Note: Descriptions are shown in the official language in which they were submitted.
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Glycosaminoglycan-antagonising MCP-1 mutants and methods of using same
The present invention relates to novel mutants of human monocyte
chemoattractant
protein 1 (MCP-1) with increased glycosaminoglycan (GAG) binding affinity and
knocked-out or reduced GPCR activity compared to wild type MCP-1, and to their
use
for therapeutic treatment of inflammatory diseases.
All chemokines, with the exception of lymphotactin and fraktaline/neurotactin
which are
members of the C and CX3C chemokine subfamily, respectively, have four
cysteines in
conserved positions and can be divided into the CXC or a-chemokine and the CC
or (3-
chemokine subfamilies on the basis of the presence or absence, respectively,
of an
amino acid between the two cysteines within the N-terminus. Chemokines are
small
secreted proteins that function as intercellular messengers to orchestrate
activation and
migration of specific types of leukocytes from the lumen of blood vessels into
tissues
(Baggiolini M., J. Int. Med. 250, 91-104 (2001)). This event is mediated by
the
interaction of chemokines with seven transmembrane G-protein-coupled receptors
(GPCRs) on the surface of target cells. Such interaction occurs in vivo under
flow
conditions. Therefore, the establishment of a local concentration gradient is
required
and ensured by the interaction of chemokines with cell surface
glycosaminoglycans
(GAGs). Chemokines have two major sites of interaction with their receptors,
one in the
N-terminal domain which functions as a triggering domain, and the other within
the
exposed loop after the second cysteine, which functions as a docking domain
(Gupta
S.K. et al., Proc.Natl.Acad.Sci., USA, 92, (17), 7799-7803 (1995)). The GAG
binding
sites of chemokines comprise clusters of basic amino acids spatially distinct
(Ali S. et
al., Biochem.J. 358, 737-745 (2001)). Some chemokines, such as RANTES, have
the
BBXB motif in the 40s loop as major GAG binding site; IL-8 interacts with GAGs
through
the C-terminal a-helix and Lys 20 in the proximal N-loop. Other chemokines,
such as
MCP-1, show a significant overlap between the residues that comprise the
receptor
binding and the GAG binding site (Lau E.K. et al., J. Biol. Chem., 279 (21),
22294-
22305 (2004)).
In the context of the chemokine-R family of cytokines, monocyte
chemoattractant
protein-1 (MCP-1) is a monocyte and lymphocyte-specific chemoattr_actant_and-
activator-
found in a variety of diseases that feature a monocyte-rich inflammatory
component,
CONFIRMATION COPY
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such as atherosclerosis (Neiken N.A. et al., J. Clin. Invest. 88, 1121-1127
(1991); Yla-
Herttuala, S., Proc. Natl. Acad. Sci USA 88, 5252-5256 (1991), rheumatoid
arthritis
(Koch A.E. et al., J. Clin. Invest. 90, 772-779 (1992); Hosaka S. et al.,
Clin. Exp.
Immunol. 97(3), 451-457 (1994), Robinson E. et al., Clin. Exp. Immunol.
101(3), 398-
407 (1995)), inflammatory bowel disease (MacDermott R.P. et al., J. Clin.
Immunol. 19,
266-272 (1999)) and congestive heart failure (Aukrust P., et al., Circulation
97, 1136-
1143 (1998), Hohensinner P.J. et al., FEBS Letters 580, 3532-3538 (2006)).
Crucially,
knockout mice that lack MCP-1 or its receptor CCR2, are unable to recruit
monocytes
and T-cells to inflammatory lesions (Grewal I.S. et al., J. Immunol. 159 (1),
401-408
(1997), Boring L. et al., J. Biol. Chem. 271 (13), 7551-7558 (1996), Kuziel
W.A., et al.,
Proc.NatI.Acad.Sci. USA 94 (22), 12053-8 (1997), Lu B., et al., J. Exp.Med.
187 (4),
601-8 (1998)); furthermore, treatment with MCP-1 neutralizing antibodies or
other
biological antagonists can reduce inflammation in several animal models
(Lukacs N.W.
et al., J.Immunol., 158 (9), 4398-4404 (1997), Flory C.M. et al., 1. Lab.
Invest. 69 (4),
396-404 (1993), Gong J.H., et al., J.Exp.Med. 186 (1), 131-7 (1997), Zisman
D.A. et al.,
J.Clin.lnvest. 99 (12), 2832-6 (1997)). Finally, LDL-receptor/MCP-1 -deficient
and apoB-
transgenic/MCP-1 -deficient mice show considerably less. lipid deposition and
macrophage accumulation throughout their aortas compared to the WT MCP-1
strains
(Alcami A. et al., J. Immunol. 160 (2), 624-33 (1998), Gosling J. et al., J.
