CA2233367A1 - Short forms of chemokine .beta.-8 - Google Patents

Abstract

Novel polypeptides of the truncated form of human Ck.beta.-8 are provided. Methods for using the polypeptides are also provided.

Description

SHORT FORl~IS OF CHEMOKINE 13--8 Background of the Invention This invention relates to polypeptide and polynucleotide sequenc~ encoding S a short form of Chemokine Beta-8 (Ck,13-8). In particular, the present invention relates to the use of the polynucleotide and polypeptide sequences, the production of the polynucleotide and polypeptide sequences, and inhibiting 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, 10 inducible, proinfl:~mmz~tory, proteins. In general, chemokines exhibit 20% to 75%
homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta. In the alpha subfarnily, the first two cysteines are separated by one amino acid 15 and are therefore referred to as the "C-X-C" subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are thus referred to as the "C-C"
subfamily. The first cysteine forms a disulfide bond with the third cysteine, and the second with the fourth, resulting in a similar tertiary structure for many of the chemokines. Structurally, many chemokines undergo proteolytic processing to yield 20 a functional, "mature" protein. This usually involves cleavage of a short leader sequence on the N-terminal portion of the protein. At least 14 distinct a-chemokines and 12 ,B-chemokines have been described at the protein and/or cDNA level.
The intercrine cytokines exhibit a wide variety of functions. One important 25 feature is their ability to stim~ tto chemotactic migration of distinct cell types such as monocytes, neutrophils, T lymphocytes, basophils, and fibroblasts. The a and ,B
subfamilies differ somewhat in their cell target selectivity. Most of the oc chemokines attract neutrophils, f1broblasts, T cells, and NK cells. ~-chemokinesprimarily attract monocytes and T lymphocytes. Many chemokines have 30 proinfl~mm~tory activity and are involved in multiple steps during an infl~mm~tory reaction. These activities include stimulation of hi~t~mine release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endoth~ l cells, enhanced binding of complement proteins, in~llf ed expression of granulocyte adhesion molecules and complement receptors, and respiratory burst.
In addition to their involvement in infl~mm~tion, certain chemokines have been S shown to exhibit other activities. For example, macrophage in~amm~tory protein (MIP-l) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells. Chemokines have been implicated in a number of physiological and disease conditions, particularly those that have an inflamm:~tt~ry component. These include but are not limited to: lymphocyte tr~fficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis. Many chemokines were initially isolated as products of an infl~mm~tory response. For example, MIP-l was origin:~lly isolated as an endotoxin-induced proinfl~mm~t~ry cytokine produced by macrophages. Other members of this subfamily have similarly been identified, based on jnflzlmm:~tion induction as well as amino acid sequell- e homology. This includes the full length as well as the shortened forms of the Ck~-8 polypeptide of the present invention.

Summary of the Invention In accordance with one aspect of the present invention, there is provided a mixture of novel polypeptides being truncated forms of human Ck,B-8 as well as biologically active and diagnostically or therapeutically useful fragml~nts, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there is provided isolated nucleic acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
In accordance with a further aspect of this invention, there is provided a process for producing such polypeptides by recombinant techniques which comprisecnltllrin~ recombinant prokaryotic and/or eukaryotic host cells, cont~ining nucleic W O 97/12041 PCTAUS96/15~92 acid sequences, under conditions promoting expression of said proteins and subsequent recovery of said proteins.
In accordance with yet a further aspect of the present invention, there is provided a process for ntili7ing such polypeptides, or polynucleotides encoding such 5 pclypeptides for therapeutic purposes, for example, to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy, and to stimlll~tP wound hP~ling.
In accordance with another aspect of the present invention, there are provided antibodies against such a polypeptide.
In accordance with a further aspect of the present invention, there are provided antagonists to such a polypeptide, which may be used to inhibit the action of such a polypeptide, for example, to treat aplastic anemia, myelodysplastic syndrome. asthma and arthritis.
In accordance with another aspect of the present invention, there is also 15 provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the Ck~-8 nucleic acid sequence.
In accordance with a further aspect of the present invention, there are provided ~ gnQ~tic assays for cletPcting diseases related to the undele~c~lc;ssion and overexpression of the polypeptide and for ~iPtpcting mutations in the nucleic acid 20 seq~çnrç~ encoding such a polypeptide.
In accordance with yet a further aspect of the present invention, there is a process for ~ltili7ing such a polypeptide, or polynucleotides encoding such a polypeptide, as research reagents for in vitro purposes related to scientific research, synthesis of DNA and m~nllf~ctnre of DNA vectors, for the purpose of developing 25 therapeutics and diagnostics for the tre~tmPnt of human disease.
These and other aspects of the present invention should be apparent to those skilled in the art from the tP~c hin~c herein.

