Amino-terminally truncated mcp-2-as chemokine antagonists

Description

The description is 14 pages long (including 4 pages of illustrations). HU 226 468 B1 The invention relates to an amino-terminally truncated human monocyte chemotactic protein 2 (MCP-2) which has chemokine antagonist activity and which lacks the amino-terminal amino acids corresponding to amino acids 1-5 of naturally occurring human MCP-2. The invention also relates to cDNA sequences encoding truncated MCP-2s, their therapeutic and diagnostic use in diseases in which antagonist activity of chemokine effects is required, and to pharmaceutical compositions containing them. Chemokines belong to a family of small proinflammatory cytokines with chemotactic and activating properties for leukocytes. Depending on the position of the first cysteine, the chemokine family can be divided into CC, CXC and C-X3-C type chemokines [Baggiolini, M. et al., Ann. Rev. Immunoi. 55, 97-179 (1994); Baggiolini, M. et al., Ann. Rev. Immunoi. 15, 675-705 (1997); Taub, D. et al., Cytokine & Growth Factor Reviews 7, 335-76 (1996)]. Many CXC-type chemokines, such as interleukin-8 (IL-8), have chemotactic effects on neutrophils, while CC-chemokines, such as monocyte chemotactic protein-3 (MCP-3), have effects on several classes of white blood cells, such as monocytes, lymphocytes, eosinophils, basophils, NK cells, and dendritic cells. The amino-terminal domain of chemokines is involved in receptor binding, and processing of the amino-terminal ends can activate or completely inactivate chemokines. The CXC-type chemokine platelet basic protein becomes neutrophil chemotactic peptide (NAP-2) only after removal of the 24 amino-terminal amino acids [Walz, A. et al., Biochem. Biophys. Res. Commun. 159, 969-75 (1989); Van Damme, J. et al., Eur. J. Immunol., 20, 2113-8 (1990)]. Removal of up to 8 amino-terminal amino acids from IL-8 results in increased chemotactic activity, but further cleavage of the Glu-Leu-Arg motif located before the first cysteine ​​in all neutrophil chemotactic CXC chemokines results in complete inactivation [Clark-Lewis, I. et al., J. Biol. Chem. 266, 23 128-23 134(1991)]. In another CXC chemokine, granulocyte chemotactic protein-2 (GCP-2), similar amino-terminal proteolysis (up to 8 amino acids) has no effect on neutrophil chemotactic activity [Proost, P. et al., Biochemistry 32, 10 170-10 177 (1993)]. Synthetic CC chemokines deficient in the amino-terminal 8-9 amino acids, MCP-1, MCP-3 and RANTES, are inactive on monocytes and can be used as receptor antagonists [Gong, J. et al., J. Biol. Chem. 271, 10 521-10 527 (1996); Gong, J. et al., J. Exp. Med. 181, 631-640 (1995)]. Extension of RANTES with a methionine completely inactivates the molecule, and Met-RANTES acts as an antagonist of the original RANTES [Proudfoot, AE et al., J. Biol. Chem. 271, 2599-2603 (1996)]. A clone of human MCP-2 (Monocyte Chemoattractant Protein-2) was isolated by differential library screening using cDNA probes from stimulated peripheral blood lymphocytes (PBL) [originally designated as HC14, Chang, HC et al., International Immunology 1(4), 388-397 (1989)] against cDNA probes from unstimulated, resting peripheral blood lymphocytes. The cDNA-derived protein sequence was identical to that of purified, native MCP-2, although a putative allelic variant was also isolated in which Gln46 replaces Lys46 [Van Coillie, E. et al., Biochem. Biophys. Res. Commun. 