Clin. Invest.
103 (6), 773-8 (1999)).
Since the first chemokines and their receptors have been identified, the
interest on
exactly understanding their roles in normal and diseased physiology has become
more
and more intense. The constant need for new anti-inflammatory drugs with modes
of
action different from those of existing drugs support the development of new
protein-
based GAG-antagonists and their use in an inflammatory set.
Since in the last years the molecular basis of the interactions of MCP-1 with
CCR2 and
GAGs have been studied in great detail, targeted engineering of the chemokine
towards
becoming an effective antagonist of MCP-1's biological action is feasible.
For this purpose several recombinant MCP-1 variants that compete with their
wild type
counterpart for glycosaminoglycan binding_and-sho.w r.educed-or-knocked-out-
activation
of.leukocytes have been generated.
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Consequently, one subject matter of the present invention is to inhibit
leukocyte, more
specifically monocyte and T cell, migration by antagonizing the GAG
interaction with an
MCP-1 -based mutant protein in the context of inflammatory or allergic
processes.
The invention is based on engineering a higher GAG binding affinity into human
MCP-1,
either by modifying the wild type GAG binding region or by introducing a new
GAG
binding region into the MCP1 protein and simultaneously knocking out or
reducing its
GPCR activity, specifically the CCR2 activity of the chemokine. This has been
successfully accomplished with a mutant MCP-1 protein wherein a region of the
MCP-1
protein is modified in a structure conserving way by introducing basic and/or
electron
donating amino acids or replacing native amino acids with basic and/or
electron
donating amino acids and optionally also modifying the N-terminal region of
said MCP-1
protein by addition, deletion and/or replacement of amino acids and,
optionally, adding
an N-terminal Methionine (M) to the mutant MCP-1 protein, resulting in partial
or
complete loss of chemotactic activity. Said inventive MCP-1 mutants can
specifically
exhibit a minimum five-fold improved Kd for standard GAGs (heparin or heparan
sulfate)
and they are deficient or reduced in inducing Calcium-release in standard
monocytic cell
culture.
MCP-1 mutant proteins showing increased GAG binding affinities and reduced
reduced
GPCR activity has not been disclosed or indicated before. US2003/0162737
describes
MCP-1 molecules with N-terminal deletions and replacements with amino acids N
or L
at selected positions 22 and 24 f the MCP-1 protein, yet these mutant proteins
did not
show the advantageous features of the inventive MCP-1 proteins. This was also
not
disclosed by Steitz S. et al (FEBS Letters, 40 (1998), pp. 158-164) who
modified only
positions 13 and 18 of the MCP-1 protein. Paavola C. et al. (J.Biol.Chem.,
1998, 273,
pp. 33157-33165) describe only MCP-1 mutants which are involved in receptor
binding
activity but did include modifications to reduce GAG binding affinity of the
mutant MCP-
1 protein.
Further, the present invention provides an isolated polynucleic acid molecule
coding for
the mutant MCP-1 protein of the invention, and a vector comprising an isolated
DNA
molecule coding for the mutant MCP-1 protein, and a recombinant cell
transfected with
the vector.
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The mutant MCP-1 protein according to the present invention can also be
formulated as
a pharmaceutical composition comprising the mutant MCP-1 protein or a
polynucleic
acid molecule coding for MCP-1 mutant protein, a vector containing an isolated
DNA
molecule coding for the MCP-1 mutant protein, and a pharmaceutically
acceptable
carrier.
Said MCP-1 mutant protein or the polynucleotide coding therefor or the vector
containing said polynucleotide can also be used for inhibiting or suppressing
the
biological activity of the respective wild type protein.
The inventive MCP-1 mutant protein according to the invention, a polynucleic
acid
coding therefor or a vector containing the polynucleotide can also be used in
a method
for preparing a medicament for the treatment of chronic or acute inflammatory
diseases
or allergic conditions. Preferably, the disease is selected from the group
comprising
rheumatoid arthritis, uveitis, inflammatory bowel disease, myocardial
infarction,
congested heart failure or ischemia reperfusion injury.
Figures:
Figure 1: Sequence of MCP-1 mutants, mutations with respect to the wild type
chemokine are underlined
Figure 2: Structural change of wtMCP-1 (Figure 2 a) and Met-MCP-1 Y13A S21 K
Q23R
(Figure 2b) upon heparan sulfate binding, as shown by far-UV CD spectroscopy
Figure 3: Scatchard plot analysis and equilibrium dissociation constants (Kd
values) of
WT MCP-1 (solid squares), Met-MCP-1 Y13A S21 K (solid triangles) and Met-MCP-1
Y13A S21 K Q23R (open circles) binding to unfractionated HS
Figure 4: Calcium influx assay induced by 20 nM wtMCP-1 and MCP-1 mutants
(20nM
each) on THP-1 cells. The changes in fluorescence emission at 495nm due to
calcium
mobilization induced by addition of chemokines are displayed: wtMCP-1 (A), Met-
MCP-
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1 Y13A S21 K (B), Met-MCP-1 Y13A S21 K Q23R (C) and Met-MCP-1 Y13A S21 K Q23R
V47K (D).