~ Detailed Description of the Invention DNA sequences encoding polypeptides structurally related to the pro-infl~nnm~tory "intercrine" chemokine ~u~elr~llily have been described. The W O 97/12041 PCT~US96/15S92 polynucleotide sequence encoding full length Ck,B-8 (120 amino acids) was derived from an aortic endothelium cDNA library. The present invention concerns novel shortened forms of Ck,B-8 cont~ining only the 82, 76. 75 or 74 C-terminal amino acids of the long form. The N-terminal amino acids as well as the 21 residue signal 5 peptide sequence are cleaved. As with the long form of Ck~-8, the first two cysteine residues in these clones are in adjacent positions placing them in the "C-C"
or beta subfamily of chemrkines. The presence of this motif distinguishes them from the a subfamily of chemokines ("CXC") in which the first two cysteine residues are separated by one amino acid.
The polynucleotide sequences encoding the mature polypeptides (SEQ ID
NOs: 1, 2, 3, and 4) may be represented in any nucleic acid form such as RNA, ssDNA, genornic DNA, etc. including additional coding and non-coding sequences associated with the polypeptide. Furthermore, due to the degeneracy of the aminoacid code, any DNA coding sequence which encodes the sarne mature polypeptides 15 shall be considered within the scope of this invention. Any variants of this sequence, allelic or non-naturally occurring shall also be incl~ l The present invention also includes polynucleotides in which the coding sequence is fused in the same reading frame to another DNA sequence which aids in e~yl~ssion or secretionof the protein from a cell or serves as a marker to identify the protein in cells (e.g., 20 HA tag).
This invention further relates to polynucleotide sequences which hybridize to the described sequence with at least 50% and preferably 70% sequence identity.
These polynucleotides encode polypeptides which preferably retain the same biological function or activity as the mature polypeptide. .AI~ern~tively, the sequence 25 may be polynucleotides which have, preferably, 50 bases that have identity to, and hybridize to the sequences of the present invention, but do not retain activity. Such sequences may be used as a probe or PCR primer.
The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. Derivatives of these polypeptides 30 may be one in which one or more amino acid substitutions are present; one in which one or more amino acids contain substituent groups; one in which the mature protein W O 97/12041 PCT~US96/15592 is fused with another compound; or one in which additional amino acids are fused to mature proteins.
The polypeptides of this invention are preferably provided in isolated form, and preferably purified to homogeneity.
S The present invention also relates to vectors which include polynucleotides of this invention, host cells which are genetically en~in~ered with this vector, and the production of polypeptides of the invention by recombinant techniques. Host cells are genetically engineered with the vectors of this invention (cloning or expression). The culture conditions for these cells in order to activate promoters, select transformants, or amplify the truncated Cki~-8 gene will be apparent to those skilled in the art.
This polynucleotide sequence may be included in any one of a variety of expression vectors so long as they are replicable and viable in the host. The polynucleotide seqnp~re inserted into the expression vector is under the control of an ~.u~.iate promoter to direct mRNA synthesis (e.g., LTR promoter). The expression vector also contains a ribosome binding site for translation initiation, a transcription terminator, enh~nrer elemPnt~ which will increase transcription (e.g., SV4û late c-nh~n~çr), and a~.upliate selectable markers to ensure m~intP~n~e of the vector in the host (e.g., ampicillin resi~t~nce gene). Host cells can be higher eukaryotic cells such as m:~mm~ n or insect cells, lower eukaryotic cells such as yeast, or prokaryotic cells such as bacteria. Introduction of this construct into the host cell can be effected by a variety of protocols. All of the aforementioned steps will be obvious to those skilled in the art. Cloning steps as well as host and vector selection are well known in the art. (Sambrook et al., Moiecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).
Proteins can be synthPsi~ecl in the host cells under the control of an applup.iate promoter. Cell-free translation systems can also be used to produce such proteins using RNAs derived from the DNA constructs of the present invention. ~Ittorn~tively, the polypeptide of this invention can be synthPtic~lly produced by conventional peptide synthP~i~Prs.

