BG102413A - Shortened forms of hemokin beta-8 - Google Patents

Abstract

The invention relates to new polypeptides of the shortenedform of human Ck -8 and to methods for the use of polypeptides.15 claims

Description

TECHNICAL FIELD

This invention relates to a polypeptide and polynucleotide sequences encoding a truncated form of chemokine beta-8 (Skr-8). Specifically, the present invention relates to the use of polynucleotide and polypeptide sequences "for the preparation of polynucleotide and polypeptide sequences and to inhibit the action of the polypeptide. Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are small, inducible, inflammatory molecules. Cytokines generally show 20% to 75% homology at amino acid levels and are characterized by four conserved cysteine residues that form two disulfide bridges. Based on the arrangement of the first two cysteine residues, the chemokines were classified into two subfamilies, alpha and beta. In the alpha subfamily, the first two cysteines are separated by one amino acid and are therefore referred to as the C-X-C subfamily. " In the subfamily beta, the two cysteines are adjacent and are therefore referred to as subfamily CC. The first cysteine forms a disulfide bridge with the third cysteine, and the second with the fourth, which results in the formation of a similar tertiary structure for many of the chemokines. For getting functional, mature

protein most chemokines undergo structural proteolytic processing. This usually involves the removal of a short leader (leading) sequence at the N-terminal region of the protein. At least 14 different α-chemokines and 12 β-chemokines have been described at the protein and / or DNA level.

Intercrine cytokines exhibit a glamorous variety of functions.

One important feature is their ability to stimulate the migration by chemotaxis of various cell types, such as monocytes, neutrophils, Tymphocytes, basophils and fibroblasts. The subfamilies a and β differ slightly in their specificity for the target cells. Most of the α-chemokines attract neutrophils, fibroblasts, T cells and NK cells. β-chemokines attract mainly monocytes and T-lymphocytes. Many chemokines have pro-inflammatory activity and are involved in numerous steps during the inflammatory response. These activities include stimulation of histamine secretion, release of lysosomal enzyme and leukotriene, increased adhesion of target immune cells to endothelial cells, enhanced complement protein binding, induction of expression of granulocyte adhesive molecules and complement receptors, as well as receptors. In addition to their involvement in inflammation, some chemokines have been shown to exhibit other functions. For example, macrophage inflammatory protein (MIP-1) is able to suppress hematopoietic stem cell proliferation, and platelet-factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL- 8) induces keratinocyte proliferation, and GRO is an autocrine growth factor for melanoma cells. Chemokines are involved in a number of physiological and disease states, and in particular those that represent the inflammatory component. These include, but are not limited to: lymphocyte trafficking, wound healing, regulation of hematopoiesis, and immunological diseases such as allergy, asthma and arthritis. Many cytokines were initially isolated as products of inflammatory response. For example, MIP-1 was isolated initially as a proinflammatory cytokine induced by

endotoxins that are produced by macrophages. Similarly, other members of this subfamily have been identified on the basis of induced inflammation and homology in amino acid sequences.

This includes full-length as well as truncated forms of the Skr ~ 8 polypeptide of the present invention.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided a mixture of novel polypeptides representing truncated forms of human Skr-8, as well as fragments, analogs and derivatives thereof, which are biologically active and useful in diagnostics and therapy.

According to another aspect of the present invention, isolated nucleic acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA, as well as fragments, analogs and derivatives thereof, are biologically active and useful in diagnosis and therapy.

According to a further aspect of this invention there is provided a method of producing such polypeptides by recombinant techniques that include the cultivation of recombinant prokaryotic and / or eukaryotic host cells containing nucleic acid sequences

......; ..: ...., under conditions conducive to protein expression and subsequent protein isolation.

According to another further aspect of the present invention there is provided a method of using such polypeptides or polynucleotides encoding such polypeptides for therapeutic purposes, for example to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy and to stimulate wound healing.

According to another aspect of the present invention are provided

antibodies against such a polypeptide.

According to a further aspect of the present invention, antagonists of such a polypeptide are provided that can be used to inhibit the action of such a polypeptide, for example, for the treatment of aplastic anemia, myelodysplastic syndrome, asthma and arthritis.