231, 726-730 (1997)]. MCP-2 can also be synthesized by solid phase chemistry [Proost, P. et al., Cytokine 7, 97-104 (1995)]. The invention is described below. Our primary objective in developing the solution according to the invention was to produce an amino-terminally truncated MCP-2 lacking the amino-terminal amino acids corresponding to amino acids 1, 1-2, 1-3, 1-4 or 1-5 of naturally occurring MCP-2, which has chemokine antagonist activity. More preferably, one of the objects of the invention is MCP-2(6-76), which is MCP-2 lacking amino-terminal amino acids 1-5, as shown in Figure 1 and SEQ ID NO:3 or 4. The amino-terminally truncated MCP-2 of the invention may be glycosylated or unglycosylated. The term "chemokine antagonist" means that it acts as an antagonist to mature, full-length, naturally occurring chemokines. Another aim of developing the solution according to the invention was to produce DNA molecules which contain DNA sequences encoding the amino-terminally truncated MCP-2 according to the invention, including substantially identical nucleotide sequences. The term "substantially identical nucleotide sequences" refers to all other nucleic acid sequences that, due to the degeneracy of the genetic code, encode the given amino acid sequences. The invention relates to expression vectors containing the above DNAs, host cells transformed with such vectors, and a method for producing the amino-terminally truncated MCP-2 of the invention by culturing the transformed cells in a suitable medium. The DNA sequence encoding the proteins of the invention can be inserted into a suitable plasmid and ligated. The resulting expression vector is introduced into a suitable host cell, which then expresses the vector(s) to produce the desired protein. Expression of any recombinant protein of the invention, as described herein, may be carried out in eukaryotic cells (e.g., yeast, insect, or mammalian cells) or prokaryotic cells. HU 226 468 B1 using appropriate expression vectors. Any method known in the art may be used. For example, DNA molecules encoding proteins obtained by any of the above-described methods can be inserted into suitably prepared expression vectors using techniques known in the art [see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, ed.: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989)]. Double-stranded cDNA can be ligated to plasmid vectors by homopolymeric end preparation or by restriction ligation using synthetic DNA linkers or by blunt-end ligation techniques: DNA ligases are used to ligate the DNA molecules and undesired linkages are avoided by alkaline phosphatase enzyme treatment. In order to express the desired protein, an expression vector must contain specific nucleotide sequences carrying transcriptional and translational regulatory information, linked to the DNA encoding the desired protein in a manner that allows gene expression and protein production. First, in order for the gene to be transcribed, it must be preceded by a promoter that is recognized by RNA polymerase, to which the polymerase binds and thus initiates the transcription process. A number of such promoters can be used, which operate with varying efficiency (strong or weak promoters). In eukaryotic hosts, different transcriptional and translational regulatory sequences can be used depending on the nature of the host. These can be derived from viral sources, such as adenovirus, bovine papillomavirus, simian virus or the like, where the regulatory signals are associated with a specific, highly expressed gene. Examples include the Herpes virus TK promoter, the SV40 early promoter, the yeast gal4 gene promoter, etc. Transcriptional initiation regulatory signals can be chosen to allow repression and activation, so that gene expression can be influenced. A DNA molecule containing a nucleotide sequence encoding a protein of the invention, to which transcriptional and translational regulatory signals are operably linked, is inserted into a vector(s) capable of introducing the desired gene sequences into the host cell. Cells stably transformed with the introduced DNA can be selected by introducing one or more markers that allow selection of host cells containing the expression vector. The marker can provide prototrophy to an auxotrophic host, can result in biocide resistance, for example to