Figure 5: Chemotaxis of THP-1 cells induced by wtMCP-1 and MCP-1 mutants at a
concentration of 10nM (error bars represent the SEM of three independent
experiments). 1 wtMCP-1, 2 Met-MCP-1, 3 Met-MCP-1 Y13A S21 K, 4 Met-MCP-1 Y13A
S21 K Q23R, 5 Met-MCP-1 Y13A S21 K Q23R V47K.
Figure 6: Dose-dependent inhibition of monocyte adhesion/efflux by Met-MCP-1
Y13AS21 KQ23R (described by the compound code PA05-008) as measured in a
murine ex vivo carotide injury model.
Figure 7: Improvement of clinical and histological scores in a rat model of
auto-immune
uveitis after treatment with Met-MCP-1 Y13AS21 KQ23R.
Figure 8: Effect of Met-MCP-1 Y13AS21 KQ23R (indicated as PA008) on ischemia
reperfusion injury in a murine myocardial infarct model.
Figure 9: Nucleotide sequences of MCP-1 Y13AS21 KV47K, MCP-1 Y13AS21 KQ23R ,
MCP-1 Y13AS21 KQ23RV47K
All dimensions specified in this disclosure are by way of example only and are
not
intended to be limiting. Further, the proportions shown in the foregoing
figures are not
necessarily to scale.
It has been shown that increased GAG binding affinity can be introduced by
increasing
the relative amount of basic and/or electron donating amino acids in the GAG
binding
region (also described in WO 05/054285, incorporated in total herein by
reference),
leading to a modified protein that acts as competitor with natural GAG binding
proteins.
This was particularly shown for interleukin-8. The specific location of GAG
binding
regions and their modification by selectively introducing at least two basic
and/or
electron donating amino acids was not disclosed for MCP-1 protein.
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Additionally, the amino terminus of MCP-1 was found to be essential for
chemokine
signalling through its GPC receptor CCR2. In order to engineer an MCP-1 -based
CCR2
antagonist, others have engineered MCP-1 in a way to completely knock-out GAG
binding and to leave CCR2 binding intact (W003084993A1). By these means, it
was
intended to block MCP-1 -mediated signalling by blocking the CCR2 receptor on
neutrophils and to prevent attachment on the endothelium via the GAG chains.
It was
therefore not obvious to turn this approach around by blocking the GAG chains
on the
endothelium (by engineering higher GAG binding affinity) and to knock out the
CCR2
binding of MCP-1.
The invention now provides a novel MCP1 mutant protein with increased GAG
binding
affinity and reduced GPCR activity compared to the wild type MCPI protein,
wherein a
region of the MCP-1 protein is modified in a structure conserving way by
insertion of at
least one basic and/or electron donating amino acids or by replacement of at
least two
amino acids preferably within the native GAG binding site or within the
structural vicinity
of a native GAG binding site by at least two basic and/or electron donating
amino acids.
According to a specific embodiment, th modified MCP-1 protein further
comprises a
further modification of at least one amino acid of the first 1 to 10 amino
acids of the N-
terminal region of said MCP-1 protein by addition, deletion and/or replacement
of at
least one amino acid residue.
If the native amino acids replaced by said basic or electron donating amino
acids are
basic amino acids, the substituting amino acids have to be more basic amino
acids or
comprise more or less structural flexibility compared to the native amino acid
residue.
Structural flexibility according to the invention is defined by the degree of
accommodating to an induced fit as a consequence of GAG ligand binding.
According to a specific embodiment of the invention the native amino acids
replaced by
basic and/or electron donating amino acids are non-basic amino acids.
According to the definition as used in the present application MCP-1 mutant
protein can
also include any parts or fragments thereof that still show chemokine activity
like
monocyte or T-cell chemotaxis and Ca-release.
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The term "vicinity" as defined according to the invention comprises amino acid
residues
which are located within the conformational neighbourhood of the GAG binding
site but
not positioned at the GAG binding sites. Conformational neighbourhood can be
defined
as either amino acid residues which are located adjacent to GAG binding amino
acid
residues in the amino acid sequence of a protein or amino acids which are
conformationally adjacent due to three dimensional structure or folding of the
protein.