According to another aspect of the present invention, nucleic acid probes are also provided, comprising nucleic acid molecules of a length sufficient to hybridize specifically with the nucleotide sequence of Skr-8.

According to a further aspect of the present invention, diagnostic tests are provided to detect diseases associated with insufficient

expression or overexpression of the polypeptide and for the detection of mutations in the nucleotide sequences encoding such polypeptides.

According to another further aspect of the present invention there is provided a method of using such a polypeptide or polynucleotides encoding such a polypeptide, such as in vitro research reagents, DNA synthesis and the production of DNA vectors, for the development of therapy and diagnostics for treatment of human disease.

These and other aspects of the present invention will become apparent to those skilled in the art from the following description.

DETAILED DESCRIPTION OF THE INVENTION

DNA sequences encoding polypeptides related to the superfamily of pro-inflammatory intercrine chemokines have been described. The polynucleotide sequence encoding full-length Skr-8 (120 amino acids) was obtained from the aortic endothelial cDNA library. The present invention relates to novel truncated forms of Ο <β-8 containing only 82, 76, 75 or 74 long-form C-terminal amino acids. The N-terminal amino acids as well as the signal peptide of 21 residues were removed. As well

in the long C1F-8 form, the first two cysteine residues in these clones are in adjacent positions, which places them in the cytokine C-C or beta subfamily.

The presence of this motif distinguishes them from the cytokine a (“CXC” subfamily, in which the first two cysteine residues are separated by one amino acid.

Polynucleotide sequences encoding mature polypeptides (SEQ ID NOs: 1, 2, 3, and 4) can be represented in the form of any nucleic acid, such as RNA, single stranded (ss) DNA, genomic DNA, etc., including additional coding and non-coding sequences associated with the polypeptide. Furthermore, as a result of the degeneracy of the amino acid code, any DNA coding sequence that encodes the same mature polypeptides will fall within the scope of this invention. All variants of this sequence, allelic or non-naturally occurring, will also be included. The present invention also includes a sequence of linked polynucleotides encoding in the same reading frame with another DNA sequence that promotes the expression or secretion of a protein from a cell or serves as a marker for the identification of a protein in cells (e.g., the HA tag).

Further, this invention relates to polynucleotide sequences that hybridize to the desired sequence with sequence identities of at least 50%, and preferably 70%. These polynucleotides encode polypeptides that are preferred to retain the same biological function or activity as the mature polypeptide. Alternatively, the sequence may be represented by polynucleotides, which preferably have 50 bases that are identical or hybridize to the sequences of the present invention but do not retain their activity. Such sequences can be used as a probe or primer for PCR (polymerase chain reaction).

The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides. Derivatives of these polypeptides may be those in which one or more amino acid substitutions are present; those in which one or more amino acids contain substituent groups; those in which the mature protein is fused to another compound; or in which additional amino acids are fused to the mature protein.

Preferably, the polypeptides of the present invention are provided in isolated form and are preferably purified to homogeneity.

The present invention also relates to vectors that include polynucleotides of the present invention, host cells obtained with this vector by genetic engineering, and to the preparation of polypeptides of the present invention by recombinant techniques. The host cells are constructed with the vectors of this invention (cloning and expression). Specialists are familiar with the culture conditions of these cells required for activation of promoters, selection of transformants, and amplification of the truncated Skr-8 gene.

Polynucleotide sequences can be incorporated into any of many expression vectors insofar as they can replicate and survive in the host. The polynucleotide sequence included in the expression vector is controlled by a suitable promoter that directs mRNA synthesis (e.g., the LTR promoter). The expression vector also contains a binding site for translation initiation ribosomes, a transcription terminator, enhancer (enhancer) elements that enhance transcription (e.g., the late SV40 inhaler), and corresponding selection markers that provide retention of the vector in the host. e.g., ampicillin resistance gene). The host cells may be cells of higher eukaryotes such as mammalian or insect cells, cells of lower eukaryotes such as yeast, and prokaryotic cells such as bacteria. The introduction of the structure into the host cell can be accomplished by different protocols. All the above mentioned steps are known to those skilled in the art. Both the cloning steps and the selection of hosts and vectors are well known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Editon, Cold Spring Harbor, N.Y., (1989).

