U.S. patent application number 13/717561 was filed with the patent office on 2013-06-13 for glycosaminoglycan-antagonising mcp-1 mutants and methods of using same.
This patent application is currently assigned to PROTAFFIN BIOTECHNOLOGIE AG. The applicant listed for this patent is Protaffin Biotechnologie AG. Invention is credited to Andreas Kungl, Anna Maria Piccinini, Christian Weber.
Application Number | 20130150303 13/717561 |
Document ID | / |
Family ID | 38720368 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130150303 |
Kind Code |
A1 |
Kungl; Andreas ; et
al. |
June 13, 2013 |
GLYCOSAMINOGLYCAN-ANTAGONISING MCP-1 MUTANTS AND METHODS OF USING
SAME
Abstract
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 their use for therapeutic treatment of inflammatory
diseases.
Inventors: |
Kungl; Andreas; (Graz,
AT) ; Piccinini; Anna Maria; (London, GB) ;
Weber; Christian; (Aachen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Protaffin Biotechnologie AG; |
Graz |
|
AT |
|
|
Assignee: |
PROTAFFIN BIOTECHNOLOGIE AG
Graz
AT
|
Family ID: |
38720368 |
Appl. No.: |
13/717561 |
Filed: |
December 17, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12670378 |
Jan 22, 2010 |
8337825 |
|
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PCT/EP2008/006298 |
Jul 31, 2008 |
|
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13717561 |
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60953140 |
Jul 31, 2007 |
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Current U.S.
Class: |
514/15.1 ;
435/29; 435/320.1; 514/16.4; 514/16.6; 514/20.8; 514/21.3; 530/300;
530/324; 536/23.5 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
31/14 20180101; C07K 14/523 20130101; A61P 9/04 20180101; A61K
38/00 20130101; A61P 1/04 20180101; A61P 29/00 20180101; G01N
33/5047 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/15.1 ;
530/300; 530/324; 536/23.5; 435/320.1; 514/21.3; 435/29; 514/16.6;
514/20.8; 514/16.4 |
International
Class: |
C07K 14/52 20060101
C07K014/52; G01N 33/50 20060101 G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 27, 2007 |
EP |
07450166.9 |
Claims
1. An MCP1 mutant protein with increased GAG binding affinity and
reduced GPCR activity compared to wild type MCP-1 protein, wherein
the MCP-1 protein is modified in a structure-conserving way by
insertion of at least one basic and/or electron donating amino acid
or replacement of at least two amino acids by at least two basic
and/or electron donating amino acids.
2. The MCP-1 mutant protein of claim 1, wherein at least one amino
acid of the first 10 amino acids of the N-terminal region of the
wild type MCP-1 protein is modified by addition, deletion and/or
replacement of at least one amino acid.
3. The MCP-1 mutant protein of claim 1, wherein the amino acids
that are replaced by the at least two basic and/or electron
donating amino acids are non-basic amino acids.
4. The MCP-1 mutant protein of claim 1, wherein the modification in
a structure-conserving way is a deviation of the modified structure
from wild type MCP1 structure of less than 30%, and preferably less
than 20% as measured by far-UV CD spectroscopy.
5. The MCP-1 mutant protein of claim 1, wherein the basic amino
acids are selected from the group consisting of R, K, and H.
6. The MCP-1 mutant protein of claim 1, wherein the electron
donating amino acids are selected from the group consisting of N or
Q.
7. The MCP-1 mutant protein of claim 1, wherein at least two amino
acids at positions 21, 23 and/or 47 are modified.
8. The MCP-1 mutant protein of claim 1, wherein the Y at position
13 is substituted by an A.
9. The MCP-1 mutant protein of claim 1, containing an N-terminal
Met.
10. The MCP-1 mutant protein of claim 1, wherein the N-terminal
amino acid residues 2-8 are deleted.
11. An MCP-1 mutant protein, it wherein the mutant protein
comprises the amino acid sequence of the general formula:
TABLE-US-00002 (M)nQ(PDAINA(Z1))mVTCC(X1)NFTN
(Z2)(Z3)I(X2)V(X3)RLASYRRITSSKCP KEAVIFKTI(X4) AKEICADPKQ
KWVQDSMDHL DKQTQTPKT
wherein Z1 is selected from the group consisting of P, A, G, L,
wherein Z2 is selected from the group consisting of R and K,
wherein Z3 is selected from the group consisting of K and R,
wherein X1 is selected from the group consisting of Y and A,
wherein X2 is selected from the group consisting of S, R, K, H, N
and Q, wherein X3 is selected from the group consisting of R, K, H,
N and Q, wherein X4 is selected from the group consisting of V, R,
K, H, N and Q, and wherein n and/or m can be either 0 or 1, and
wherein at least two of the positions X2, X3 or X4 are
modified.