antibiotics, heavy metals such as copper or the like. The selectable marker gene can be linked directly to the DNA of the gene sequences to be expressed or can be introduced into the same cell by cotransfection. Additional elements may also be required for optimal synthesis of the proteins of the invention. Important factors in selecting a particular plasmid or viral vector include: the ease with which recipient cells containing the vector can be recognized and selected from recipient cells that do not contain the vector, the desired copy number of the vector in a particular host, and whether it is desirable for the vector to be able to shuttle between host cells of different species. Once the vector(s) or construct containing the DNA sequence for expression are prepared, the DNA construct(s) can be introduced into a suitable host cell by any of a number of suitable means, such as transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, etc. Host cells can be prokaryotic or eukaryotic. Eukaryotic hosts, such as mammalian cells such as human, monkey, mouse, and Chinese hamster ovary (CHO) cells, are preferred because they provide the opportunity for post-translational modification of protein molecules, such as conformational changes or glycosylation at appropriate sites. Yeast cells also perform post-translational modifications, such as glycosylation. A variety of recombinant DNA strategies utilize strong promoter sequences and high copy number plasmids that can be used to produce the desired protein in yeast. Yeast cells recognize leader sequences on cloned mammalian gene products and select peptides (i.e., prepeptides) bearing the leader sequences. After introduction of the vector(s), the host cells are cultured in a selective medium that selects for the growth of cells containing the vector. Expression of the cloned gene sequence(s) results in the production of the desired proteins. The amino-terminally truncated MCP-2 of the invention can be prepared by any well-known method in the art, preferably by well-established chemical synthesis methods using automated solid phase peptide synthesizers, followed by chromatographic purification. Chemokines of the invention can be synthesized, for example, by Fmoc (9-fluoroenylmethoxycarbonyl), tBoc (t-butoxycarbonyl) or any other similar chemical synthesis with or without appropriate side chain protecting groups on the various amino acids. Amino acids with or without appropriate side chain protecting groups are pre-activated - for example, with HBTU-HOBt [2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate / 1-hydroxybenzotriazole] - and coupled to the growing peptide chain. Before the next amino acid is added, the protecting group (e.g. Fmoc) is removed from the α-amino group. After synthesis, all protecting groups are removed, the intact, full-length peptide is purified and chemically or enzymatically folded (e.g., by forming disulfide bridges between cysteines) according to the chemokines of the invention. Purification of natural, synthetic or recombinant proteins can be carried out by any known method suitable for this purpose, i.e. extraction, small-scale separation, HU 226 468 Β1 pás, chromatography, electrophoresis or the like according to conventional methods [see, for example, Proost, P. et al., Methods: A companion to Methods in Enzymol. 