The term "adjacent" according to the invention is defined as lying within the
cut-off
radius of the respective amino acid residues to be modified of not more than
20nm,
preferably 15nm, preferably 10nm, preferably 5nm.
To be able to perform their biological function, proteins fold into one, or
more, specific
spatial conformations, driven by a number of non-covalent interactions such as
hydrogen bonding, ionic interactions, Van der Waals' forces and hydrophobic
packing.
Three dimensional structure can be determined by known methods like X-ray
crystallography or NMR spectroscopy.
Identification of native GAG binding sites can be determined by mutagenesis
experiments. GAG binding sites of proteins are characterized by basic residues
located
at the surface of the proteins. To test whether these regions define a GAG
binding site,
these basic amino acid residues can be mutagenized and decrease of heparin
binding
affinity can be measured. This can be performed by any affinity measurement
techniques as known in the art.
Rational designed mutagenesis by insertion or substitution of basic or
electron-donating
amino acids can be performed to introduce foreign amino acids in the vicinity
of the
native GAG binding sites which can result in an increased size of the GAG
binding site
and in an increase of GAG binding affinity.
The GAG binding site or the vicinity of said site can also be determined by
using a
method as described in detail in US 6107565 comprising:
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(a) providing a complex comprising the protein and the GAG ligand molecule,
for
example heparan sulfate (HS), heparin, keratin sulfate, chondroitin sulfate,
dermatan
sulfate and hyaluronic acid etc. bound to said protein;
(b) contacting said complex with a cleavage reagent like a protease, e.g.
trypsin,
capable of cleaving the protein, wherein said GAG ligand molecule blocks
protein
cleavage in a region of the protein where the GAG ligand molecule is bound,
and
whereby said protein is cleaved in regions that are not blocked by said bound
GAG
ligand molecule; and
(c) separating and detecting the cleaved peptides, wherein the absence of
cleavage
events in a region of the protein indicates that said GAG ligand molecule is
bound to
that region. Detection can be for example by LC-MS, nanoHPLC-MS/MS or Mass
Spectrometric Methods.
A protocol for introducing or improving a GAG binding site is, for example,
partially
described in WO 05/054285 and can be as follows:
- Identify a region of the protein which is involved in GAG binding
- Design a new GAG binding site by introducing (replacement or insertion) at
least one
basic or electron donating amino acids, preferably Arg, Lys, His, Asp and Gin
residues
at any position or by deleting at least two amino acids which interfere with
GAG binding
- Check the conformational stability of the resulting mutant protein in silica
- Provide the wild type protein cDNA (alternatively: purchase the cDNA)
- Use this as template for PCR-assisted mutagenesis to introduce the above
mentioned
changes into the amino acid sequence
- Subclone the mutant gene into a suitable expression system (prokaryotic or
eukaryotic
dependent upon biologically required post-translational modifications)
- Expression, purification and characterization of the mutant protein in vitro
Criterion for an increased GAG binding affinity: KdGAG(mutant) < 10uM.
- Check for structural conservation by far-UV CD spectroscopy or 1-D NMR
spectroscopy.
A deviation of the modified structure as measured by far-UV CD spectroscopy
from wild
type MCP-1 structure of less than 30%, preferably less than 20%, preferably
less than
10% is defined as structure conserving modification according to the
invention.
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According to an alternative embodiment, the structure conserving modification
is not
located within the N-terminus of the MCP1 protein.
The key residues relating to the GAG binding domain of wtMCP-1 are S21, Q23
and/or
V47. According to the invention, the MCP-1 mutant protein may contain at least
two
amino acid modifications within at least two amino acid residues at positions
21, 23
and/or 47.
The modifications can be, for example, a substitution of, or replacement by,
at least two
basic or electron donating amino acids. Electron donating amino acids are
those amino
acids which donate electrons or hydrogen atoms (Droenstedt definition).
Specifically,
these amino acids can be N or Q. Basic amino acids can be selected from the
group
consisting of R, K and H.
According to a further embodiment of the invention, R at amino acid position
18 can by
modified by K, and/or K19 position can be modified by R and/or P8 can be
modified by
any amino acid substitution to at least partially decrease receptor binding of
the
modified MCP-1.
Alternatively, the MCP-1 mutant protein of the invention is characterized in
that Y at
position 13 is further substituted by any amino acid residue, preferably by
A..
Y13 and R18 were shown to be also critical residues for signalling, and the
replacement
of these residues by other amino acid residues gave rise to a protein unable
to induce
chemotaxis. Two-dimensional 1 H-15N HSQC spectra recorded on both deletion and
substitution MCP-1 variants revealed that these mutations do not generate
misfolded
proteins (Chad D. Paavola et al., J. Biol. Chem., 273 (50), 33157-33165
(1998)).
