HK1032425B - Amino-terminally truncated mcp-2 as chemokine antagonists - Google Patents

Description

Amino-terminally truncated MCP-2 as chemokine antagonists

Technical Field

The present invention relates to amino-terminally truncated MCP-2 lacking the NH 2-terminal amino acid corresponding to amino acid residues 1, 1-2, 1-3, 1-4 or 1-5 of naturally occurring MCP-2 and having chemokine antagonistic activity, cDNA sequences encoding these MCP-2, their use in the treatment and/or diagnosis of diseases in which antagonism of the chemokine action is desired, and pharmaceutical compositions comprising them.

Background

Chemokines constitute a small family of proinflammatory cytokines with leukocyte chemotactic and activating properties. The chemokine family can be divided into C-C, C-X-C and C-X according to the position of the first cysteine₃C chemokines (Baggiolini M. et al, 1994; Baggiolini M. et al, 1997 and Taub D. et al, 1996).

Many C-X-C chemokines, such as interleukin-8 (IL-8), are chemotactic for neutrophils, while C-C chemokines, such as monocyte chemotactic protein-3 (MCP-3), are active on a variety of leukocytes including monocytes, lymphocytes, eosinophils, basophils, NK cells, and dendritic cells.

The amino-terminal region of chemokines is involved in receptor binding, and amino-terminal processing can activate or render the chemokine completely inactive.

The C-X-C chemokine, platelet basic protein, only becomes a neutrophil chemotactic peptide (NAP-2) after removal of the amino-terminal 24 residues (Walz A. et al, 1989 and Van Damme J. et al, 1990).

Deletion of the 8 amino-terminal residues of IL-8 results in increased chemotactic activity, but further cleavage of the Glu-Leu-Arg motif preceding the first Cys in all neutrophil chemotactic C-X-C chemokines leads to complete inactivation (Clark-Lewis I. et al, 1991).

Another C-X-C chemokine, granulocyte chemotactic protein-2 (GCP-2), undergoes similar amino-terminal proteolysis (8 amino acids cleaved) but has no effect on neutrophil chemotactic activity (Proost P et al, 1993 a).

The synthetic C-C chemokines MCP-1, MCP-3 and RANTES, which lack 8 to 9 amino terminal amino acids, are inactive on monocytes and are used as receptor antagonists (Gong J. et al, 1996; and Gong J. et al, 1995).

Extension of RANTES by one methionine will result in complete inactivation of the molecule, Met-RANTES appears to be a true RANTES antagonist (probfoot a.e. et al, 1996).

Clones of human MCP-2 (monocyte attractant protein-2) have been isolated by differential laboratory screening using cDNA probes derived from stimulated, quiescent Peripheral Blood Lymphocytes (PBLs) (originally designated "HCl 4", Chang H.C. et al, 1989). The cDNA derived protein sequence is the same as the purified natural MCP-2; however, putative allelic variants were also isolated (in which Gln46 replaced Lys46) (Van Coollie et al, 1997).

MCP-2 was also synthesized using solid phase chemistry (Proost P. et al, 1995).

Description of the invention

The primary object of the present invention is an amino-terminally truncated MCP-2 lacking the amino-terminal amino acid corresponding to amino acid residues 1, 1-2, 1-3, 1-4 or 1-5 of naturally occurring MCP-2 and having chemokine antagonist activity.

More specifically, one object of the present invention is MCP-2(6-76), which is MCP-2 lacking amino terminal amino acids 1-5, as shown in FIG. 1 and SEQ ID NO: 3 or SEQ ID NO: 4, respectively.

Such amino-terminally truncated MCP-2 of the present invention may be in a glycosylated form or in an unglycosylated form.

The term "chemokine antagonist" refers to a "mature, naturally occurring antagonist of the full-length chemokine".

Another object of the present invention is a DNA molecule comprising a DNA sequence encoding the amino-terminally truncated MCP-2 of the present invention, wherein the DNA sequence comprises substantially the same nucleotide sequence.