12. The MCP-1 mutant protein of claim 1, wherein the protein has it
is an amino acid sequence selected from the group consisting of SEQ
ID NO:5, and SEQ ID NO:6.
13. An isolated polynucleic acid molecule which encodes the MCP-1
mutant protein of claim 1.
14. The isolated polynucleic acid molecule of claim 13, wherein the
nucleotide sequence is selected from the group consisting of SEQ ID
No. 7, SEQ ID No. 8 or SEQ ID No. 9 and at least a part
thereof.
15. The isolated polynucleic acid molecule of claim 13, wherein the
polynucleic acid molecule is in a vector.
16. The isolated polynucleic acid of claim 15, wherein the vector
is transfected into a recombinant cell.
17. A pharmaceutical composition which comprises a protein
according to claim 1 and a pharmaceutically acceptable carrier.
18. A method for inhibiting or suppressing the biological activity
of the wild type MCP-1 protein in vitro comprising using the MCP-1
mutant protein of claim 1 in a diagnostic assay.
19. A method for treating chronic or acute inflammatory disease or
autoimmune conditions comprising administering the MCP-1 mutant
protein of claim 1 to a patient in need thereof.
20. The method of claim 19, wherein the inflammatory disease is
selected from the group consisting of rheumatoid arthritis,
uveitis, inflammatory bowel disease, myocardial infarction,
congested heart failure and ischemia reperfusion injury.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of, and claims the
benefit of priority under 35 U.S.C. .sctn.120 from, U.S. patent
application Ser. No. 12/670,378, filed on Jan. 22, 2010 and
entitled GLYCOSAMINOGLYCAN-ANTAGONISING MCP-1 MUTANTS AND METHODS
OF USING SAME, which is the U.S. national stage of International
Patent Application No. PCT/EP2008/006298, filed on Jul. 31, 2008,
which claims the benefit of priority under 35 U.S.C. .sctn.120 from
U.S. Patent Application No. 60/953,140, filed on Jul. 31, 2007 and
entitled GLYCOSAMINOGLYCAN-ANTAGONISING MCP-1 MUTANTS AND METHODS
OF USING SAME. This application also claims the benefit of priority
under 35 U.S.C. .sctn.119 from European Patent Application No.
07450166.9, filed on Sep. 27, 2007 and entitled
GLYCOSAMINOGLYCAN-ANTAGONISING MCP-1 MUTANTS AND METHODS OF USING
SAME. The disclosures of the foregoing applications are
incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0002] The entire content of a Sequence Listing titled
"Sequence_Listing.txt," created on Dec. 17, 2012 and having a size
of 8 kilobytes, which has been submitted in electronic form in
connection with the present application, is hereby incorporated by
reference herein in its entirety.
FIELD OF THE INVENTION
[0003] 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.
[0004] 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 .alpha.-chemokine and
the CC or .beta.-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 .alpha.-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)).
[0005] In the context of the chemokine-.beta. family of cytokines,
monocyte chemoattractant protein-1 (MCP-1) is a monocyte and
lymphocyte-specific chemoattractant and activator found in a
variety of diseases that feature a monocyte-rich inflammatory
component, such as atherosclerosis (Nelken 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. Natl. 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. Invest. 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)).
[0006] 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.
[0007] 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.
[0008] For this purpose several recombinant MCP-1 variants that
compete with their wild type counterpart for glycosaminoglycan
binding and show reduced or knocked out activation of leukocytes
have been generated.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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
[0016] FIG. 1: Sequence of MCP-1 mutants, mutations with respect to
the wild type chemokine are underlined
[0017] FIG. 2: Structural change of wtMCP-1 (FIG. 2a) and Met-MCP-1
Y13A S21K Q23R (FIG. 2b) upon heparan sulfate binding, as shown by
far-UV CD spectroscopy
[0018] FIG. 3: Scatchard plot analysis and equilibrium dissociation
constants (Kd values) of WT MCP-1 (solid squares), Met-MCP-1 Y13A
S21K (solid triangles) and Met-MCP-1 Y13A S21K Q23R (open circles)
binding to unfractionated HS
[0019] FIG. 4: Calcium influx assay induced by 20 nM wtMCP-1 and
MCP-1 mutants (20 nM each) on THP-1 cells. The changes in
fluorescence emission at 495 nm due to calcium mobilization induced
by addition of chemokines are displayed: wtMCP-1 (A), Met-MCP-1
Y13A S21K (B), Met-MCP-1 Y13A S21K Q23R (C) and Met-MCP-1 Y13A S21K
Q23R V47K (D).