Furthermore, the N-terminal methionine reduces the binding affinity of MCP-1
for CCR2
on THP-1 cells (Hemmerich S. et al, Biochemistry 38 (40), 13013-13025 (1999))
so that
the chemotactic potency of [Met]-MCP-1 is approximately 300-fold lower than of
the wild
type (Jarnagin K. et al., Biochemistry 38, 16167-16177 (1999)). This is in
contrast to the
potent receptor antagonist [Met]-RANTES which does not induce chemotaxis but
binds
with high affinity to the receptor.
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Therefore, according to an altemative embodiment of the invention, the MCP-1
mutant
protein may contain an N-terminal Met. MCP-1 variants retaining the N-terminal
methionine appear to have an increased apparent affinity for heparin (Lau E.K.
et al., J.
Biol. Chem. 279 (21), 22294-22305 (2004)).
According to the present invention, the N-terminal region of the wild type MCP-
1 region
that can be modified comprises the first 1 to 10 N-terminal amino acids. The
inventive
MCP-1 mutant protein can also have the N-terminal amino acid residues 2-8
deleted.
Truncation of residues 2-8 ([1+9-76]hMCP-1) produces a protein that cannot
induce
chemotaxis.
Specifically, MCP-1 mutant protein can be selected from the group of Met-MCP-1
Y13A
S21 K V47K, Met-MCP-1 Y13A S21 K Q23R and Met-MCP-1 Y13A S21 K Q23R V47K.
In order to knock out GPCR activity and at the same time to improve affinity
for GAGs,
minimizing the number of modifications as far as possible, site-directed MCP-1
mutants
were designed using bioinformatical and biostructural tools. This means, since
the
structure of wtMCP-1 is known, mutants were rationally designed. This means
for
knocking-in higher GAG binding affinity, that more GAG binding sites are
introduced into
the already existing GAG binding domain by replacing amino acids which are not
directly involved in GAG binding, which are structurally less important, and
which are
solvent exposed by vicinity to basic amino acids such as K or R. By doing so,
special
attention was drawn to conserving the specific GAG, interaction sites of MCP-
1, i.e.
those amino acids responsible for hydrogen bonding and van der Waals contacts
with
the GAG ligand, as well as the overall fold of the chemokine to preserve the
ability of
the chemokine to penetrate chemokine networks which relies on protein-protein
interactions contained in-the -surface-of -MCI?-1-.
The amino acid sequence of the modified MCP-1 molecule can be described by the
general formula:
(M),Q(PDAINA(Z1))mVTCC(X1)NFTN (Z2)(Z3)I(X2)V(X3)RLASYRRITSSKCP
KEAVIFKTI(X4) AKEICADPKQ KWVQDSMDHL DKQTQTPKT
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wherein Z1 is selected from the group of P and A, G, L, preferably it is A,
wherein Z2 is selected from the group of R and K,
wherein Z3 is selected from the group of K and R,
wherein Xl is selected from the group consisting of Y and A, preferably it is
A,
wherein X2 is selected from the group consisting of S, R, K, H, N and Q,
preferably it is
K,
wherein X3 is selected from the group consisting of R, K, H, N and Q,
preferably it is R,
wherein X4 is selected from the group consisting of V, R, K, H, N and Q,
preferably it is
K,
and wherein n and/or m can be either 0 or 1 and wherein at least two of
positions X2,
X3 or X4 are modified.
A further aspect of the present invention is an isolated polynucleic acid
molecule which
codes for the inventive protein as described above.
Specifically, an isolated polynucleic acid molecule comprising a nucleotide
sequence of
SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9 or at least part thereof is
covered, too.
The polynucleic-acid may be DNA or RNA. Thereby the modifications which lead
to the
inventive MCP-1 mutant protein are carried out on DNA or RNA level. This
inventive
isolated polynucleic acid molecule is suitable for diagnostic methods as well
as gene
therapy and the production of inventive MCP-1 mutant protein on a large scale.
Alternatively, the isolated polynucleic acid molecule hybridizes to the above
defined
inventive polynucleic acid molecule under stringent conditions. Depending on
the
hybridisation conditions, complementary duplexes form between the two DNA or
RNA
r-nolecules, either by perfectly matching or-also-by-compr-ising-mismatched
bases-(see
Sambrook et al., Molecular Cloning: A laboratory manual, 2"d ed., Cold Spring
Harbor,
N.Y. 1989). Probes greater in length than about 50 nucleotides may accomplish
up to
25 to 30% mismatched bases. Smaller probes will accomplish fewer mismatches.
The
tendency of a target and probe to form duplexes containing mismatched base
pairs is
controlled by the stringency of the hybridization conditions which itself is a
function of
factors, such as the concentrations of salt or formamide in the hybridization
buffer, the
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temperature of the hybridization and the post-hybridization wash conditions.
