can be immediately trapped by the
covalently linked ligand-binding domains of gp130 before dissociation occurs.
Our inhibitor strategy is also applicable to other cytokines that
signal via heteromeric receptor complexes. Indeed, in a recent publication Economides et al. (32) used a similar
approach to create so called "cytokine traps" as highly potent
inhibitors for IL-1, IL-4, and IL-6. In their study the complete
ectodomains of the respective receptor subunits including the regions
dispensable for ligand binding were fused to the Fc part of human IgG.
This results in dimerization of the receptor chains by disulfide bond formation of the Fc parts. In the case of the IL-6 inhibitor, this
leads, besides the desired sgp130-Fc/sIL-6R
-Fc heterodimers, to the
formation of gp130-Fc/gp130-Fc and sIL-6R
-Fc/sIL-6R
-Fc homodimers. As a consequence, before application, the heterodimer must
be separated from the homodimers. On the XG-1 myeloma cells an
IC50 of 50 pM sgp130-Fc/sIL-6R
-Fc was
determined for the neutralization of 2.5 pM (0.05 ng/ml)
IL-6 (32). On our Ba/F3-gp130-IL6R cells treated with 45 pM
IL-6, the IC50 of fp stalk-49 is 90 pM. If one
takes into account the molar ratio between the IL-6 concentration in
the two different proliferation assays and the IC50 of the respective inhibitor, it turns out that fp stalk-49 is about 10-fold more potent than sgp130-Fc/sIL-6R
-Fc. These findings suggest that
the more structure-based approach presented in our study confirms the
validity of the basic concept and furthermore leads to optimized
inhibitory fusion proteins.
We conclude that appropriate fusion of the ligand-binding domains
of soluble receptor proteins leads to cytokine inhibitors of
extraordinary activity which might be of considerable therapeutical value for the development of new anti-cytokine therapies.