"substantially identical nucleotide sequences" include all other nucleic acid sequences which, by virtue of the degeneracy of the genetic code, are also capable of encoding a given amino acid sequence.

The present invention also includes an expression vector containing the above DNA, a host cell transformed with the vector, and a method for preparing these amino-terminally truncated MCP-2 of the present invention by culturing the transformed cell in a suitable medium.

The DNA sequence encoding the protein of the present invention may be inserted into and ligated with an appropriate plasmid. Once the expression vector is formed, it is introduced into a suitable host cell, which in turn expresses the vector to produce the desired protein.

Expression of any of the recombinant proteins of the invention mentioned herein can be carried out in eukaryotic cells (such as yeast, insect or mammalian cells) or prokaryotic cells using a suitable expression vector. Any method known in the art may be used.

For example, a DNA molecule encoding a protein obtained by any of the methods described above can be inserted into an appropriately constructed expression vector using techniques well known in the art (see Sambrook et al, 1989). Double-stranded cDNA is ligated into plasmid vectors using homopolymer tailing, restriction ligation using synthetic DNA linkers, or blunt end ligation techniques. DNA molecules were ligated with DNA ligase, and undesired ligation was avoided by alkaline phosphatase treatment.

In order to express the desired protein, the expression vector should also contain a specific nucleotide sequence containing transcriptional and translational regulatory information linked to the DNA encoding the desired protein, thereby allowing the gene to express and produce the protein. First, in order for a gene to be transcribed, it must be preceded by a promoter recognized by RNA polymerase, which binds to the polymerase, thereby initiating the transcription process. Various promoters (strong and weak promoters) with different work efficiencies can be employed.

For eukaryotic hosts, different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived from viruses such as adenovirus, bovine papilloma virus, simian virus, etc., wherein the regulatory signals are associated with specific genes having high expression levels. Examples are the TK promoter of herpes virus, SV40 early promoter, yeast gal4 gene promoter, etc. The transcriptional initiation regulatory signal may be selected to have repression and activation effects that can regulate gene expression.

A DNA molecule comprising a nucleotide sequence encoding a protein of the invention is inserted into a vector, which is capable of integrating a desired gene sequence into a host cell, in operative association with transcriptional and translational regulatory signals.

Cells stably transformed with the introduced DNA may also be selected by introducing one or more markers that allow the selection of host cells containing the expression vector. The marker may also provide phototrophic, microbiocidal resistance to an auxotrophic host, such as an antibiotic, or a heavy metal such as copper, and the like. The selectable marker gene may be directly linked to the DNA gene sequence to be expressed or introduced into the same cell by co-transfection. Additional elements may also be required in order to optimize the synthesis of the proteins of the invention.

Important factors for the selection of a particular plasmid or viral vector include: ease of identifying and selecting recipient cells containing a vector from recipient cells not containing a vector; the number of copies of the vector desired in a particular host; and whether it is desirable for the vector to be "shuttled" between different host cell species.

Once a vector or construct containing a DNA sequence has been prepared for expression, the DNA construct may be introduced into a suitable host cell by a variety of suitable methods including: transformation, transfection, coupling, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, and the like.

The host cell may be a prokaryotic cell or a eukaryotic cell. Eukaryotic hosts, such as mammalian cells, e.g., human, monkey, mouse, and Chinese Hamster Ovary (CHO) cells, are preferred because they provide post-translational modifications to the protein molecule, including proper folding or glycosylation at the correct site. Yeast cells can also be subjected to post-translational peptide modifications including glycosylation. There are many recombinant DNA strategies that employ strong promoter sequences and high copy number plasmids that can be used to produce the desired protein in yeast. Yeast recognizes the leader sequence on the cloned mammalian gene product and secretes a peptide carrying the leader sequence (i.e., a pre-peptide).

After introduction of the vector, the host cells are grown in a selective medium that allows selective growth of the cells containing the vector. Expression of the cloned gene sequence results in production of the desired protein.

The amino-terminally truncated MCP-2 of the present invention may be prepared by other methods well known in the art, in particular by established chemical synthesis methods: and (3) performing chromatographic purification by using an automatic solid-phase peptide synthesizer.