W O 97/12041 PCT~US96/lS592 Following transformation or transfection of a suitable host strain and growth of the host strain to an a~ iate cell density, the selected promoter is induced by a~ o~iate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, 5 disrupted by physical or chrmic~l means, and purified to homogeneity by a variety of methods (e.g., chromatography, HPLC) Depending upon the host cell employed, the polypeptide may be modified in some manner (e.g., glycosylation).
Polypeptides of the invention may also include an initial methionine residue.
The long form of Ck~-8 is a chemoattractant for leukocytes and therefore 10 may be employed in a variety of immunoregulatory and infl~mm~t-)ry functions as well as a number of disease conditions. This protein is expressed predominantly in tissues of hemopoietic origin. One important biological effect of the Ck,13-8 polypeptide on leukocytes is stim~ tion of the mobilization of Ca++ reserves. This mobilization pertains to functional activation of the cell. It has been found that 15 stim~ tion of differentiated EOL-3 cells with a mixture cont~ining four shortened forms of Ck,B-8 (SEQ ID NOS: 1-4) results in an imm~ te increase in intracellular Ca+~ which is dose dependent. When tested individually, the short form having SEQ ID NO: 4 was the most active. In contrast, the long form of Ckl3-8 was approximately 1000 times less potent than the short forms at mobilizing Ca++. The 20 potency of the short forms of the present invention may have implications for the effectiveness of therapeutic applications of this chemokine including, but not limited to, protection of bone marrow stem cells from chemotherapeutic agents during chemotherapy, removal of leukrmic cells, stiml-l~tion of an immnne response, regulation of hematopoiesis and lymphocyte tr~fficking, treatment of psoriasis, solid 25 tumors, enhancing host defenses against resistant chronic and acute infection, and ctim~ tion of wound hr~ling.
The polynucleotides and polypeptides of the present invention may be used as a research reagents, in synthesis of DNA and man--f~rt--re of DNA vectors, and for the purpose of developing therapeutics and diagnostics for the treatment of 30 human disease (e.g., in the expansion of imm~tllre hematopoietic progenitor cells).
Fragments of this truncated Ck,B-8 polynucleotide sequence may also be used as a W O 97/12041 PCTrUS96/15592 hybridization probe to isolate other genes which have a high seqll~n-~e ~imil~rity.
Techniques for these kinds of ~ GlhllGnts are known to those skilled in the art.This invention is also related to the use of these sequences as part of a diagnostic assay for det~cting ~ e:~cçs or susceptibility to ~lice~ces related to the presence of mutations in the nucleic acid sequences. Such tlice~ces are related to under-expression of the chemokine polypeptides. For example, individuals carrying mutations in the Ck,13-8 gene may be detected at the DNA level by a variety of techniques such as PCR. Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fr~gm~nts in gels with or without denaturing agents. The detection of specific DNA sequence may be achieved by methods such as hybridization, R~ase protection, chPmir~l cleavage, direct DNA sequencin~, RFLP analysis, and Southern blotting of genomicDNA. Mutations can also be ~i~t~ct~d by in situ analysis.
The present invention also relates to a diagnostic assay for detecting altered levels of Ck~-8 protein in various tissues since an over-expression of the proteins cu~ alcd to normal control tissue s~mrles may detect the p.Gsellce of a disease or susceptibility to a disease, for example, a tumor. Assays used to detect levels of Ck~-8 protein in a sample derived from a host are well known to those of skill in the art and include radioimmunoassays, colll~GLilive-binding assays, Western blot analysis, ELISA assays and "sandwich" assays.
This invention provides a method for iclentific~tion of the receptors for the chemokine polypeptides. The gene encoding the receptor can be identified by numerous mPtho-lc known to those of skill in the art including ligand panning and FACS sorting.
An alternative approach for receptor i~l~ntifi~iqtion involves photo:~ffinity linking the labeled polypeptides with the cell membrane or extract preparations that express the receptor molecule. The labeled complex can be icol~terl and subjected to protein microseq~l~oncing The amino acid sequence obtained would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.