10, 82 (1996)]. A further purification method which is advantageously applicable to purify the protein of the invention is affinity chromatography based on the affinity for monoclonal antibodies or heparin which bind the target protein and which can be carried out by immobilizing the method on a gel matrix in a column. The impure preparations containing the proteins are passed through the column. The protein binds to the column via the heparin or specific antibody, while the contaminants pass through the column. After washing, the protein is released from the gel by changing the pH or ionic strength. The amino-terminally truncated MCP-2 of the invention can be used in the therapy and / or diagnosis of diseases in which antagonist activity of chemokine effects is required. Such diseases include inflammatory diseases, diseases related to angiogenesis or hematopoiesis, tumors, infectious diseases such as HIV, autoimmune diseases, atherosclerosis, respiratory diseases and skin disorders. According to a further aspect of the invention, the invention relates to the use of the protein of the invention for the manufacture of a medicament for the treatment of the above-mentioned diseases. The medicament is preferably prepared in the form of a pharmaceutical composition comprising the proteins of the invention in association with one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions form yet another aspect of the invention. In a further embodiment of the invention, there is provided a method for treating the above-mentioned diseases, comprising administering a therapeutically active amount of the amino-terminally truncated MCP-2 of the invention to a patient at risk of developing such diseases or to a patient already showing symptoms. The invention is described by the following examples, which are not intended to limit the invention in any way. In the examples, reference is made to the figures described below. The following is a description of the figures. Figure 1 depicts the amino acid sequence of MCP-2 and its known variant. Signal sequences are indicated in italics, while C-amino acids are indicated in bold. Arrows indicate the first amino acids of the amino-terminally truncated MCP-2(6-76) of the invention. The amino acid that is different in the MCP-2 variant is underlined. Figure 2 shows an SDS-PAGE image of MCP2(6-76) truncated from its amino terminus: Lane 1: native MCP-2(1-76), 100 ng / lane, Lane 2: natural MCP-2(1-76), 30 ng / lane, Lane 3: natural MCP-2(6-76), 30 ng / lane, Lane 4: synthetic MCP-2(1-76), 60 ng / lane The gels were run under reducing conditions and the proteins were stained with silver. Figure 3 shows a comparison of the chemotactic activity of modified MCP-2 forms. The chemotactic activity of intact, natural (nat) and synthetic (syn) MCP-2(1-76), amino-terminally truncated, natural MCP-2(6-76), and carboxy-terminally truncated synthetic MCP-2(1-74) was tested on THP-1 cells. The results are the mean Cl ±SEM of four or more independent experiments. Figure 4 shows that native MCP-2 is a weaker agonist than MCP-1 in calcium mobilization in monocytes. Intact MCP-2 (15, 50, and 150 ng / ml) dose-dependently increases [Ca2+]i in THP-1 cells. Representative results from one of two experiments are shown. The following are examples. Example 1: MCP-2 truncated at its amino terminus Materials and methods Chemoklin- and immunoassay MCP-2 was synthesized and purified as described above (Proost, P. et al., Cytokine 7, 97-104 (1995)). The specific anti-human MCP-2 antibody was prepared from mouse and affinity purified on a Sepharose column to which synthetic MCP-2 was bound under the conditions recommended by the manufacturer (CNBr activated Sepharose-4B, Pharmacia, Uppsala, Sweden). ELISA plates were coated with affinity purified human anti-MCP-2 antibody and biotinylated anti-MCP-2 was used to bind the antibody. Detection was performed with peroxidase-labeled streptavidin and TMB. The detection level in the MCP-2 ELISA was approximately 0.1 ng / ml. Production and purification of MCP-2 Monocyte chemotactic proteins were purified from conditioned medium derived from peripheral blood mononuclear cells from 132 blood products obtained from the Antwerp and Leuven Blood Transfusion Centers [Proost, P. et al., Methods: A companion to Methods in Enzymol. 