For example, the chemokines of the invention may be synthesized using Fmoc (9-fluorenylmethoxycarbonyl), tBoc (t-butyloxycarbonyl) or any other similar chemical synthesis with or without suitable side chain protecting groups on the different amino acids. Amino acids with or without suitable side chain protecting groups are pre-activated (e.g., with HBTU/HOBt [2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyl-ureido hexafluorophosphate/1-hydroxybenzotriazole) and coupled to the growing peptide chain. The protecting group (e.g., Fmoc) is removed from the alpha amino group prior to addition of the next residue. After synthesis all protecting groups are removed, the whole full-length peptide is purified and the peptide is folded chemically or enzymatically (including disulfide bond formation between cysteines) into the corresponding chemokine of the invention.

Natural, synthetic or recombinant proteins can be purified by any method known for purification, i.e., any conventional method, including extraction, precipitation, chromatography, electrophoresis, etc. (see, e.g., Proost P. et al, 1996). Another preferred purification method for purifying the protein of the invention is affinity chromatography, which utilizes the affinity of monoclonal antibodies or heparin that bind to the target protein and are produced and immobilized on a gel matrix contained in a column. Passing an impure preparation containing the protein through the column. Proteins will be bound to the column by heparin or specific antibodies, while impurities will pass through the column. After washing the column, the protein is eluted from the gel by changing the pH or ionic strength.

The amino-terminally truncated MCP-2 of the present invention may be used for the treatment and/or diagnosis of diseases where chemokine antagonism is desired. Examples of such diseases include: inflammatory diseases, angiogenesis and hematopoiesis related diseases, tumors, infectious diseases (including HIV), autoimmune diseases, atherosclerosis, lung diseases and skin diseases.

Thus, in a further aspect, the invention provides the use of a protein of the invention in the manufacture of a medicament for the treatment of a disease as described above.

The medicament is preferably in the form of a pharmaceutical composition comprising a protein of the invention together with one or more pharmaceutically acceptable carriers and/or excipients. These pharmaceutical compositions form a further aspect of the invention.

A further aspect of the present invention is a method for the treatment of the above-mentioned diseases, which comprises administering to a subject at risk of developing these diseases or a subject already exhibiting these pathological phenomena a pharmaceutically active amount of an amino-terminally truncated MCP-2 of the present invention.

The invention will now be described by way of the following examples, which should not be construed as limiting the invention in any way. The embodiments will be described with reference to the drawings described below.

Brief Description of Drawings

FIG. 1: it shows the amino acid sequence of MCP-2 and known variants thereof. The signal sequence is in italics and the C-terminal residues are in bold. The arrow indicates the first amino acid of the amino-terminally truncated MCP-2(6-76) of the present invention. The underlining is a different amino acid than in the MCP-2 variant.

FIG. 2: SDS-PAGE of amino-terminally truncated MCP-2 (6-76):

lane 1: native MCP-2(1-76, 100 ng/lane);

lane 2: native MCP-2(1-76, 30 ng/lane);

lane 3: native MCP-2(6-76, 30 ng/lane); and

lane 4: synthetic MCP-2(1-76, 60 ng/lane).

The gel was electrophoresed under reducing conditions and the proteins were stained with silver.

FIG. 3: it shows a comparison of the chemotactic potency of modified MCP-2.

Intact native (nat) and synthetic (syn) MCP-2(1-76), amino-terminally truncated native MCP-2(6-76) and carboxy-terminally truncated synthetic MCP-2(1-74) were tested for chemotactic activity on THP-1 cells. Results are presented as mean of CI ± SEM of four or more separate experiments.

FIG. 4: native MCP-2 is a weaker agonist for calcium flux in monocytes compared to MCP-1. Intact MCP-2 increases [ Ca ] in THP-1 cells with increasing doses (15, 50 and 150ng/ml)²⁺]_i. The figure shows the results of a typical one of the two experiments.