Proteins can be synthesized in the host cell under the control of a suitable promoter. For the production of such proteins, extracellular (/ h vitro) translation systems can also be used using RNAs derived from the DNA constructs of the present invention. Alternatively, the polypeptide of the present invention can be synthetically produced by conventional peptide synthesizers.

After transformation or transfection of a suitable host and culturing the host strain to the appropriate cell density, the selected promoter is induced in appropriate ways (e.g., temperature change or chemical induction) and the cells are further cultured. Cells are typically harvested by centrifugation, destroyed by physical or chemical means, and purified to homogeneity by various methods (e.g., chromatography, HPLC). Depending on the host used, the polypeptide may be modified in any way (e.g., glycosylated). The polypeptides of the invention may also include an initial methionine residue.

The long form of Skr-8 is a chemoattractant for leukocytes and can therefore be used in various immunoregulatory and inflammatory functions, as well as in a number of disease states. The protein is mainly expressed in tissues of hematopoietic origin. One important biological effect of the Skr-8 polypeptide on leukocytes is to stimulate the mobilization of Ca ⁺⁺ reserves. This mobilization is inherent in the functional activation of the cell. Stimulation of different EOL-3 cells with a mixture containing four truncated forms of SkP-8 (SEQ ID NOS: 1-4) was found to result in immediate dose-dependent increase in intracellular Ca ⁺⁺ . In individual testing, the form with SEQ ID NO was the most active. 4. In contrast, the long form of Skr-8 was approximately 1000 times less active than the truncated Ca ⁺⁺ mobilization forms. The high activity of the truncated forms of the present invention can effectively render the therapeutic use of this chemokine including, but not limited to, the prevention of bone marrow stem cells from chemotherapy, the removal of leukemic cells, stimulation of the immune response, regulation of heme and lymphocytic trafficking, treatment of psoriasis, solid tumors, enhancement of host host defense against resistant chronic or acute infections, and stimulating the healing of pa us.

The polynucleotides and polypeptides of the present invention can be used as research reagents, for DNA synthesis and the production of DNA vectors, and for the purposes of therapy and diagnosis in the treatment of human disease (e.g., in the expansion of immature hematopoietic parental cells). fragments of this truncated Skr-8 polynucleotide sequence can also be used as a hybridization probe to isolate other genes that are very similar in sequence. The techniques for this type of experimentation are well known to those skilled in the art.

This invention also relates to the use of these sequences as part of a diagnostic test for the detection of diseases or the susceptibility to diseases associated with the presence of mutations in the nucleotide sequences. Such diseases are associated with insufficient expression of chemokine polypeptides. For example, individuals carrying mutations in the Skr-8 gene can be detected at the DNA level by various techniques such as PCR. Genetic testing based on differences in DNA sequence can be achieved by detecting changes in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Detection of a specific DNA sequence can be achieved by methods such as hybridization, PHase protection, chemical degradation, direct DNA sequencing, RFLP (restriction fragment length polymorphism) analysis and genomic DNA transfer (Southern blotting). Mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic test for detecting altered levels of Skr-8 protein in different tissues, in that the overexpression of proteins compared to samples from normal control tissues can detect the presence of a disease or a predisposition to disease, for example. The tests used to determine the levels of Skr-8 protein in a host sample are well known to those skilled in the art and include radioimmunoassay, competitive binding assays, Western blot analysis, ELISA (enzyme- linked immunosorbent assay) and sandwich assays.

This invention provides a method for identifying receptors for chemokine polypeptides. The receptor encoding gene can be identified by various methods known to those skilled in the art, including panning of ligands and sorting by FACS (fluorescence activated cell sorting).

An alternative approach for the identification of receptors involves the photo affinity binding of labeled polypeptides to the cell membrane or the extraction of preparations that express the receptor molecule. The labeled complex can be isolated and subjected to protein microsequencing. The resulting amino acid sequence could be used to design a set of degenerate oligonucleotide probes to screen a cDNA library for identification of genes encoding putative receptors.