10, 82 (1996)]. Red blood cells and granulocytes were removed by sedimentation in hydroxyethyl starch (Fresenius AG, Bad Homburg, Germany) and gradient centrifugation in sodium metrizoate solution (Lymphoprep; Nyegaard, Oslo Norway). Mononuclear cells (60x109 cells) (5x106 cells / ml) were incubated with 10 pg / ml ConA and 2 pg / ml LPS. The conditioned medium was collected after 48-120 hours and kept at -20 °C until purification. HU 226 468 B1 Native MCP-2 was purified using the four-step purification procedure described above (Proost, P. et al., Methods: A companion to Methods in Enzymol. 10, 82 (1996)). Briefly, the conditioned medium was concentrated on a controlled pore size glass filter or silica and partially purified by affinity chromatography on a heparin Sepharase column (Pharmacia). Fractions with MCP-2 immunological activity were further purified by Mono S (Pharmacia) cation exchange chromatography and eluted with a sodium chloride 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 proteins were eluted with an acetonitrile gradient. Biochemical characterization of MCP forms by SDS-PAGE, amino acid sequencing and mass spectrometry The purity of the column fractions was checked by SDS-PAGE under reducing conditions on a Tris / tricine gel [Proost, P. et al., Methods: A companion to Methods in Enzymol. 10, 82 (1996)]. The proteins were stained with silver and the following relative molecular markers (Mr) were used: OVA (Mr 45000), carbonic anhydrase (Mr 31000), soybean-derived trypsin inhibitor (Mr 21500), β-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 fluid 477A / 120A protein sequencer (Perkin-Elmer) using N-methylpiperidine as the linker base. The protected proteins were cleaved between aspartic acid and proline in 75% formic acid for 50 h. The formic acid digest was sequenced without further purification. The Mr value of MCP-2 was determined using matrix-assisted laser desorption ionization / time of flight-mass spectrometry, MALDI / TOF-MS-spectrometry (Micromass TofSpec, Manchester, UK). Alphacyano-hydroxycinnamic acid and cytochrome C were used as matrix and internal standards, respectively. Detection of chemotactic activity The chemotactic ability of MCP-2 was measured on freshly purified monocytes (2*10® cells / ml) or monocytic THP-1 cells (0.5*10® cells / ml, 2 days after subculture) in a Boyden microchamber using 5 μιτι pore size polycarbonate membranes treated with poly(vinylpyrrolidone). 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 at 37 °C for 2 hours, cells were fixed and stained with Diff-Quick stain solution (Harleco, Gibbstown, NJ) and cells migrating through the membranes were counted microscopically in ten oil immersion fields at 500x magnification. The chemotactic index (Cl) of the samples was measured in triplicate in each chamber. - the number of cells migrating towards the sample was calculated as the excess of the number of cells migrating towards the control medium [Van Damme, J. et al., J. Exp. Med. 176, 59(1992)]. In desensitization experiments, cells were incubated with a biologically inactive chemokine variant for 10 min at 37 °C before being added to the upper well of a Boyden microchamber. The percentage inhibition of Cl was calculated using the chemotactic index of HBSS-treated cells towards the sample as a reference value. Detection of intracellular Ca2+ concentration Intracellular calcium concentrations ([Ca2t]) were measured as previously described [Wuyts, A. et al., Biochemistry 36, 2716-2723 (1997)]. Purified monocytes or THP-1 cells (107 cells / ml) were incubated in medium containing Eagle's Minimum Essential Medium (EMEM, Gibco) + 0.5% FCS with fura-2 fluorescent indicator (fura / 2AM 2.5 μιτιοΙ, Molecular Probes Europe BV, Leiden, The Netherlands) and 0.01% Pluronic F-127 (Sigma, St Louis MO). After incubation at 37 °C for 30 min, the cells were washed twice and resuspended at a concentration of 10® cells / ml in HBSS containing 1 mM Ca2+ and 0.1% FCS (buffered to pH 7.4 with 10 mM HEPES / NaOH). The cells were equilibrated at 37 °C for 10 min before fura-2 fluorescence was measured using an LS50B luminospectrophotometer (Perkin-Elmer). Fluorescence was measured at 510 nm upon excitation at 340 and 380 nm. [Ca2+]r was calculated from the Grynkiewicz equation [Grynkiewicz et al., J. Biol. Chem. 260, 3440 (1985)]. To determine Rmgx, cells were digested with 50 pmol digitonin. The pH was then adjusted to 8.5 with 20 mM Tris and R^m was obtained by adding 10 mM EGTA to the digested cells. The K( / 224 nmol. In desensitization experiments, monocytes or THP-1 cells were first stimulated with buffer, chemokine, or various concentrations of chemokine antagonist. As a second stimulus, MCP-2 was applied at a concentration that induced a significant increase in [Ca2+]j after pre-stimulation with buffer. The second stimulus was applied 2 min after the addition of the first stimulus. The percent inhibition of the increase in [Ca2+]j to the second stimulus was calculated by comparing the signal after pre-stimulation with chemokine or chemokine antagonist with the signal after addition of buffer. Results Isolation of post-translationally modified MCP-2 forms A specific and sensitive ELISA method was used to detect different forms of MCP-2 produced by peripheral blood mononuclear cells stimulated with mitogen and endotoxin. The ?(10.o / 22.s) conditioning medium was purified according to the conventional isolation procedure [Proost, P. et al., Methods: A companion to Methods in Enzymol. 10, 82 (1996)], including controlled poly HU 226 468 Β1 by binding to a pore-sized glass filter and heparin Sepharose chromatography. Subsequently, FPLC Mono S cation exchange chromatography purification was performed, followed by a subsequent purification step using C-8 RP HPLC. Molecular weights were determined by SDS-PAGE and MALDI / TOFMS. Different forms of MCP-2 were isolated: in addition to the original 7.5 kDa MCP-2(1-76), a 7 kDa MCP-2 form truncated at its amino terminus, lacking five amino acids [MCP-2(6-76), was purified to homogeneity by RP-HPLC and identified by amino acid sequence analysis (Figure 2). MALDI / TOF-MS (Table I) yielded a molecular weight of 8881 Da for intact MCP-2 (theoretical Mr 8893 Da), while a molecular weight of 8365 Da was measured for MCP-2(6-76), confirming the absence of the five amino-terminal amino acids (theoretical Mr 8384 Da). Functional comparison of these natural MCP-2 forms in a THP-1 chemotaxis assay revealed that intact MCP-2 was active at concentrations as low as 5 ng / ml, while truncated MCP-2(6-76) showed no chemotactic activity over a concentration range of 0.6-60 ng / ml (Figure 3). Intact, natural MCP-2 was compared with synthetic MCP-2(1-76) and a carboxy-terminally truncated synthetic form [Proost, P.et al., Cytokine 7, 97-104 (1995)], in which two amino acids are missing [MCP-2(1-74)]. The minimally effective chemotactic concentration of these forms was found to be 5 ng / ml (Figure 3). In the chemotactic assay, the specific activities of native, intact MCP-1 and MCP-2 were comparable [Van Damme, J. et al., J. Exp. Med. 176, 59 (1992)], but the calcium mobilization capacity of MCP-2 is still questionable. In calcium mobilization experiments, the minimally effective dose of both natural and synthetic MCP-2(1-76) was tenfold higher than that of natural, intact MCP-1(1-76) (Figure 4), while MCP-2(6-76) remained inactive. However, intact MCP-2 (50 ng / ml) was able to desensitize to MCP-2 (15 ng / ml) and MCP-3 (10 ng / ml), representing 52% and 45% inhibition of chemotaxis, respectively. Because of the low specific activity of MCP-2 in calcium assays, desensitization of MCP-2(6-76) chemotaxis was performed in a Boyden microchamber. Since intact MCP-2 has been reported to cross-desensitize with active MCP-1, MCP-2, and MCP-3 in the monocyte chemotaxis assay [Sozzani, S. et al., J. Immunol. 