By applying
well known principles that occur in the formation of hybrid duplexes,
conditions having
the desired stringency can be achieved by one skilled in the art by selecting
from
among a variety of hybridization buffers, temperatures and wash conditions.
Thus,
conditions can be selected that permit the detection of either perfectly
matching or
partially matching hybrid duplexes. The melting temperature (Tm) of a duplex
is useful
for selecting appropriate hybridisation conditions. Stringent hybridization
conditions for
polynucleotide molecules over 200 nucleotides in length typically involve
hybridizing at a
temperature 15-25 C below the melting temperature of the expected duplex. For
olignucleotide probes over 30 nucleotides which form less stable duplexes than
longer
probes, stringent hybridization usually is achieved by hybridizing at 5 to 10
C below the
Tm. The Tm of a nucleic acid duplex can be calculated using a formula based on
the
percent G+C contained in the nucleic acids and that takes chain lengths into
account,
such as the formula
Tm = 81.5-16.6(log[NA+])+ 0.41 (% G+C) - (600/N), where N = chain length.
A further aspect relates to a vector comprising an isolated DNA molecule
according to
the present invention, as defined above. The vector comprises all regulatory
elements
necessary for efficient transfection as well as efficient expression of
proteins. Such
vectors are well known in the art and any suitable vector can be selected for
this
purpose.
A further aspect of the present invention relates to a recombinant cell which
is
transfected with an inventive vector as described above. Transfection of cells
and
cultivation of recombinant cells can be performed as well known in the art.
Such a
recombinant cell as well as any descendant cell therefrom comprises the
vector.
Thereby, a cell line is provided which-expresses-the-MCP=1-mutantprotein-
either-
continuously or upon activation.depending on the vector.
A further aspect of the invention relates to a pharmaceutical composition
comprising a
MCP-1 mutant protein, a polynucleic acid or a vector according to the present
invention,
as defined above, and a pharmaceutically acceptable carrier. Of course, the
pharmaceutical composition may further comprise additional substances which
are
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usually present in pharmaceutical compositions, such as salts, buffers,
emulgators,
coloring agents, etc.
A further aspect of the present invention relates to the use of the MCP-1
protein, a
polynucleic acid or a vector according to the present invention, as defined
above, in a
method for either in vivo or in vitro inhibiting or suppressing the biological
activity of the
respective wild type protein. As mentioned above, the MCP-1 mutant protein of
the
invention will act as an antagonist whereby the side effects which occur with
known
recombinant proteins will not occur with the inventive MCP-1 mutant protein.
In this
case this will particularly be the biological activity involved in
inflammatory reactions.
Therefore, a further use of the MCP-1 protein, a polynucleic acid or a vector
according
to the present invention, as defined above, is in a method for producing a
medicament
for the treatment of an inflammatory condition. In particular, it will act as
antagonist
without side effects and will be particularly suitable for the treatment of
inflammatory
diseases or conditions, either of chronic or acute nature. Therefore, a
further aspect of
the present invention is also a method for the treatment of inflammatory
diseases or
allergic conditions, wherein the MCP-1 mutant protein according to the
invention, the
isolated polynucleic acid molecule or vector according to the present
invention or a
pharmaceutical preparation according to the invention is administered to a
patient.
More specifically, the inflammatory diseases or allergic conditions are
respiratory
allergic diseases such as asthma, allergic rhinitis, COPD, hypersensitivity
lung
diseases, hypersensitivity pneumonitis, interstitial lung disease, (e.g.
idiopathic
pulmonary fibrosis, or associated with autoimmune diseases), anaphylaxis or
hypersensitivity responses, drug allergies and insect sting allergies;
inflammatory bowel
diseases, such as Crohn's disease and ulcerative colitis;
spondyloarthropathies,
--scleroder-ma; psoriasis-and-inflammatory-dermatoses-such-as-dermatitis,-
eczema,-
atopic dermatitis, allergic contact dermatitis, uticaria; vasculitis;
autoimmune diseases
with an aetiology including an inflammatory component such as arthritis (for
example
rheumatoid arthritis, arthritis chronica progrediente, psoriatic arthritis and
arthritis
deformans) and rheumatic diseases, including inflammatory conditions and
rheumatic
diseases involving bone loss, inflammatory pain, hypersensitivity (including
both airways
hypersensitivity and dermal hypersensitivity) and allergies. Specific auto-
immune
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14
diseases include autoirmmune hematological disorders (including e.g. hemolytic
anaemia, aplastic anaemia, pure red cell anaemia and idiopathic
thrombocytopenia),
systemic lupus erythromatosus, polychondritis, Wegener granulomatosis,
dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis,
Steven-Johnson
syndrome, autoimmune inflammatory bowel disease (including e.g. ulcerative
colitis,
Crohn's disease and Irritable Bowel Syndrome), autoimmune thyroiditis,
Behcet's
disease, endocrine ophthalmopathy, Graves disease, sarcoidosis, multiple
sclerosis,
primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I),
uveitis (anterior and
posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis,
interstitial lung
fibrosis, and glomerulonephritis (with and without nephrotic syndrome, e. g.