This invention provides a method of screening compounds for the purpose of identifying agonists and antagonists of the chemokine polypeptide of the present invention. Chemotaxis can be tested by placing cells that are chemically attracted by these polypeptides onto the top of a filter with pores large enough to pass the cells (5 mm). Solutions of potential agonists or antagonists are placed in the lower chamber so that cells migrate or are prevented from migrating through the membrane for a period of time. Alternatively, the C0-8 receptors could be incubated with a labeled polypeptide in the presence of a compound. The ability of a compound to block this interaction or to interact with the receptor in the absence of a polypeptide could be measured.

Examples of potential truncated Skr-8 antagonists include antibodies, oligonucleotides that bind to polypeptides, or polypeptides that bind to the wild-type polypeptide receptor but have no biological activity. Antisense technology used to control gene expression could also be a potential antagonist. The suitability of the aforementioned technologies should be apparent to those skilled in the art.

Antagonists can be used to treat infectious diseases such as silicosis, sarcoidosis, idiopathic pulmonary fibrosis, idiopathic hyper-eosinophilic syndrome and endotoxic shock - all

I by suspending the synthesis of the polypeptide of the present invention. Antagonists can also be used to treat atherosclerosis by stopping infiltration of monocytes into the arterial wall. The antagonists can be used to treat allergic reactions mediated by histamine and various immunological disorders affecting the dermis.

Antagonists may also be used to treat bone marrow failure in aplastic anemia or myelodysplastic syndrome. They can also be used to treat asthma and allergy, as well as fibrosis of the underlying basement membrane, which is characteristic of the asthmatic lung.

Chemokine polypeptides, agonists and antagonists may be used with a suitable pharmaceutical carrier, such as saline, buffered saline, dextrose, water, glycerol, ethanol, or combinations thereof. The composition must be appropriate to the mode of administration. The invention also provides a pharmaceutical kit or kit comprising one or more packaging filled with one or more of the ingredients of the pharmaceutical compositions of this invention.

pharmaceutical preparations may be suitably administered

such as by local, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal or intradermal routes in an amount effective to treat a specific indication. These applications should be apparent to those skilled in the art.

Chemokine polypeptides, agonists or anatagonists that are polypeptides can be used in accordance with the present invention by expressing such polypeptides in vivo. This type of gene therapy is well known in the art. For example, patient cells may be manipulated ex vivo with DNA or RNA encoding truncated forms of the C0-8 polypeptide, and the manipulated cells expressing these polypeptides may be introduced into the patient. Similarly, it is possible for cells to be manipulated in vivo for expression of the polypeptide in vivo.

As is known in the art, it is possible to use retroviral particles containing RNA encoding the polypeptide of the present invention. Retroviral plasmid vectors are used to transduce packaging cell lines (e.g., PE501) through various methods (e.g., electroporation) to form producing cell lines. In a preferred embodiment, the retroviral expression vector comprising

the polynucleotide sequence is under the control of a promoter, such as LTR (long terminal repeat) and contains a marker for drug resistance selection (e.g., peo). The preparation of these vectors and derived cell lines should be known to those skilled in the art, as will be apparent from the techniques contained herein. The producing cell line forms infectious particles from the retroviral vector that contain the nucleotide sequence (s) encoding the polypeptides. Such retroviral vector particles can then be used to transduce eukaryotic cells, for example, fibroblasts or endothelial cells, both in vitro and in vivo. The transduced eukaryotic cells will then express the nucleotide sequence (s) encoding the protein.

The sequences of the present invention are also valuable for the identification of chromosomes. The sequences target specifically and can hybridize at a specific locus to the individual chromosome. Mapping DNA into chromosomes is an important step in finding a correlation between genes and a disease. One technique known to those skilled in the art involves the preparation of primers used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids that contain the human gene corresponding to the primer will give an amplified fragment. Other mapping strategies known to those skilled in the art include, but are not limited to: hybridization in situ, screening with flow-sorted chromosomes, sublocalization into fragments of specific chromosomes using the same primers, and selection through hybridization to construct cDNA libraries specific to chromosomes. For precise chromosomal localization in one step, fluorescence in situ hybridization (FISH) of scattered metaphase cDNA clones can be used

chromosomes.