152, 3615 (1994)], we investigated whether native, inactive MCP-2(6-76) could also desensitize to MCP-1, MCP-2, MCP-3, and RANTES (Table II). Preincubation of THP-1 cells with 100 ng / ml inactive MCP-2(6-76) significantly inhibited chemotaxis induced by 10 ng / ml MCP-1 (63%), 5 ng / ml MCP-2 (75%), 30 ng / ml MCP-3 (62%) and 100 ng / ml RANTES (75%). Furthermore, chemotaxis induced by three times lower concentrations of the respective MCPs was completely (91-100%) inhibited by 100 ng / ml MCP-2(6-76).Furthermore, MCP-2(6-76) was able to significantly inhibit the chemotactic activity induced by MCP-1 (3 ng / ml), MCP-2 (1.5 ng / ml) or MCP-3 (10 ng / ml) and RANTES (30 ng / ml) even at a concentration of 10 ng / ml. In summary, MCP-2(6-76) is naturally occurring, inactive as a chemoattractant, and antagonizes several CC chemokines, most notably MCP-3. Table / Biochemical characterization of natural forms of MCP-2. Amino-terminal amino acid sequence analysis of natural MCP isoforms purified by C-8 RP-HPLC and comparison of experimental (SDS-PAGE and MALDI / TOF-MS) and theoretical Mr values MCP form Aminoterminal sequence Mr(Da) theoretically unglycosylated SDS-PAGE MALDI / TOF-MS MCP-2(1-76) protected 8893 7500 8881 MCP-2(2-76) SIPITCC 8384 7000 8365 Table II MCP-2(6-76) desensitizes the monocyte chemotactic response to MCP-1, MCP-2, MCP-3 and RANTES in a microchamber Chemokine3 Concentration Antagonism of the chemotactic responseb>c Percentage inhibition of chemotaxis buffer 100 ng / ml MCP-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 HU 226 468 Table Β1 / / (continued) Chemokine3 Concentration Antagonism of the chemotactic responseb·c Percentage inhibition of chemotaxis buffer 100 ng / ml MCP-2(6-76) MCP-2 5 36.0112.6 10.816.1 7518.0 1.5 6.711.4 1.510.3 9117.0 MCP-3 30 13.210.4 6.014.0 62131 10 3.OH ,5 <1 10010.0 RANTES 100 6.310.8 2.611.3 75119 30 4.0l0.8 1.510.3 77116 buffer 10 ng / ml MCP-2(6-7 6) MCP-1 10 12.712.3 10,513.8 2411.8 3 7,510.0 3,010.3 6914.0 MCP-2 5 38,015.3 27,214.9 3016.0 1.5 18,314.6 9,211.4 45123 MCP-3 30 13,211.9 8,011.0 37119 10 7,711.4 1,710.3 9016.0 RANTES 100 5,510.6 5.8l0.9 1717.0 30 3,210.7 2,510.5 39118 3MCP-1, MCP-2, MCP-3 or RANTES were added as chemoattractants to the lower wells.bThe upper wells of the microchamber were filled with THP-1 cells preincubated with MCP-2(6-76) or buffer.cMean Cl values ​​±SEM of three independent experiments. References Baggiolini M. et al., Ann. Port. Immunoi., 55, 97-179, 1994. 35 Baggiolini M. et al., Ann. Port. Immunol., 15, 675-705, 1997. Chang HC et al., International Immunology, 1(4), 388-397, 1989. Clark-Lewis I. et al., J. Bioi. Chem., 266, 40 23 128-23 134, 1991. De Meester I. et al., J. Immunol. Methods 189, 99-10 526, 1996. Deng H. etal., Naturé., 381, 661-666, 1996. Gong J et al. J. Exp. Med., 181, 631-640, 1995. 45 Gong J. et al., J. Biol. Chem. 271, 10 521-10 527, 1996. Grynkiewicz G. et al., J. Biol. Chem., 260, 3440, 1985. Proost P. et al., Biochemistry, 32, 10 170-10 177, 50 1993a. Proost P. et al., J. Immunoi., 150, 1000-1010, 1993. Proost P. et al., Cytokine, 7, 97-104, 1995. Proost P. et al., Methods: A companion to Methods in Enzymol., 10, 82, 1996. Proudfoot AE et al., J. Bioi. Chem., 271, 2599-2603, 1996. 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, 1383-1388, 1997. Sozzani S. et al., J. Immunoi., 152, 3615, 1994. Taub D. et al., Cytokine & Growth Factor Reviews, 7, 335-76, 1996. Van Coillie E. et al., Biochem. Biophys, Rés. Commun., 231, 726-730, 1997. Van Damme J. et al., Eur. J. Biochem., 181, 337-344, 1989. Van Damme J. et al., Eur. J. Immunoi., 20, 2113-8, 1990. Van Damme J. et al., J. Exp. Med., 176, 59, 1992. Walz A. et al., Biochem. Biophys, Rés. Commun., 159, 969-75, 1989. Wuyts A., et al., Biochemistry 36, 2716-2723, 1997. SEQUENTIALIST DATA FOR SEQUENCING IDENTIFICATION NUMBER 1: SEQUENCE CHARACTERISTICS: LENGTH: 99 amino acids