Examples

Example 1: amino-terminally truncated MCP-2

Materials and methods

Chemokines and immunoassays

MCP-2 was synthesized and purified as described previously (Proost P. et al, 1995).

Specific anti-human MCP-2 antibodies were obtained from mice and affinity purified on a Sepharose column (CNBr activated Sepharose4B, pharmacia, Uppsala, Sweden) and the synthesized MCP-2 was coupled to the column using the conditions provided by the manufacturer.

ELISA plates were coated with affinity purified anti-human MCP-2 and biotinylated anti-MCP-2 was used as the capture antibody. Detection was performed with peroxidase-labeled streptavidin and TMB. The limit of detection of MCP-2 ELISA was approximately 0.1 ng/ml.

Production and purification of MCP-2

Monocyte chemotactic protein was purified from monocyte-derived conditioned medium obtained from 132 donors from Antwerp and Leuven transfusion centers (Proost P. et al, 1996).

Erythrocytes and granulocytes were removed by precipitation in hydroxyethyl starch (Fresenius AG, Bad Homburg, Germany) and gradient centrifugation in sodium 3-acetamido-5-acetylmethylamino-2, 4, 6-triiodobenzoate solution (sodium metrioate, Lymphoprep; Nyegaard, Oslo Norway).

Monocytes (60X 10) were incubated with 10. mu.g/ml Con A and 2. mu.g/ml LPS⁹A cell). After 48 to 120 hours, the conditioned medium was collected and placed at-20 ℃ until purification.

Native MCP-2 was purified using a four-step purification procedure as previously described (Proost P. et al, 1996).

Briefly, conditioned media was concentrated on glass or silicic acid of controlled pore size and partially purified by affinity chromatography on a heparin-Sepharose column (Pharmacia).

The fraction containing MCP-2 immunoreactivity was further purified by Mono S (Pharmacia) cation exchange chromatography and eluted with a NaCl gradient at pH 4.0.

Native MCP-2 was purified to homogeneity by RP-HPLC on a C-8 Aquapore RP-300 column (Perkin Elmer, Norwalk CT) equilibrated with 0.1% trifluoroacetic acid (TFA). The protein was eluted with a gradient of acetonitrile.

Biochemical characterization of MCP-forms by SDS-PAGE, amino acid sequence analysis and mass spectrometry

The purity of the column fractions was checked by SDS-PAGE on Tris/tricine gels under reducing conditions (Proost P. et al, 1996). Proteins were silver stained and labeled with the following relative molecular weights (Mr): OVA (Mr 45000), carbonic anhydrase (Mr 31000), soybean trypsin inhibitor (Mr 21500), beta-lactoglobulin (Mr 18400), lysozyme (Mr 14400) and aprotinin (Mr 6500).

The amino-terminal sequence of the purified chemokines was determined by Edman degradation on a pulsed liquid 477A/120A protein sequencer (Perkin Elmer) using N-methylpiperidine as coupling base. The blocked protein was cleaved between Asp and Pro by placing it in 75% formic acid for 50 hours. The formate digest was sequenced without further purification. Mr of MCP-2 was determined by matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF-MS) (Micromass TofSpec, Manchester, UK). Alpha-cyano-4-hydroxycinnamic acid and cytochrome C were used as matrix and internal standards, respectively.

Detection of chemotactic Activity

Testing of MCP-2 on freshly purified monocytes (2X 10) in a Boyden microchamber with a polyvinylpyrrolidone (PVP) membrane with a pore size of 5 μm⁶Cells/ml) or monocytic THP-1 cells (0.5X 10)⁶Cells/ml; 2 days after the transfer).

The samples and cells were diluted in HBSS (Life technologies/Gibco BRL, Paisley, Scotland) supplemented with 1 mg/ml human serum albumin (Red Cross Belgium). After incubation for 2 hours at 37 ℃, cells were fixed, stained with Diff-Quick staining solution (Harleco, gibbstown. nj), and cells migrating through the membrane were counted by microscope in 10 oil-immersed fields at 500 x magnification.