Once a sequence is mapped to a precise chromosomal locus, its physical location can be correlated with genetic mapping data. The link between genes and diseases can be identified by a linkage analysis. Any difference in cDNA or genomic sequences between affected and unaffected individuals may be an indication of disease. For example, a mutation in DNA that is found only in the affected individuals may be the cause of the disease.

The polypeptides, their fragments or other derivatives or their expressing cells can be used as an immunogen to produce antibodies against them. These antibodies may be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single-stranded, and humanized antibodies, as well as Fab fragments or the product of a library expressing Fab. Various methods known in the art can be used to prepare such antibodies or fragments. Polyclonal antibodies prepared by standard methods can be used to isolate polypeptides from tissues expressing these polypeptides. For the production of monoclonal antibodies, any technique that provides the production of antibodies from continuous cell line cultures may be used. The techniques described for the preparation of single-chain antibodies can be adapted to produce single-chain antibodies against the immunogenic peptides of this invention. Transgenic mice may also be used to express humanized antibodies against immunogenic polypeptides of this invention.

The present invention will be described below with reference to the following examples; however, it should be understood that the present invention is not limited to such examples. In order to facilitate the understanding of the following examples, some commonly encountered methods and / or terms will be briefly described.

The starting plasmids can either be purchased, or are generally available without restriction, or can be constructed from accessible plasmids in accordance with published methods. In addition, plasmids equivalent to those described are known in the art and are known to those of ordinary skill in the art.

The various restriction enzymes used to digest DNA are commercially available, and their reaction conditions, cofactors and other requirements should be known to those of ordinary skill in the art. Separation of the degraded fragments by size is performed using an 8% polyacrylamide gel.

Oligonucleotides are either single-stranded polydeoxynucleotides or two complementary polydeoxynucleotide chains that can be chemically synthesized. Such synthetic oligonucleotides do not have 5'-phosphate and will therefore not be ligated with another oligonucleotide without the addition of phosphate with ATP (adenosine triphosphate) in the presence of kinase. A synthetic oligonucleotide will be ligated with a fragment that has not been dephosphorylated. The transformation is performed unless otherwise noted, as described in the method of Graham, F. and Van der Eb, A., Virology 1973, 52, 465-457.

The following examples are for illustrative purposes only and are not intended to limit the invention.

EXAMPLES FOR THE IMPLEMENTATION OF THE INVENTION

Example 1: Bacterial Expression and Purification of Ck3-b

The DNA sequence encoding Ckp-8, ATCC # 75676, was initially amplified by using PCR primers corresponding to the 5'- and 3'-terminal sequences of the processed Skr-8 protein (minus the signal peptide sequence) and vector sequences. from the 3'-end of the Skr-8 gene. The 5'-oligonucleotide primer has the sequence 5'-TCAGGATCCGTCACAAAAGATGCAGA-3 '(SEQ ID NO: 5) and the 3'-primer has the sequence 5'CGCTCTAGAGTAAAACGACGGCCAGT-3' (SEQ ID NO: 6). These primers contain, respectively, restriction sites for BamHl and Xaa], which are used to clone in the polylinker region of the ampicillin resistant bacterial expression vector PQE-9 (Qiagen, Inc., Chatsworth, CA). The amplified sequences were ligated into PQE-9 in a frame with the sequence encoding the histidine tag and the ribosome binding site (RBS). Ligation was used to transform £ coli strain M15 / rep 4 (Qiagen) containing multiple copies of plasmid pREP4. pREP4 expresses the lacl repressor and also confers kanamycin resistance. Transformants are selected for their ability to grow on LB plates (Luria / Bertani culture medium) with ampicillin / kanamycin added. Clones containing the desired construct (isolated and confirmed by restriction assay) were cultured overnight in liquid LB medium to which both Amp (100 μg / ml) and Cap (25 μg / ml) were added. This culture is used to inoculate a large culture at a ratio of 1: 100 to 1: 250. Cells were cultured to an optical density of 600 between 0.4 and 0.6. IPTG (isopropyl-B-O-thiogalacto-pyranoside) was then added to a final concentration of 1 mM. The cells were then cultured for an additional 3 to 4 hours, then harvested by centrifugation. The cell pellet was dissolved in the chaotropic substance 6 M guanidine HCl, lysed and chromatographed on a nickel-chelate column under conditions that allowed the tight binding of proteins containing the 6-His plug (Hochuli, E. etal. J. Chromatography 1984 , 411,177-184). The truncated Ckp-8 (95% pure) was eluted from the column in 6 M guanidine HCl pH 5 and for renaturation purposes was brought to 3 M guanidine HCI, 100 mM sodium phosphate, 10 mM glutathione (reduced) and 2 mM glutathione (oxidized) ). After incubation in this solution for 12 hours, the protein was dialyzed against 10 mM sodium phosphate.