W O 97/12041 PCTAUS96/1~592 This invention provides a method of screening compounds to identify agonists and antagonists to the chemokine polypeptide of the present invention.
Chemotaxis may be assayed by placing cells chemoattracted by these polypeptides on top of a filter with pores large enough to admit the cells (5 mm). Solutions of 5 potential agonists or antagonists are placed in a bottom chamber so that cellsmigrate or are prevented from migrating through the membrane over a period of time. Alternatively, the receptors of Ck~-8 would be incubated with a labeled polypeptide in the presence of a compound. The ability of the compound to eitherblock this interaction or interact with the receptor in the absence of polypeptide 10 could be measured.
Examples of potential truncated Ck~-8 antagonists include antibodies, oligonucleotides which bind to the polypeptides or polypeptides which bind to the receptor of the wild-type polypeptide, but fail to retain biological activity.
,~nticence technology used to control gene expression can also be a potential 15 antagonist. The availability of the aforementioned technologies should be obvious to those skilled in the art.
Antagonists may be employed to treat infectious flice~cec such as silicosis, sarcoidosis, idiopathic pulmonary fibrosis, idiopathic hyper-eosinophilic syndrome, and endotoxic shock- all by preventing production of the polypeptide of the present 20 invention. The antagonists may also be employed for treating atherosclerosis, by preventing monocyte infiltration in the artery wall. Antagonists may be employedto treat hict~mine-mediated allergic reactions and various immunological disorders involving the dermis.
The antagonists may also be used to treat chronic and acute infl~mm~tion by 25 preventing the attraction of monocytes to the wound area. They may also be used to treat infl~mm~ts)ry pnlmon~ry disease, general infl~mm~tion, and rheumatoid arthritis by preventing monocyte influx into affected areas.
Antagonists may also be employed to treat cases of bone marrow failure in aplastic anemia or myelodysplastic syndrome. They may also be used to treat 30 asthma and allergy as well as subepithelial basement membrane fibrosis which is a feature of the ~cthm~tic lung.

W O 97/12041 PCT~US96/15S92 The chemokine polypeptide and agonists and antagonists may be employed in combination with a suitable ph~ eutic~l carrier such as saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of a-imini~tration. The invention also provides a 5 ph~rrn~eutic~l pack or kit comprising one or more cont~inçrs filled with one or more of the ingredients of the pharmaceutical compositions of this invention. The polypeptides, agonists, and antagonists may be employed in conjunction with other theldl)euLic compounds.
The ph~ eutical compositions may be a-lmini~t~red in a convenient 10 manner such as by topical, intravenous, intraperitoneal, intr~mnCcul~r~ intratumor, sllbc-lt~n~ous, intranasal, or intradermal routes in an amount which is effective for treating the specific indication. These applications should be evident to those skilled in the art.
The chemokine polypeptides, agonists, or antagonists which are 15 polypeptides, may be employed in accordance with the present invention by expression of such polypeptides in vivo. This type of gene therapy is well known in the art. For example, cells from a patient may be engineered with DNA or RNA
encoding the short form of Ck~-8 polypeptide ex vivo and the en~in~red cells expressing these polypeptides provided to the patient. Similarly, cells may be 20 en~in~çred in vivo for expression of a polypeptide in vivo.
As known in the art, the use of a retroviral particle conr~ining RNA
encoding the polypeptide of the present invention may be used. Retroviral plasmid vectors are used to tran~cillçe packaging cell lines (e.g., PE501) by various means (e.g., electroporation) to form producer cell lines. In a plefel,~,d embodiment, the 25 retroviral expression vector cont~ining the polynucleotide sequences, is under the control of a promoter such as LTR and contains a selectable drug resi~t:~nce marker (e.g., neo). The preparation of these vectors and rçsnltin~ cell lines should befamiliar to those skilled in the art and from the techniques contained herein. The producer cell line generates infectious retroviral vector particles which include the 30 nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed to transduce eukaryotic cells, for example, fibroblasts or en~cth~ l cells, either in l~itro or in vivo. The trzmcr~ ed eukaryotic cells will then express the nucleic acid sequence(s) encoding the protein.
The sequences of the present invention are also valuable for chromosome ntifi~tion. The sequences are specifically targeted to and can hybridize with a 5 particular location on an individual chromosome. The mapping of DNA to chromosomes is an important step in correlating genes associated with disease. One technique known to those of skill in the art involves preparing primers used for PCR
screening of somatic cell hybrids cont~inin~ individual human chromosomes. Only those hybrids cont~ining the human gene corresponding to the primer will yield an 10 amplified fragment. Other mapping strategies known to those of skill in the art include. but are not limited to: in situ hybridization, prescreening with labeled flow-sorted chromosomes, sublocalization to fragments of specific chromosomes using the same PCR primers, and preselection by hybridization to construct chromosome specific-cDNA libraries. Fluorescence in situ hybridization (FISH) of15 cDNA clones to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence can be correlated with genetic map data. The relationship between genes and disease can be identified through linkage analysis.
20 Any differences in the cDNA or genomic ~ sequences between affected and unaffected individuals can be indicative of disease. For example, a mutation in the DNA found only in the affected individuals may be the causative agent of the disease.
The polypeptides, their fragments or other derivatives, or analogs thereof, or 25 cells expressing them can be used as an immunogen to produce antibodies thereto.
These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain and h~-m~ni7e-1 antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and 30 fr~gm~n~. Polyclonal antibodies prepared by standard procedures can be used to isolate the polypeptides from tissue expressing that polypeptide. For preparation of WO 97/12041 PCT~US96/15592 monoclonal antibodies, any technique which provides antibodies produced by continuous cell lines cultures can be used. Techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies to immunogenic polypeptides of this invention. Also, transgenic mice may be used toS express hllm~ni7~1 antibodies to imrnunogenic polypeptides of this invention.
The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. In order to facilitate underst~n~ling of the followingexamples certain frequently occurring methods andlor terms will be briefly described.
The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be app~c;llt to the ordinarily skilled artisan.
The various restriction enzymes used herein for "digestion" of DNA are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. Sizeseparation of the cleaved fr~gm~ntc is pe,rol",ed using 8% polyacrylarnide gel.
Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chPmir~lly synthPci~e~l Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated. Unless otherwise stated, transformation was performedas described in the method of Graham, F. and Van der Eb, A., Virology 1973, 52, 456457.
The following exarnples are provided for illustrative purposes only and are not inten~le~l to limit the invention.