Chemotaxis Index (CI) was calculated for samples (in triplicate in each chamber) based on the ratio of the number of cells migrated into the sample to the number of cells migrated into control medium (Van Damme J. et al, 1992).

For desensitization experiments, cells were incubated with the biologically inactive chemokine variant at 37 ℃ for 10 minutes prior to addition to the upper row of wells of the Boyden microchamber. The CI of HBSS-treated cells versus the CI of the sample was used as a reference value and% inhibition of CI was calculated.

Intracellular Ca²⁺Detection of concentration

Intracellular calcium concentration ([ Ca ] was determined as described previously (Wuyts A. et al, 1997)²⁺]_i). Purified monocytes or THP-1 cells (10. mu.M) were incubated in Eagle Limit essential Medium (EMEM, Gibco) + 0.05% FCS containing The fluorescent indicators fura-2(fura-2/AM 2.5. mu.M; Molecular Probes Europe BV, Leiden, The Netherlands) and 0.01% Pluronic F-127(Sigma, St Louis MO)⁷Cells/ml).

After 30 minutes at 37 ℃ the cells were washed twice and 10⁶Cell/ml concentration resuspended in 1mM Ca²⁺And 0.1% FCS in HBSS (10 mM Hepes/NaOH pH7.4 as buffer). Cells were equilibrated at 37 ℃ for 10 min, and fura-2 fluorescence was measured in an LS50B luminescence spectrophotometer (Perkin Elmer).

After excitation at 340 and 380nm, fluorescence was detected at 510 nm. Calculated using the Grynkiewicz equation (Grynkiewicz et al, 1985) [ Ca²⁺]_i. To measure R_maxCells were lysed with 50 μ M digitonin. Subsequently, pH was adjusted to 8.5 with 20mM Tris, and 10mM EGTA was added to the lysed cells to obtain R_min. K used_d224 nM.

For desensitization experiments, monocytes or THP-1 cells were first stimulated with buffer, varying concentrations of chemokines or chemokine antagonists. As a second stimulus, MCP-2 was used at a concentration that was induced by pre-stimulation with buffer [ Ca ]²⁺]_iA significantly increased concentration. The second stimulus was applied 2 minutes after the addition of the first stimulus. Calculating [ Ca ] in response to the second stimulus by comparing the signal after pre-stimulation with a chemokine or chemokine antagonist with the signal after addition of buffer²⁺]_iPercent inhibition increased.

Results

Isolation of post-translationally modified MCP-2

Different forms of MCP-2 produced by mitogen and endotoxin stimulated peripheral blood mononuclear cells were followed up by a specific, sensitive ELISA.

The conditioned medium was purified according to standard separation procedures (Proost P. et al, 1996) which included adsorption onto pore size controlled glass and heparin Sepharose chromatography.

Subsequently, FPLC mono S cation exchange chromatography purification was performed followed by further purification using C-8 RP HPLC. The molecular weight was determined by SDS-PAGE and MALDI/TOF-MS.

The different forms of MCP-2 are obtained by separation: in addition to authentic 7.5kDa MCP-2(1-76), 7kDa amino-truncated MCP-2[ MCP-2(6-76) ] lacking 5 residues was purified to homogeneity by RP-HPLC and identified by amino acid sequence analysis (FIG. 2). MALDI/TOF-MS (Table I) determined the molecular weight of intact MCP-2 to be 8881Da (theoretical relative molecular weight of 8893Da) and MCP-2(6-76) to be 8365Da, confirming the 5 amino-terminal amino acid deletions (theoretical relative molecular weight of 8384 Da). A functional comparison of these native forms of MCP-2 in the THP-1 chemotaxis assay showed that intact MCP2 was still active at 5ng/ml, whereas truncated MCP-2(6-76) was not chemotactic when tested at concentrations ranging from 0.6 to 60ng/ml (FIG. 3). The potency of intact native MCP-2 was also compared to synthetic MCP-2(1-76) and carboxyl-terminally truncated synthetic MCP-2[ MCP-2(1-74) ] (Proost P. et al, 1995) lacking 2 residues.