Type: amino acid HOW MANY THREADS: HU 226 468 B1 TOPOLOGY: linear MOLECULAR: protein HYPOTHETICAL: no FEATURES: NAME / DESIGNATION: protein LOCATION: 1...76 DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 1: Met Lys Val Ser -20 Down Down Leu Leu Cys -15 Leu Leu Leu Met Down -10 Down Thr Phe Ser Pro -5 Gin Gly Leu Down Gin 1 Pro Asp Ser Val 5 Ser Ile Pro Ile Thr 10 Cys Cys Phe Asn Val 15 Ile Asn Arg Lys Ile 20 Pro Ile Gin Arg Leu 25 Glu Ser Tyr Thr Arg 30 Ile Thr Asn Ile Gin 35 Cys Pro Lys Glu Down 40 Val Ile Phe Lys Thr 45 Lys Arg Gly Lys Glu 50 Val Cys Under Asp Pro 55 Lys Glu Arg Trp Val 60 Arg Asp Ser Met Lys 65 His Leu Asp Gin Ile 70 Phe Gin Asn Leu Lys 75 Pro DATA OF SEQUENCE IDENTIFICATION NUMBER 2: SEQUENCE CHARACTERISTICS: LENGTH: 99 amino acids TYPE: amino acid NUMBER OF STRANDS: TOPOLOGY: linear MOLECULAR: protein HYPOTHETICAL: no PROPERTIES: NAME / DESIGNATION: protein LOCATION: 1...76 DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 2: Met Lys Val Ser -20 Down Down Leu Leu Cys -15 Leu Leu Leu Met Down -10 Down Thr Phe Ser Pro -5 Gin Gly Leu Down Gin 1 Pro Asp Ser Val 5 Ser Ile Pro Ile Thr 10 Cys Cys Phe Asn Val 15 Ile Asn Arg Lys Ile 20 Pro Ile Gin Arg Leu 25 Glu Ser Tyr Thr Arg 30 Ile Thr Asn Ile Gin 35 Cys Pro Lys Glu Down 40 Val Ile Phe Lys Thr Gin Arg Gly Lys Glu Val Cys Under Asp Pro Lys Glu 50 55 HU 226 468 B1 Arg Trp Val Arg Asp Ser Met Lys His Leu Asp Gin Ile Phe Gin Asn 60 65 70 Leu Lys Pro 75 DATA OF SEQUENCE IDENTIFICATION NUMBER 3: SEQUENCE CHARACTERISTICS: LENGTH: 71 amino acids Type: amino acid HOW MANY THREADS: TOPOLOGY: linear MOLECULE TYPE: protein HYPOTHETICAL: no DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 3: Ser 1 Ile Pro Ile Thr 5 Cys Cys Phe Asn Val 10 Ile Asn Arg Lys Ile 15 Pro Ile Gin Arg Leu Glu Ser Tyr Thr Arg Ile Thr Asn Ile Gin Cys Pro 20 25 30 Lys Glu Under Val Ile Phe Lys Thr Lys Arg Gly Lys Glu Val Cys Under 35 40 45 Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met Lys Leu His Asp Gin 50 55 60 Ile Phe Gin Asn Leu Lys Pro 65 70 DATA OF SEQUENCE IDENTIFICATION NUMBER 4: SEQUENCE CHARACTERISTICS: LENGTH: 71 amino acids TYPE: amino acid NUMBER OF STRANDS: TOPOLOGY: linear MOLECULAR: protein HYPOTHETICAL: no DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 4: Ser 1 Ile Pro Ile Thr Cys 5 Cys Phe Asn Val 10 Ile Asn Arg Lys Ile 15 Pro Ile Gin Arg Leu Glu Ser Tyr Thr Arg Ile Thr Asn Ile Gin Cys Pro 20 25 30 Lys Glu Under Val Ile Phe Lys Thr Gin Arg Gly Lys Glu Val Cys Under 35 40 45 Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met Lys Leu His Asp Gin 50 55 60 Ile Phe Gin Asn Leu Lys Pro 65 70 HU 226 468 B1

Claims

CLAIMS 1. An amino-terminally truncated human monocyte chemotactic protein 2 (MCP-2) having chemokine antagonist activity and lacking the amino-terminal amino acids corresponding to amino acids 1-5 of naturally occurring human MCP-2.

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

4.

3. The amino-terminally truncated MCP-2 of claim 1 or 2, which is in a glycosylated form.

4. A DNA molecule according to any one of claims 1-3, comprising DNA sequences encoding MCP-2 truncated from its amino terminus, or a nucleotide sequence substantially identical thereto.

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 a protein according to any one of claims 1-3, characterized in that the cells according to claim 6 are cultured in a suitable culture medium.

8. A protein according to any one of claims 1-3 for use as a medicament.

9. Use of a protein according to any one of claims 1-3 in the manufacture of a medicament for the therapy and / or diagnosis of diseases requiring antagonist activity of chemokine effects.

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

11. A pharmaceutical composition comprising a protein according to any one of claims 1-3 in combination with one or more pharmaceutically acceptable carriers and / or excipients.