EXAMPLES
F.Y~mple 1: B~ I ;al Expression and Puriffcation of Ck,l~-8 The DNA sequence encoding Ck,13-8, ATCC# 75676 was initially arnplified using PCR primers corrcsponding to the 5' and 3' end sequences of the processed S Ck,B-8 protein (minus the signal peptide sequence) and the vector sequçn- çs 3' to the Ck,l~-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 respectively contain BamHI and XbaI restriction sites used for cloning into 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 frame with the sequence encoding for the hi.~ti~line tag and the ribosome binding site (RBS). This ligation was used to Llal1srollll E. coli strain M15/rep 4 (Qiagen) which contains mllltiple copies of plasmid pREP4. pREP4 expresses the lacI repressor and also confers kanamycin resi~t~nre Transforrnants were sçlecteci by their ability to grow on LB plates supplemented with ampicillin/kanamycin. Clones cont~ining the desired constructs (isolated and confirmed by restriction analysis) were grown overnight in liquid culture in LB
media supplemented with both Amp (100 ~g/ml) and Kan (25 ,ug/ml). This culture was used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density 600 of between 0.4 and 0.6. IPTG
(isopropyl-B-D-thiogalacto-pyranoside) was then added to a hnal concentration of I
mM. Cells were then grown an extra 3 to 4 hours, then harvested by centrifugation.
The cell pellet was solubilized in the chaotropic agent 6 M Gu:~ni~iine HCl, clarified, and subjected to chromatography on a Nickel-Chelate column under conditions thatallow for tight binding by proteins cont~ining the 6-His tag (Hochuli, E. et al. J.
Chromatography 1984, 411, 177-184). Truncated Ck~-8 (95% pure) is eluted from the column in 6 M gu~ni~line HCl pH 5 and for the purposes of renaturation adjusted to 3 M gu~ni-line HCl, 100 mM sodium phosphate, 10 mM gl~lt~thione (reduced) and 2 mM glutathione (oxidized). After incubation in this solution for 12 hours, the protein was dialyzed to 10 mM sodium phosphate.