The lowest effective chemotactic concentration of these forms was also found to be 5ng/ml (FIG. 3). Although the specific activities of native intact MCP-1 and MCP-2 were comparable in chemotaxis assays (Van Damme J, et al, 1992), the ability of MCP-2 to mobilize calcium remains to be debated.

However, at Ca²⁺In migration experiments, the minimal effective dose of natural or synthetic MCP-2(1-76) was 10 times higher than that of natural intact MCP-1(1-76) (FIG. 4), while MCP-2(6-76) was still inactive.

Nevertheless, intact MCP-2(50ng/ml) desensitized MCP-2(15ng/ml) and MCP-3(10ng/ml) to 52% and 45% inhibition of chemotaxis, respectively.

Due to MCP-2 being in Ca²⁺The specific activity in the assay was low, so desensitization of MCP-2(6-76) to chemotaxis was performed in a Boyden microchamber. It was reported that intact MCP-2 cross-desensitized to active MCP-1, MCP2 and MCP 3 in monocyte chemotaxis assays (Sozzani S. et al, 1994), so we investigated whether native inactive MCP-2(6-76) could also desensitize MCP-1, MCP-2, MCP-3 and RANTES (Table II). Pre-incubation of THP-1 cells with 100ng/ml inactive MCP-2(6-76) has significantly inhibited chemotaxis induced by 10ng/ml MCP-1 (63%), 5ng/ml MCP-2 (75%), 30ng/ml MCP-3 (62%) and 100ng/ml RANTES (75%). In addition, chemotaxis by each MCP at a concentration 3-fold lower was completely (91-100%) inhibited by 100ng/ml MCP-2 (6-76). Furthermore, MCP-2(6-76) still significantly inhibited MCP-1(3ng/ml), MCP-2(1.5ng/ml) or MCP 3(10ng/ml) and RANTES (30ng/ml) induced chemotactic activity at concentrations as low as 10 ng/ml. In conclusion, MCP-2(6-76) is a naturally occurring, inactive chemoattractant and antagonizes several C-C chemokines, with the most prominent effect on MCP-3.

TABLE I

Biochemical characterization of native MCP-2. Amino-terminal amino acid analysis and comparison of the experimental (SDS-PAGE and MALDI/TOF-MS) and theoretical relative molecular weights of C-8 RP-HPLC-purified native MCP-isoforms

MCP forms	Amino terminal sequence	Relative molecular weight (Da)
							Theoretical value of unglycosylation	SDS-PAGE	MALDI/TOF-MS
MCP-2(1-76)	Sealing of	8893	7500	8881
					MCP-2(2-76)	SIPITCC	8384	7500	8365

TABLE II

MCP-2(6-76) desensitizes monocyte chemotactic responses of MCP-1, MCP-2, MCP-3 and RANTES in the microchamber.

Chemotactic factora	Concentration of	Antagonism of chemotactic responseb，c		Inhibition of chemotaxis%
							Buffer solution	100ng/mlMCP-2(6-76)
MCP-1	10	22.3±7.9	8.3±3.8	63±21
						3	15.0±8.0	1.3±0.3	99±1.0
MCP-2	5	36.0±15.6	10.8±6.1	75±8.0
						1.5	6.7±1.4	1.5±0.3	91±7.0
MCP-3	30	13.2±0.4	6.0±4.0	62±31
						10	3.0±1.5	＜1	100±0.0
RANTES	100	6.3±0.8	2.6±1.3	75±19
						30	4.0±0.8	1.5±0.3	77±16
		Buffer solution	100ng/mlMCP-2(6-76)
					MCP-1	10	12.7±2.3	10.5±3.8	24±1.8
	3	7.5±0.0	3.0±0.3	69±4.0
					MCP-2	5	38.0±5.3	27.2±4.9	30±6.0
	1.5	18.3±4.6	9.2±1.4	45±23
					MCP-3	30	13.2±1.9	8.0±1.0	37±19
	10	7.7±1.4	1.7±0.3	90±6.0
					RANTES	100	5.5±0.6	5.8±0.9	17±7.0
	30	3.2±0.7	2.5±0.5	39±18

^aMCP-1, MCP-2, MCP-3 or RANTES is added to the lower row of wells as a chemical attractant.