Example 2: Expression of recombinant Ckb-8 in COS cells

The CMV-Ckp-8 HA expression plasmid was obtained from the pcDNAI / Amp vector (Invitrogen) containing the ampicillin resistance gene and the CMV promoter, followed by a polylinker region, an SV40 intron, and a polyadenylation site. A DNA fragment encoding the truncated sequence of Skr-8 and the HA fusion fused to the 3'-end is cloned into the polylinker region of the vector; thus, the expression of the recombinant protein is driven by the CMV promoter. The HA attachment corresponds to an epitope derived from the hemagglutinin protein of influenza virus, as described previously (I. Wilson et al., Cell 1984, 37: 767). The fusion of the HA strand with the target protein makes it easy to detect the recombinant protein with an antibody that recognizes the HA epitope.

For the expression of recombinant truncated forms of Skr-8, COS cells are transfected with the expression vector by the DEAE-dextran method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ( 1989). Cells were labeled 8 hours with ³⁵ 8-cysteine two days after transfection. The culture media were then collected and the cells lysed with detergent (RIPA buffer (150 mM NaCI, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7 , 5) (Wilson, I. et al., ID. 1984, 37: 767) Both cell lysate and culture media were precipitated with a monoclonal antibody specific for HA. The precipitated proteins were analyzed on 15% SDS-PAGE gels. .

Example 3: Expression and Purification of a Short Form Chemokine Skr-8 Using a Baculovirus Expression System

BR9 cells were infected with recombinant baculovirus engineered to express cDNA for Ckp-8. The cells were infected with a 10 liter culture at MOI 2 and cultured under low serum conditions at 28 ° C for 72-96 hours. Cell debris from the infected culture is removed by low-speed centrifugation. A cocktail of protease inhibitors was added to the supernatant to a final concentration of 20 μg / ml (1 μg / ml leupeptin, 1 μg / ml E-64 and 1 mM EDTA). These particular culture conditions (i.e., low serum content) lead to the production of some truncated, 2H-end forms of the Skr-8 polypeptide. The level of Skr-8 in the supernatant was monitored by applying 20-30 µl of the supernatant on a 15% SDS-PAGE gel. Truncated forms of Skr-8 were detected as visible bands corresponding to expression levels of several mg per liter. Shortened Skr-8 was further purified by a three step process: 1) Heparin-binding affinity chromatography. The baculovirus culture pad was mixed with 1/3 volume buffer containing 100 mM HEPES / MEM / NaOAc pH 6 and filtered through a 0.22 μm membrane. The sample was then applied to a heparin-binding column (HEI poros 20, ΒΙΟ-Perceptive System Inc.). The truncated forms of Ckp-8 elute at approximately 300 mM NaCI in a linear gradient of 50 to 500 mM NaCI at 50 mM

HEPES / MES / NaOac at pH 6; 2) Cation exchange chromatography. The heparin-rich protein was subjected to 5-fold dilution with buffer containing 50 mM HEPES / MES / NaOAc pH 6. The resulting mixture was then applied to a cation exchange column (S / M poros 20, ΒΙΟ-Perceptive System Inc.). Truncated Ckp-8 was eluted at 250 mM NaCI in a linear gradient from 25 to 300 mM NaCI in 50 mM HEPES / MES / NaOAc at pH 6; 3) Gel filtration. After cation exchange chromatography, the shortened Ckp-8 was further purified by gel filtration (HW50, TOSO HAAS, 1.4 x 45 cm). The amino acid sequencing of the purified preparation shows a seed of at least four predominant sequences corresponding to the four truncated forms of the protein (SEQ ID NOS: 1, 2, 3 and 4).