-W O 97/12041 PCT~US96/15592 Example 2: Expression of recombinant Ck~-8 in COS cells The expression of plasmid, CMV-Ck~-8 HA is derived from a vector pcDNAI/Amp (Invitrogen) cont~ining the ampicillin reci~t~nce gene and the CMV
promoter fol1Owed by a polylinker region, SV40 intron, and polyadenylation site. A
5 DNA fragment encoding the truncated Ck~-8 sequences and an HA tag fused in frame to the 3' end is cloned into the polylinker region of the vector; therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson et al.~ Cell 1984, 37:767). The infusion of HA tag 10 to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
For expression of the recombinant short forms of Ck,B-8, COS cells are transfected with the expression vector by DEAE- Dextran method (J. Sambrook, E.
Fritsch, T. M~ni~tic. Molecular Cloning: A Laboratory Manual, Cold Spring HarborlS Laboratory Press, (1989)). Cells are labeled for 8 hours with 35S- cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 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 are precipitated with a HA specific monoclonal antibody. Proteins 20 precipitated are analyzed on 15% SDS-PAGE gels.

Example 3: Expl~ ~n and Purification of Short Form of Che-l-tl-ine Ck~-8 using a Baculovirus Expression System SF9 cells were infected with a recombinant baculovirus dç~ n~d to express 25 the Ck,~-8 cDNA. Cells were infected in a 10 liter culture at an MOI of 2 andcultured under low serum conditions at 28~C for 72-96 hours. Cellular debris from the infected culture was removed by low speed centrifugation. Protease inhibitorcocktail was added to the supernatant at a final concentration of 20 ~Lg/ml ( 1, Lg/ml leupeptin, 1 ,ug/ml E-64 and 1 mM EDTA). These particular culture conditions 30 (i.e., low serum) produced several NH2-terminal truncated forms of the Ck~-8 polypeptide. The level of the Ck,B-8 in the supernatant was monitored by loading W O 97/12041 PCT~US96/15592 20-30 ,ul of supern~t~nt on a 15% SDS-PAGE gel. The shortened forms of Ck~-8 were detected as visible bands, corresponding to an expression level of several mg per liter. Trllnc~te~l Ckl3-8 was further purified through a three-step purification procedure: 1) Heparin binding affinity chromatography. Supernatant of baculovirus culture was mixed with l/3 volume of buffer cont~ining 100 mM
HEPES/MEM/NaOAc pH 6 and filtered through 0.22 ,um membrane. The sample was then applied to a heparin binding column (HE1 poros 20, BIo-pe,eeplive System Inc.). The short forms of Ck,B-8 was eluted at approximately 300 mM NaCl in a linear gradient of 50 to 500 mM NaCl in 50 mM HEPES/MES/NaOac at pH 6;

2) Cation exchange chromatography. The protein enriched from heparin chromatography was subjected to a 5-fold dilution with a buffer cont~ining 50 mMHEPES/MES/NaOAc pH 6. The resultant mixture was then applied to a cation exchange column (S/M poros 20, BIO-Perceptive System Inc.). Truncated Ck,~-8 was eluted at 250 mM NaCl in a linear gradient of 25 to 300 mM NaCl in 50 mM
HEPES/MES/NaOAc at pH 6; 3) Size exclusion chromatography. Following the cation exchange chromatography, truncated Ck~-8 was further purified by applyingto a size exclusion column (HW50, TOSO HAAS, 1.4 x 45 cm.). Amino Acid sequencing of the purified sample showed a mixture of at least four dorninant sequences corresponding to the four truncated forms of the protein (SEQ ID NOs: l, 2, 3, and 4).

F.Y~mrle 4: Stirnulation of EOL-3 cells and PBLs with a ~ lur~ comprising short forms of Ck,B-8 EOL-3 cells were grown under standard growth conditions and differentiated for two weeks with l ~M Na Butyrate. PBLs (peripheral blood leukocytes) were obtained from human donors by vena puncture. PBLs were purified by standard techniques. Both cell ~nc~al~-Lions were separately loaded with FURA-2 (1 ~M) for 45 minutes. Cells at lxlO6/ml (2 ml total) were transferred to plastic cuvettes. The fluorescent ~ecL,v~hotometer was adjusted to give a baseline recording before the short forms (871a, 871b, 871c, 871RP) or long form (889) of Ckl3-8 was added at a concentration of 0.33 to 33 nM. The increase in fluorescence was monitored with W O 97/12041 PCT~US96/15592 the spectrophotometer. The change in free Ca++ inside the cell was calculated byfrst lysing the cells with Triton xlOO in the presence of excess Ca++ (to obtainFmax) and then adding 5 mM EGTA, to obtain Fmin. The ratio of free Ca++ can be calculated from the min and max values. MCP-1 (monocyte chemotactic protein-1) 5 is a known chemokine and was used as a positive control.