including
idiopathic nephrotic syndrome or minimal change nephropathy); graft rejection
(e.g. in
transplantation including heart, lung, combined heart- lung, liver, kidney,
pancreatic,
skin, or corneal transplants) including allograft rejection or xenograft
rejection or graft-
versus-host disease, and organ transplant associated arteriosclerosis;
atherosclerosis;
cancer with leukocyte infiltration of the skin or organs; stenosis or
restenosis of the
vasculature, particularly of the arteries, e.g. the coronary artery, including
stenosis or
restenosis which results from vascular intervention, as well as neointimal
hyperplasia;
and other diseases or conditions involving inflammatory responses including
ischemia
reperfusion injury, hematologic malignancies, cytokine induced toxicity (e.g.
septic
shock or endotoxic shock), polymyositis, dermatomyositis, and granulomatous
diseases
including sarcoidosis.
Preferably, the inflammatory disease is selected form the group comprising
rheumatoid
arthritis, uveitis, inflammatory bowel disease, myocardial infarction,
congested heart
failure or ischemia reperfusion injury.
The following examples describe the invention in more detail without limiting
the scope
_af_the-invention.-
EXAMPLES
The carotide injury model as well as the animal models used for the present
invention
were performed in the laboratories of Prof. Christian Weber
(Universitatsklinikum
Aachen).
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Structural analysis of MCP-1 mutants upon GAG binding
Analysis of secondary structural elements of MCP-1 mutants by far-UV CD
spectroscopy showed that the overall ratio of alpha/beta/turns was conserved
during
protein design. Furthermore, protein unfolding studies showed that
particularly Met-
MCP-1 Y13AS21 KQ23R exhibited very similar unfolding transition parameters
compared to the wild-type protein, indicating similar stability of these
protein variants.
Also the small secondary structural change induced by HS binding found for
wtMCP-1
was reproduced in the MCP-1 mutants (as exemplified by the comparison of wtMCP-
1
and Met-MCP-1 Y13AS21 KQ23R in Figure 1). However, the stability of both
proteins
was significantly improved in the presence of HS as determined by temperature-
induced
unfolding studies. This means that contrary to other chemokines, HS impacts
the fold of
MCP-1 variants stronger than their secondary structure. This may be partly due
to the
elongated, partially unstructured form of MCP-1 in the absence of GAGs which
experiences a structure-induction upon GAG binding, leading to a more compact
fold
and, thus, to greater stability.
Increase in GAG binding affinity
We have determined the increased GAG binding affinity by surface plasmon
resonance
(SPR) using a Biacore 3000 system. The immobilization of biotinylated HS onto
a
streptavidin coated CM4 sensor chip was performed according to an established
protocol (28). The actual binding interactions were recorded at 25 C in PBS pH
7.4
containing 0.01 % (v/v) P20 surfactant (BlAcore AB). 2.5 min injections of
different
protein concentrations at a flow rate of 60pl/min were followed by 5 min
dissociation
periods in buffer and a pulse of 1 M NaCI for complete regeneration. The
maximum
response signals of protein binding to the HS surface, corresponding to the
plateaus of
the respective sensograms, were used for Scatchard plot analysis and the
calculation of
equilibrium dissociation constants (Kd values). In Figure 3 the Scatchard
plots of
wtM-CP-1-and-two-mutants-ar-e-displayed.. wtMCP=1-gave-a-Kd value-of 1;26-pM,-
Me# -
MCP-1 Y13A S21 K yielded 676 nM, and Met-MCP-1 Y13A S21 K Q23R gave 152 nM.
This means that in the latter mutant the affinity for HS has been improved by
a factor of
>8. The Met-MCP-1 Y13AS21 KV47K mutant did not exhibit any improvement in
affinity
for the natural HS ligand.
Knock-out of GPCR activity
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In order to obtain dominant-negative MCP-1 mutants, the GPCR activity of MCP-1
has
been knocked out in addition to the improved GAG binding affinity. This was
done by
replacing the tyrosine residue at position 13 by an alanine residue and by
keeping the
N-terminal methionine residue. This led to a complete knock-out of MCP-1-
related
CCR2 activity, as exemplified by the complete absence of Ca influx and Thp-1
chemotaxis in the case of the Met-MCP-1 Y13A S21 K Q23R mutant (Figs. 4 & 5).