[0020] FIG. 5: Chemotaxis of THP-1 cells induced by wtMCP-1 and
MCP-1 mutants at a concentration of 10 nM (error bars represent the
SEM of three independent experiments). 1 wtMCP-1, 2 Met-MCP-1, 3
Met-MCP-1 Y13A S21K, 4 Met-MCP-1 Y13A S21K Q23R, 5 Met-MCP-1 Y13A
S21K Q23R V47K.
[0021] FIG. 6: Dose-dependent inhibition of monocyte
adhesion/efflux by Met-MCP-1 Y13AS21KQ23R (described by the
compound code PA05-008) as measured in a murine ex vivo carotide
injury model.
[0022] FIG. 7: Improvement of clinical and histological scores in a
rat model of auto-immune uveitis after treatment with Met-MCP-1
Y13AS21KQ23R.
[0023] FIG. 8: Effect of Met-MCP-1 Y13AS21KQ23R (indicated as
PA008) on ischemia reperfusion injury in a murine myocardial
infarct model.
[0024] FIG. 9: Nucleotide sequences of MCP-1 Y13AS21KV47K, MCP-1
Y13AS21KQ23R, MCP-1 Y13AS21KQ23RV47K
[0025] 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.
[0026] 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.
[0027] Additionally, the amino terminus of MCP-1 was found to be
essential for chemokine signaling 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 (WO03084993A1). By these means, it was
intended to block MCP-1-mediated signaling 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.
[0028] The invention now provides a novel MCP1 mutant protein with
increased GAG binding affinity and reduced GPCR activity compared
to the wild type MCP1 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.
[0029] According to a specific embodiment, the 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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 20 nm, preferably 15 nm,
preferably 10 nm, preferably 5 nm.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] The GAG binding site or the vicinity of said site can also
be determined by using a method as described in detail in U.S. Pat.
No. 6,107,565 comprising:
[0039] (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;
[0040] (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
[0041] (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.
[0042] A protocol for introducing or improving a GAG binding site
is, for example, partially described in WO 05/054285 and can be as
follows: [0043] Identify a region of the protein which is involved
in GAG binding [0044] 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 Gln residues at any
position or by deleting at least two amino acids which interfere
with GAG binding [0045] Check the conformational stability of the
resulting mutant protein in silica [0046] Provide the wild type
protein cDNA (alternatively: purchase the cDNA) [0047] Use this as
template for PCR-assisted mutagenesis to introduce the above
mentioned changes into the amino acid sequence [0048] Subclone the
mutant gene into a suitable expression system (prokaryotic or
eukaryotic dependent upon biologically required post-translational
modifications) [0049] Expression, purification and characterization
of the mutant protein in vitro Criterion for an increased GAG
binding affinity: K.sub.d.sup.GAG(mutant).ltoreq.10 uM. [0050]
Check for structural conservation by far-UV CD spectroscopy or 1-D
NMR spectroscopy.
[0051] 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.
[0052] According to an alternative embodiment, the structure
conserving modification is not located within the N-terminus of the
MCP1 protein.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] Y13 and R18 were shown to be also critical residues for
signaling, and the replacement of these residues by other amino
acid residues gave rise to a protein unable to induce chemotaxis.
Two-dimensional 1H-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)).
[0058] 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.
[0059] Therefore, according to an alternative 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)).
[0060] 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.
[0061] Specifically, MCP-1 mutant protein can be selected from the
group of Met-MCP-1 Y13A S21K V47K, Met-MCP-1 Y13A S21K Q23R and
Met-MCP-1 Y13A S21K Q23R V47K.
[0062] 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 MCP-1.
[0063] The amino acid sequence of the modified MCP-1 molecule can
be described by the general formula:
TABLE-US-00001 (M).sub.nQ(PDAINA(Z1)).sub.mVTCC(X1)NFTN
(Z2)(Z3)I(X2)V(X3)RLASYRRITSSKCP KEAVIFKTI(X4) AKEICADPKQ
KWVQDSMDHL DKQTQTPKT
[0064] wherein Z1 is selected from the group of P and A, G, L,
preferably it is A,
[0065] wherein Z2 is selected from the group of R and K,
[0066] wherein Z3 is selected from the group of K and R,
[0067] wherein X1 is selected from the group consisting of Y and A,
preferably it is A,
[0068] wherein X2 is selected from the group consisting of S, R, K,
H, N and Q, preferably it is K,
[0069] wherein X3 is selected from the group consisting of R, K, H,
N and Q, preferably it is R,
[0070] wherein X4 is selected from the group consisting of V, R, K,
H, N and Q, preferably it is K,
[0071] 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.