Example 4: Stimulation of EOL-3 cells and PBLs with a mixture containing truncated forms of Skr-8

EOL-3 cells were cultured under standard culture conditions and differentiated for 2 weeks in 1 μM Na-butyrate. PBLs (peripheral blood leukocytes) are obtained from human donors via venous puncture. PBLs were purified by standard methods. The two cell preparations were individually loaded with FURA-2 (1 μM) for 45 minutes. Cells at a concentration of 1x10 ⁶ / ml (2 ml total) were transferred to plastic cuvettes. The fluorescence spectrophotometer was zeroed to give a baseline before the addition of truncated forms (871a, 871b, 871 c, 871RP) or of the long form (899) of Skr-8 to a concentration of 0.33 to 33 nm. The fluorescence increase is monitored with a spectrophotometer. The change in free Ca ⁺⁺ inside the cell was calculated by first lysing the cells with Triton X10O in the presence of excess Ca ⁺⁺ (to obtain Fmax) and then adding 5 mM EGTA to obtain Fmin . The fraction of free Ca ⁺⁺ can be calculated from the min and max values. MCP-1 (monocytic chemotaxis protein-1) is a known chemokine and is used as a positive control.

LIST OF SEQUENCES (1) GENERAL INFORMATION:

(i) APPLICANTS: John White, Edward Appelbaum, Daniel O'Shannessy,

James Allan Fornwald and Kevin O'Donnel (ii) TITLE HA OF THE INVENTION: SHORT CHEMOKIN FORMS

BETA-8 (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESS:

(B) STREET: 709 Swedeland Road (C) CITY: King of Prussia (D) STATE: PA (E) COUNTRY: USA (F) CODE: 19406 (v) COMPUTER READING FORM:

(A) MEDIA TYPE: Diskette, 3.5 inches, 1.44 Mb memory (B) COMPUTER: IBM 486 (C) OPERATION SYSTEM: WINDOWS FOR WORKGROUPS (D) PROGRAM: WORDPERFECT 5.1 (vi) DETAILS OF THIS APPLICATION :

(A) APPLICATION NUMBER: not yet given (B) SUBMISSION DATE: NOW (C) CLASSIFICATION:

(vii) PRIORITY REQUEST DATA:

(A) APPLICATION NUMBER:

(B) DATE OF SUBMISSION:

(viii) INFORMATION ABOUT THE AUTHORITY / AGENT:

(A) NAME: William T. Nap (B) REGISTRATION NUMBER: 34 344 (C) INDICATOR / REGISTER NUMBER: P50381 (ix) TELECOMMUNICATION INFORMATION:

(A) PHONE: (610) 270-5219 (B) PHONE: (610) 270-5090 (2) SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 82 (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1

GLU ASN PRO VAL LEU LEU ASP ARG PHE HIS ALA THR SER ALA ASP

5 1015

CYS CYS ILE SER TYR THR PRO ARG SER ILE PRO CYS SER LEULE

2530

GLU SER TYR PHE GLU THR ASN SER GLU CYS SER LYS PRO GLY VAL 35 4045

ILE PHE LEU THR LYS LYS GLY ARG ARG PHE CYS ALA ASN PRO SER 55 5560

ASP LYS GLN VAL GLN VAL CYS MET ARG MET LEU LYS LEU ASP THR 65 7075

ARG ILE LYS THR ARG LYS ASN (2) SEQ ID NO: 2:

(i) HA SEQUENCE CHARACTERISTICS:

(A) LENGTH: 77 (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2

LEU ASP ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR

5 1015

THR PRO ARG SER ILE PRO CYS SER LEU LEU GLU SER TYR PHE GLU 20 2530

THR ASN SER GLU CYS SER LYS PRO GLY VAL ILE PHE LEU THR LYS 35 4045

LYS GLY ARG ARG PHE CYS ALA ASN PRO SER ASP LYS GLN VAL GLN

5560

VAL CYS MET ARG MET LEU LYS LEU ASP THR ARG ILE LYS THR ARG

7075

LYS ASN (2) SEQ ID NO: 3:

(i) HA SEQUENCE CHARACTERISTICS:

(A) LENGTH: 76 (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3

ASP ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR THR 15 10 15

PRO ARG SER ILE PRO CYS SER LEU LEU GLU SER TYR PHE GLU THR 20 2530

ASN SER GLU CYS SER LYS PRO GLY VAL ILE PHE LEU THR LYS LYS 35 4045

GLY ARG ARG PHE CYS ALA ASN PRO SER ASP LYS GLN VAL GLN VAL 50 5560

CYS MET ARG MET LEU LYS LEU ASP THR ARG ILE LYS THR ARG LYS 65 7075

ASN (2) SEQ ID NO: 4:

(i) HA SEQUENCE CHARACTERISTICS:

(A) LENGTH: 75 (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4

ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR THR PRO

5 1015

ARG SER ILE PRO CYS SER LEU LEU GLU SER TYR PHE GLU THR ASN 20 2530

SER GLU CYS SER LYS PRO GLY VAL ILE PHE LEU THR LYS LYS GLY 35 4045

ARG ARG PHE CYS ALA ASN PRO SER ASP LYS GLN VAL GLN VAL CYS 50 5560

MET ARG MET LEU LYS LEU ASP

7075 (2) SEQ ID NO: 5:

(i) HA SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 (B) TYPE: amino acid (C) CHAIN: single stranded (D) TOPOLOGY: linear (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5

TCAGGATCCG TCACAAAAGA TGCAGA (2) SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 (B) TYPE: amino acid (C) CHAIN: single-stranded (D) TOPOLOGY: linear (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6 CGCCTCAG

Claims (15)

Patent Claims A polypeptide comprising the sequence of SEQ ID NO: 1. A polypeptide comprising the sequence of SEQ ID NO: 2. A polypeptide comprising the sequence of SEQ ID NO: 3. 4. The polypeptide; comprising the sequence of SEQ ID NO: 4. 5. A composition comprising a mixture of the polypeptides of claims 1, 2, 3 and 4. A vector comprising a polynucleotide encoding the polypeptide of claim 1,2,3 or 4. 7. A host cell, characterized in that it is genetically engineered by the vector of claim 6. A method for producing a polypeptide of claim 1, 2, 3 or 4, characterized in that it comprises the expression of a host cell polypeptide that is genetically engineered by a vector comprising a plinucleotide encoding the polypeptide of claim 1 , 2,3 or 4. The polypeptide agonist of claim 1, 2, 3 or 4. The polypeptide antagonist of claim 1, 2, 3 or 4. A method of treating a patient in need of Skr-8, comprising administering to the patient an effective amount of the polypeptide of claim 1, 2, 3 or 4. A method for treating a patient in need of Skr-8, comprising administering to the patient an effective amount of the preparation of claim 5. A method of treating a patient in need of Skr-8, comprising administering to the patient an effective amount of the antagonist of claim 10. A method for identifying antagonists and agonists of a polypeptide of claims 1, 2, 3 or 4, characterized in that it comprises combining the selected test cells, the compound being screened and the polypeptide of claim 1, 2. 3 or 4; and determining whether the seeding is an effective agnoist or antagonist based on the test cell response. 15. A method for diagnosing a disease or susceptibility to a disease associated with the lower expression of a polypeptide of claims 1, 2, 3 or 4, characterized in that it determines whether there is a mutation in the nucleic acid sequence encoding the polypeptide . <0 claim 1, 2, 3 or 4; and determining whether the seeding is an effective agnoist or antagonist based on the test cell response. 15. A method for diagnosing a disease or susceptibility to a disease associated with the lower expression of a polypeptide of claims 1, 2, 3 or 4, characterized in that it determines whether there is a mutation in the nucleic acid sequence encoding the polypeptide .