The
inability of this mutant to activate its high-affinity GPC receptor on target
monocyte cells
is expected to lead, in combination with the increased GAG binding affinity,
to a potent
inhibitor of MCP-1 activity in vivo.
Inhibition of cell migration
The effect of Met-MCP-1 Y13A S21 K Q23R on monocyte migration was investigated
in
an ex vivo model. For this purpose, apolipoprotein E-deficient (Apoe)-/- mice
were
subjected to wire-induced endothelial denudation injury after 1 week of
atherogenic diet
(1). After 24 hours carotid arteries were isolated for ex vivo perfusion as
described (1).
Carotid arteries were preperfused at 5 NI/min with Met-MCP-1 Y13A S21 K Q23R
at a
concentration of 1, 5 or 10 Ng/mI for 30 min. Mono Mac 6 cells (11 06/ml) were
labeled
with calcein-AM, washed twice and perfused through the carotid artery.
Adhesive
interactions with the injured vessel wall were recorded using stroboscopic
epifluorescence illumination (Drelloscop 250, Drello) and an Olympus BX51
microscope
after 10 min of perfusion. By this means, a concentration dependent inhibition
of
monocyte adhesion by Met-MCP-1 Y13AS21 KQ23R was observed (see Figure 6).
Inhibition/improvement of auto-immune uveitis
Lewis rats were immunized into both hind legs with a total volume of 200 NI
emulsion
containing 15 pg PDSAg (retinal peptide) in complete Freund's adjuvant,
fortified with
Mycobacterium tuberculosis strain H37RA (BD, Heidelberg, Germany) to a final
-concentr-ation-of-2.5-mg/mh-1-00-Ng-Met-MCP--1 Y1-3AS21-KQ23R-mutant--
dissolved-in- - -
0.5 ml PBS (or PBS only as control) was applied i. p. daily from day 1 after
active
immunization until day 19. The time course of disease was determined by daily
examination of animals with an ophthalmoscope. Uveitis was graded clinically
as
described (Gong J.H. and Clark-Lewis I., J. Exp.Med. 181 (2), 631-640 (1995)))
and the
average clinical score of all eyes is shown per group and day. As can be seen
from
Figure 7, the Met-MCP-1 Y13AS21 KQ23R mutant had a significant impact on the
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17
progression of the disease. Since uveitis is characterized by occular
accumulation of T-
cells and monocytes which finally lead to blindness, the therapeutic effect of
Met-MCP-1
Y13AS21 KQ23R can be assigned to its inhibition of the migration of CCR2-
activated
leukocytes which mainly constitute monocytes and basophils.
Inhibition/improvement of myocardial infarction
C57/B6 mice were intubated under general anaesthesia (100mg/kg ketamine and
10mg/kg xylasine, intraperitoneal) and positive pressure ventilation was
maintained with
oxygen and isofluran 0.2% using a rodent respirator. Hearts were exposed
through a
left toracotomy and MI was produced by suture occlusion of the left anterior
descending
artery (LAD) over a two mm silicon tube. The suture was opened after 30 min by
cutting
the silicon tube and reperfusion was re-established. In sham-operated mice,
the suture
was left open during the same time. The muscle layer and skin incision were
closed with
a silk suture. Animal experiments were approved by local authorities and
complied with
German animal protection law.
Met-MCP-1 Y13AS21 KQ23R was dissolved in PBS at 100 pg/mI. Mice were treated
intraperitoneally with 100 pl each during ischemia (10 min after ligation), 2
hours after
reperfusion, and every day until the end point. Control mice were treated in
the same
way with vehicle.
At indicated time points, mice were anesthetized and the heart function was
analyzed
using a Langendorff apparatus (Hugo Sachs Elektronik-Harvard Apparatus) and
HSE
Isoheart software under constant perfusion pressure (100 mmHg) and electrical
stimulation to assure a constant heart rate (600 bpm). The coronary flow,
developed
pressure, the increase (dP/dtmax) and decrease (dP/dtmin) in left ventricular
pressure
were measured without or with dobutamin (300 pmol in bolus). The measured
-----par-ameter-s-ar-e-displayed-in-F-igur-e-8-(upper-panel).-At the-end,-the-
heart-s-wer-e-fixed-in
distension with 10% formalin and cut into 5 pm serial slices.
Serial sections (10-12 per mouse, 400 pm apart, until mitral valve) were
stained with
Gomori's 1 step trichrome stain. The infarction area was determined on every
section .
using Diskus software (Hilgers) and express as percent from total left
ventricular volume
(see Figure 8, lower panel).