[0072] A further aspect of the present invention is an isolated
polynucleic acid molecule which codes for the inventive protein as
described above.
[0073] 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.
[0074] 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.
[0075] 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
molecules, either by perfectly matching or also by comprising
mismatched bases (see Sambrook et al., Molecular Cloning: A
laboratory manual, 2.sup.nd 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 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.degree.
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.degree. 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.sup.+])+0.41 (% G+C)-(600/N), where N=chain
length.
[0076] 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.
[0077] 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 mutant protein either continuously or upon activation
depending on the vector.
[0078] 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 usually present in pharmaceutical
compositions, such as salts, buffers, emulgators, coloring agents,
etc.
[0079] 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.
[0080] 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.
[0081] 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, scleroderma; 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 diseases include autoimmune
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.
[0082] 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.
[0083] The following examples describe the invention in more detail
without limiting the scope of the invention.
EXAMPLES
[0084] 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).
[0085] Structural Analysis of MCP-1 Mutants Upon GAG Binding
[0086] 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
Y13AS21KQ23R 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
Y13AS21KQ23R in FIG. 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.
[0087] Increase in GAG Binding Affinity
[0088] 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.degree.
C. in PBS pH 7.4 containing 0.01% (v/v) P20 surfactant (BIAcore
AB). 2.5 min injections of different protein concentrations at a
flow rate of 60 .mu.l/min were followed by 5 min dissociation
periods in buffer and a pulse of 1M NaCl 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 FIG. 3 the Scatchard plots
of wtMCP-1 and two mutants are displayed. wtMCP-1 gave a Kd value
of 1.26 .mu.M, Met-MCP-1 Y13A S21K yielded 676 nM, and Met-MCP-1
Y13A S21K 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 Y13AS21KV47K mutant did not exhibit any improvement in
affinity for the natural HS ligand.
[0089] Knock-Out of GPCR Activity
[0090] 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
S21K 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.
[0091] Inhibition of Cell Migration
[0092] The effect of Met-MCP-1 Y13A S21K 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 .mu.l/min with Met-MCP-1 Y13A S21K Q23R at a
concentration of 1, 5 or 10 .mu.g/ml for 30 min. Mono Mac 6 cells
(1'10.sup.6/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 Y13AS21KQ23R was observed (see FIG. 6).
[0093] Inhibition/Improvement of Auto-Immune Uveitis
[0094] Lewis rats were immunized into both hind legs with a total
volume of 200 .mu.l emulsion containing 15 .mu.g PDSAg (retinal
peptide) in complete Freund's adjuvant, fortified with
Mycobacterium tuberculosis strain H37RA (BD, Heidelberg, Germany)
to a final concentration of 2.5 mg/ml. 100 .mu.g Met-MCP-1
Y13AS21KQ23R 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 FIG. 7, the Met-MCP-1 Y13AS21KQ23R mutant had a
significant impact on the 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 Y13AS21KQ23R can be assigned to its inhibition of the
migration of CCR2-activated leukocytes which mainly constitute
monocytes and basophils.
[0095] Inhibition/Improvement of Myocardial Infarction
[0096] C57/B6 mice were intubated under general anaesthesia (100
mg/kg ketamine and 10 mg/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.
[0097] Met-MCP-1 Y13AS21KQ23R was dissolved in PBS at 100 .mu.g/ml.
Mice were treated intraperitoneally with 100 .mu.l 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.
[0098] 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 .mu.mol in bolus). The measured parameters are
displayed in FIG. 8 (upper panel). At the end, the hearts were
fixed in distension with 10% formalin and cut into 5 .mu.m serial
slices.
[0099] Serial sections (10-12 per mouse, 400 .mu.m 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 FIG. 8, lower panel).