^bTHP-1 cells previously incubated with MCP-2(6-76) or buffer are added to the upper row of wells of the microchamber

^cMean of CI. + -. SEM for 3 independent experiments

Reference to the literature

Baggiolini m. et al, ann.rev.immunol., 55, 97-179, 1994.

Baggiolini m. et al, ann. rev. immunol. 15, 675-705, 1997.

Chang H.C. et al, International Immunology, 1(4), 388-.

Clark-Lewis I, et al, J.biol.chem., 266, 23128-.

De Meester i. et al, j.immunol. methods 189, 99-10526, 1996.

Deng H. et al, Nature, 381, 661- & 666, 1996.

Gong J. et al J.exp.Med, 181, 631-640, 1995.

Gong J. et al, J.biol.chem.271, 10521-10527, 1996.

Grynkiewicz g. et al, j.biol.chem., 260, 3440, 1985.

Proost P, et al, Biochemistry, 32, 10170-.

Proost P. et al, J.Immunol., 150, 1000. supplement 1010, 1993.

Proost P. et al, Cytokine, 7, 97-104, 1995.

Pro st p. et al, Methods: a company to Methods in enzymol, 10, 82, 1996.

Proudfoot A.E. et al, J.biol.chem., 271, 2599-.

Sambrook et al, molecular cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold

Spring Harbor，NY，1989

Schols D. et al, J.Exp.Med., 186, 1383-.

Sozzani s. et al, j.immunol., 152, 3615, 1994.

Taub D. et al, Cytokine & Growth Factor Reviews, 7, 335-76, 1996.

Van Coillie e et al, biochem. biophysis. res. commun., 231, 726-730, 1997.

Van Damme J. et al, Eur. J.biochem., 181, 337-344, 1989.

Van Damme j. et al, eur.j.immunol., 20, 2113-8, 1990.

Van Damme j. et al, j.exp.med., 176, 59, 1992.

Walz a. et al, biochem. biophysis. res. commun., 159, 969-75, 1989.

Wuyts A, et al, Biochemistry 36, 2716-2723, 1997.

Sequence listing (1) general information:

(i) the applicant:

(A) name: APPLIED RESEARCH SYSTEMS ARS HOLDING N.V.

(B) Street: 14 JOHN B. GORSIRAWEG

(C) City: CURACAO

(E) The state is as follows: THE NETHERLANDS ANTILLES

(F) And (3) post code: is not provided with

(G) Telephone: 639300

(H) Electric transmission: 614129

(I) Telegraph:

(ii) the invention name is as follows: amino-terminally truncated MCP-2 as chemokine antagonists

(iii) Number of sequences: 4

(v) A computer-readable form:

(A) type of recording medium: flexible disk

(B) A computer: IBM PC compatible

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patent in Release #1.0, Version #1.30(EPO) (2) SEQ ID NO: 1, information:

(i) sequence characteristics:

(A) length: 99 amino acid

(B) Type (2): amino acids

(C) Chain type:

(D) topological structure: linearity

(ii) Molecular type: protein

(iii) Suppose that: is not provided with

(ix) Is characterized in that:

(A) name/Key: protein

(B) Position: 1..76

(xi) Description of the sequence: SEQ ID NO: 1: met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu Met Ala Ala Thr

-20 -15 -10Phe Ser Pro Gln Gly Leu Ala Gln Pro Asp Ser Val Ser Ile Pro Ile

-5 1 5Thr Cys Cys Phe Asn Val Ile Asn Arg Lys Ile Pro Ile Gln Arg Leu10 15 20 25Glu Ser Tyr Thr Arg Ile Thr Asn Ile Gln Cys Pro Lys Glu Ala Val

30 35 40Ile Phe Lys Thr Lys Arg Gly Lys Glu Val Cys Ala Asp Pro Lys Glu

45 50 55Arg Trp Val Arg Asp Ser Met Lys His Leu Asp Gln Ile Phe Gln Asn

60 65 70Leu Lys Pro

75(2) SEQ ID NO: 2, information:

(i) sequence characteristics:

(A) length: 99 amino acid

(B) Type (2): amino acids

(C) Chain type:

(D) topological structure: linearity

(ii) Molecular type: protein

(iii) Suppose that: is not provided with

(ix) Is characterized in that:

(A) name/Key: protein

(B) Position: 1..76

(xi) Description of the sequence: SEQ ID NO: 2: met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu Met Ala Ala Thr

-20 -15 -10Phe Ser Pro Gln Gly Leu Ala Gln Pro Asp Ser Val Ser Ile Pro Ile

-5 1 5Thr Cys Cys Phe Asn Val Ile Asn Arg Lys Ile Pro Ile Gln Arg Leu10 15 20 25Glu Ser Tyr Thr Arg Ile Thr Asn Ile Gln Cys Pro Lys Glu Ala Val

30 35 40Ile Phe Lys Thr Gln Arg Gly Lys Glu Val Cys Ala Asp Pro Lys Glu

45 50 55Arg TrP Val Arg Asp Ser Met Lys His Leu Asp Gln Ile Phe Gln Asn

60 65 70Leu Lys Pro

75(2) SEQ ID NO: 3, information:

(i) sequence characteristics:

(A) length: 71 amino acid

(B) Type (2): amino acids

(C) Chain type:

(D) topological structure: linearity

(ii) Molecular type: protein

(iii) Suppose that: is not provided with

(xi) Description of the sequence: SEQ ID NO: 3:

Ser Ile Pro Ile Thr Cys Cys Phe Asn Val Ile Asn Arg Lys Ile Pro

1 5 10 15

Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr Asn Ile Gln Cys Pro

20 25 30

Lys Glu Ala Val Ile Phe Lys Thr Lys Arg Gly Lys Glu Val Cys Ala

35 40 15

Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met Lys His Leu Asp Gln

50 55 60

Ile Phe Gln Asn Leu Lys Pro

6570 (2) SEQ ID NO: 4:

(i) sequence characteristics:

(A) length: 71 amino acid

(B) Type (2): amino acids

(C) Chain type:

(D) topological structure: linearity

(ii) Molecular type: protein

(iii) Suppose that: is not provided with

(xi) Description of the sequence: SEQ ID NO: 4: ser Ile Pro Ile Thr Cys Cys Phe Asn Val Ile Asn Arg Lys Ile Pro 151015 Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr Asn Ile Gln Cys Pro

20 25 30Lys Glu Ala Val Ile Phe Lys Thr Gln Arg Gly Lys Glu Val Cys Ala

35 40 45Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met Lys His Leu Asp Gln

50 55 60Ile Phe Gln Asn Leu Lys Pro65 70

Claims (10)

1. An amino-terminally truncated MCP-2 lacking amino-terminal amino acids corresponding to amino acid residues 1-5 of naturally occurring MCP-2 and having chemokine antagonistic activity.

2. The amino-terminally truncated MCP-2 according to claim 1, having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

3. Amino-terminally truncated MCP-2 according to any one of the preceding claims, in glycosylated form.

4. A DNA molecule comprising a DNA sequence encoding the amino-terminally truncated MCP-2 of claim 1.

5. An expression vector comprising the DNA molecule of claim 4.

6. A host cell comprising the expression vector of claim 5.

7. A recombinant method for producing the amino-terminally truncated MCP-2 of claim 1, comprising culturing the host cell of claim 6 in a culture medium.

8. Use of the amino-terminally truncated MCP-2 according to claim 1 for the manufacture of a medicament for the treatment or diagnosis of a disease requiring an activity antagonistic to the action of chemokines.

9. Use according to claim 8 for the manufacture of a medicament for the treatment of inflammatory diseases, HIV infection, angiogenesis and hematopoiesis related diseases or tumors.

10. A pharmaceutical composition comprising the amino-terminally truncated MCP-2 of claim 1, and a pharmaceutically acceptable carrier and/or excipient.