W O 97/12041 PCT~US96/15592 ~UU~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS: John White, Edward Appelbaum, Daniel O'Sh~nn~ssy, James Allan Fornwald and Kevin O'Donnell (ii) TITLE OF INVENTION: Short Forms of Chemokine Beta-8 iii) NUMBER OF ~yu~S: 6 (iv) CORRESPONDENCE ADDRESS:
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(A) TELEPHONE: (610) 270-5219 (B) TELEFAX: (610) 270-5090 (2) INFORMATION FOR SEQ ID NO: 1:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 82 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear W O 97/12041 PCT~US96/15592 (xi) ~ U~N~ DESCRIPTION: SEQ ID NO: 1:
GLU ASN PRO VAL LEU LEU ASP ARG PHE HIS ALA THR SER ALA ASP

CYS CYS ILE SER TYR THR PRO ARG SER ILE PRO CYS SER LEU LEU

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

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

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

ARG ILE LYS THR ARG LYS ASN

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 77 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear xi) ~Qu~N~: DESCRIPTION: SEQ ID NO: 2:
LEU ASP ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR

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

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

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

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

LYS ASN
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 76 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear J

WO 97/12041 PCT~US96/15592 (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO: 3:
ASP ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR THR
l 5 l0 15 PRO ARG SER ILE PRO CYS SER LEU LEU GLU SER TYR PHE GLU THR

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

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

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

ASN
t2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) ~U~N~'~ DESCRIPTION: SEQ ID NO: 4:
ARG PHE HIS ALA THR SER ALA ASP CYS CYS ILE SER TYR THR PRO
l 5 l0 15 ARG SER ILE PRO CYS SER LEU LEU GLU SER TYR PHE GLU THR ASN

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

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

MET ARG MET LEU LYS LEU ASP THR ARG ILE LYS THR ARG LYS ASN

2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No WO 97/12041 PCT~US96/15592 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TCAGGATCCG T~A~A~.A TGCAGA 26 (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) BENGTH: 26 (B) TYPE: Nucleic Acid (C) STRA~~ S: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Claims (15)

What is cl~imt-.d is:

1. A polypeptide comprising ~EQ ID NO: 1.

2. A polypeptide comprising SEQ ID NO: 2.

3. A polypeptide comprising SEQ ID NO: 3.

4. A polypeptide comprising SEQ ID NO: 4.

5. A composition comprising a mixture of the polypeptides of claims 1, 2, 3, and 4.

6. A vector comprising a polynucleotide encoding polypeptide of claim 1, 2, 3, or4.

7. A host cell g~netic~.~lly engineered with a vector of claim 6.

8. A process for producing a polypeptide of claim 1, 2, 3, or 4 comprising e~.t;ssillg the polypeptide from a host cell genetically engineered with a vector comprising a polynucleotide encoding the polypeptide of claim 1, 2, 3, or 4.

9. An agonist for the polypeptide of claim 1, 2, 3 or 4.

10. An antagonist for the polypeptide of claim 1, 2, 3, or 4.

11. A method for the treatment of a patient having a need of Ck,B-8 comprising ~rlmini~t~ring to a patient an effective amount of the polypeptide ofclaim 1, 2, 3, or 4.

12. A method of treatment of a patient having a need of Ck.beta.-8 comprising administering to a patient an effective amount of the composition of claim 5.

13. A method for the treatment of a patient having a need to inhibit Ck.beta.-8 comprising administering to a patient an effective amount of an antagonist of claim 10.

14. A method for identifying antagonists and agonists of a polypeptide of claim 1, 2, 3, or 4 comprising combining selected test cells, a compound to be screened and a polypeptide of claim 1, 2, 3, or 4; and determining if the compound is an effective agonist or antagonist based upon the response of the test cells.

15. A process for diagnosing a disease or a susceptibility to a disease related to the underexpression of a polypeptide of claim 1, 2, 3 or 4 comprisingdetermining if there is a mutation in the nucleic acid sequence encoding the polypeptide.