Sequence CWU 1
1
10176PRTHomo sapiens 1Gln Pro Asp Ala Ile Asn Ala Pro Val Thr Cys
Cys Tyr Asn Phe Thr 1 5 10 15 Asn Arg Lys Ile Ser Val Gln Arg Leu
Ala Ser Tyr Arg Arg Ile Thr 20 25 30 Ser Ser Lys Cys Pro Lys Glu
Ala Val Ile Phe Lys Thr Ile Val Ala 35 40 45 Lys Glu Ile Cys Ala
Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met 50 55 60 Asp His Leu
Asp Lys Gln Thr Gln Thr Pro Lys Thr 65 70 75
277PRTArtificialmodified MCP-1 protein 2Met Gln Pro Asp Ala Ile Asn
Ala Pro Val Thr Cys Cys Tyr Asn Phe 1 5 10 15 Thr Asn Arg Lys Ile
Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile 20 25 30 Thr Ser Ser
Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val 35 40 45 Ala
Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser 50 55
60 Met Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr 65 70 75
377PRTArtificialmodified MCP-1 protein 3Met Gln Pro Asp Ala Ile Asn
Ala Pro Val Thr Cys Cys Ala Asn Phe 1 5 10 15 Thr Asn Arg Lys Ile
Lys Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile 20 25 30 Thr Ser Ser
Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val 35 40 45 Ala
Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser 50 55
60 Met Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr 65 70 75
477PRTArtificialmodified MCP-1 protein 4Met Gln Pro Asp Ala Ile Asn
Ala Pro Val Thr Cys Cys Ala Asn Phe 1 5 10 15 Thr Asn Arg Lys Ile
Lys Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile 20 25 30 Thr Ser Ser
Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Lys 35 40 45 Ala
Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser 50 55
60 Met Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr 65 70 75
577PRTArtificialmodified MCP-1 protein 5Met Gln Pro Asp Ala Ile Asn
Ala Pro Val Thr Cys Cys Ala Asn Phe 1 5 10 15 Thr Asn Arg Lys Ile
Lys Val Arg Arg Leu Ala Ser Tyr Arg Arg Ile 20 25 30 Thr Ser Ser
Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val 35 40 45 Ala
Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser 50 55
60 Met Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr 65 70 75
677PRTArtificialmodified MCP-1 protein 6Met Gln Pro Asp Ala Ile Asn
Ala Pro Val Thr Cys Cys Ala Asn Phe 1 5 10 15 Thr Asn Arg Lys Ile
Lys Val Arg Arg Leu Ala Ser Tyr Arg Arg Ile 20 25 30 Thr Ser Ser
Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Lys 35 40 45 Ala
Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser 50 55
60 Met Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr 65 70 75
7234DNAArtificialnucleotide sequence of modified MCP-1 protein
Y13AS21KV47K 7atgcaaccgg acgctatcaa cgcaccggtt acttgttgtg
cgaacttcac caaccgtaag 60atcaaagttc agcgtctggc tagctaccgt cgtatcacga
gctctaaatg cccgaaagaa 120gctgttatct tcaaaaccat caaagctaaa
gaaatctgcg cggatccgaa acagaaatgg 180gttcaggact ctatcgacca
cctggacaaa cagacccaga ccccgaagac ctga
2348234DNAArtificialnucleotide sequence of modified MCP-1 protein
Y13AS21KQ23R 8atgcaaccgg acgctatcaa cgcaccggtt acttgttgtg
cgaacttcac caaccgtaag 60atcaaagttc gccgtctggc tagctaccgt cgtatcacga
gctctaaatg cccgaaagaa 120gctgttatct tcaaaaccat cgttgctaaa
gaaatctgcg cggatccgaa acagaaatgg 180gttcaggact ctatcgacca
cctggacaaa cagacccaga ccccgaagac ctga
2349234DNAArtificialnucleotide sequence of modified MCP-1 protein
Y13AS21KQ23RV47K 9atgcaaccgg acgctatcaa cgcaccggtt acttgttgtg
cgaacttcac caaccgtaag 60atcaaagttc gccgtctggc tagctaccgt cgtatcacga
gctctaaatg cccgaaagaa 120gctgttatct tcaaaaccat caaagctaaa
gaaatctgcg cggatccgaa acagaaatgg 180gttcaggact ctatcgacca
cctggacaaa cagacccaga ccccgaagac ctga 2341077PRTArtificialmutated
MCP-1 protein 10Met Gln Pro Asp Ala Ile Asn Ala Xaa Val Thr Cys Cys
Xaa Asn Phe 1 5 10 15 Thr Asn Xaa Xaa Ile Xaa Val Xaa Arg Leu Ala
Ser Tyr Arg Arg Ile 20 25 30 Thr Ser Ser Lys Cys Pro Lys Glu Ala
Val Ile Phe Lys Thr Ile Xaa 35 40 45 Ala Lys Glu Ile Cys Ala Asp
Pro Lys Gln Lys Trp Val Gln Asp Ser 50 55 60 Met Asp His Leu Asp
Lys Gln Thr Gln Thr Pro Lys Thr 65 70 75
* * * * *