CA2217216A1 - Human chemokine beta-13 - Google Patents

Abstract

Human chemokine polypeptides and DNA (RNA) encoding such chemokine polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such chemokine polypeptides for the treatment of leukemia, tumors, chronic infections, auto-immune disease, fibrotic disorders, wound healing and psoriasis. Antagonists against such chemokine polypeptides and their use as a therapeutic to treat rheumatoid arthritis, auto-immune and chronic and acute inflammatory and infective diseases, allergic reactions, prostaglandin-independent fever and bone marrow failure are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and altered concentrations of the polypeptides. Also disclosed are diagnostic assays for detecting mutations in the polynucleotides encoding the chemokine polypeptides and for detecting altered levels of the polypeptide in a host.

Description

WO 96~9521 PCTAU~9 ~uman Ch~~kine Beta-13 This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention has been putatively identified as a human ch~~okine polypeptides, sometimes hereinafter referred to as human ~h~ok;ne beta-13 (Ck~-13).
The invention also relates to ~nh; h; ting the action of such polypeptides.
~ h~m~kines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are 8-10 kd in size. In general, ~h~7kine8 _Yh;h; t 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 subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the "C-X-C"
subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the W O 96~9521 PCT~U5~J/~/~5S

l'C-C~' sub~amily. Thus far, at least nine different members of this family have been identified in h~ nQ
The intercrine cytokines ~xhi h; t a wide variety of ~unctions. A h~l 1 ~k feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutroph;lr, T lymphocytes, basophils and fibroblasts. Many ~ ok;n~s have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include st;m~ tion of hist~m;n~
release, lysosomal enzyme and leukotriene release, increased adherence of target ; ~ lnP cells to endoth~ cells, F-nh~nced h;n~;ng of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvF t in inflammation, certain l-h- l_; n~Q have been shown to ~Yh;h; t other activities. For example, macrophage inflammatory protein 1 (MIP-1) is able to ~ ess hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent ;nh;h;tor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GR0 is an autocrine growth $actor for ~~1 ~n-_ cells.
In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound h~l ;ng, hematopoietic regulation and ;mmnnological disorders such as allergy, asthma and arthritis.
Members of the "C-C~ branch exert their effects on the following cells: eo6inoph;l6 which de6troy parasites to lessen parasitic infection and cause chronic inflammation in the airways of the respiratory system; monocytes and macrophages which suppress tumor formation in vertebrates; T
lymphocytes which attract T cells and basophils which release hist~m;ne which plays a role in allergic inflammation.

W O 96~9521 PCT/U'jS/~ S

While m~mh~r5 of the C-C branch act pr~ i n;~nt 1y on I ~ntlclear cells and ~e~s of the C-X-C branch act pr~ n~ntly on neutrophils a distinct rh~mo~ttractant property cannot be assigned to a rh~mokine based on this guiA~l, n~, Some rh~m~kines from one family show characteristics of the other.
The polypeptide of the present invention has the conserved cysteine "C-C" region, and has amino acid sequence homology to known chemokines.
In accordance with one aspect of the present invention, there are provided novel polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or the~euLically useful fra~_ t~, analogs and derivatives thereof.
In ac~G~ ~ ~ce with another aspect of the present invention there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to ~pecifically hybridize to nucleic acid Se~l~nC~S ~nroA~ n~ the polypeptide of the present invention.
In ac~oL~,ce with yet a further aspect of the present invention, there is provided a process for proAllr~ ng such polypeptide by recombinant techniques which c~..~ises culturing recombinant prokaryotic and/or eukaryotic host cells, cont~;n;ng a nucleic acid 6e~uence ~nroA;ng a polypeptide of the present invention, under conditions promoting expression of said protein and subsequent recovery of said protein.
In accordance with yet a further a8pect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides ~nro~; ng such polypeptides .

W O 96~9521 PCT~US95/07294 for therapeutic purpo8es, for example, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-tmm~lne diseases, parasitic infections, p80ria8i8, to regulate hematopoiesis, to st;m~llAte growth factor activity, to ~nh;h;t angiogenesis and to ~ro,.,oLe wound h~l;ng, In accor~Ance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to ;nh;h;t the action of such polypeptides, for example, in the treatment of certain auto-;~nn~ digeases, atherosclerosis, chronic inflammatory and infectious diseases, hist~m;ne and Ig~-mediated allergic reactions, prostaglAn~;n-indep~n~nt fever, bone marrow failure, silicosis, sarcoidosis, rheumatoid arthritis, and hyper-eosinophilic syndrome.
In accordance with another aspect of the present invention there is provided a method of diagnosing a disease or a susceptibility to a disease related to a mutation in the nucleic acid _equences of the present invention and to altered levels of the protein encoded by such nucleic acid sequences.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides ~nco~;ng such polypeptides, for in vit~o purposes related to scientific research, synthesis of DN~ and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the t~rh;n~s.
herein.
The following drawings are illustrative of ~mho~;m~n~s of the invention and are not meant to limit the scope of the invention as ~ncoTrAssed by the ~l~;~e, W O 96~95Zl PCT~US95/07294 Figure 1 displays the cDNA sequence and correspondiny deduced amino acid sequence of Ck~-13. The initial 28 amino acids represent the leader sequence such that the putative mature polypeptide comprises 65 amino acids. The st~n~rd one-letter a~breviations for amino acids are used.
Sequencing was per~ormed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).
Figure 2 displays the amino acid sequence homology between Ck~-13 (top) and human MIP-la polypeptide (bottom).
In accordance with an aspect of the present invention, there are provided isolated nucleic acid8 (polynucleotides) which Pncn~ for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone(s) deposited as ATCC Deposit No. 97113 on April 28, 1995.
Polynucleotides encoding Ck~-13 have been isolated from an activated monocyte cDNA library. Ck~-13 is a ~ .-~e of the C-C branch of chem~ktne It ~ont~tn~ an open r~A~ing frame enco~tng a protein of 93 amino acid residues of which approximately the first 28 amino acids residues are the putative leader sequence such that the mature protein comprises 65 amino acids. The protein has structural homology to chemokine polypeptides and the homology to the hn~-n MIP-la polypeptide with 33~ t~ntity and 53~ 5tm~l~rity over the entire sequence is u~ed solely as an example.
The four spatially conserved cysteine residues in che~okines are found in the polypeptide of the present invention as can be seen in Figure 1.
The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single str~n~ may be the coding strand or non-co~tng (anti-sense) strand. The coding sequence which ~nco~ the mature polypeptides may be identical to the coding sequence shown in Figures 1 tSEQ ID

CA 022l72l6 l997-l0-02 WO 96~9521 PCTAJS95/07294 NO:1) or that of the deposited clone(s) or may be a different coding sequence which coding sequence, as a result of the re~ln~ncy or degeneracy o~ the genetic code, encodes the same mature polypeptides as the DNA of Figure 1 (SEQ ID NO:1) or the deposited cDNA.
The polynucleotides which ~ncoA~ for the mature polypeptide of Figure 1 (SEQ ID NO:2) or ~or the mature polypeptide encoded by the deposited cDNA may include: only the roA; ng sequence for the mature polypeptide; the coding sequence for the mature polype _de and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional ro~; ng sequence) and non-coding sequence, such as introns or non-co~ ng sequence 5~ and/or 3~ of the roA; n~ sequence for the mature polypeptides.
Thus, the term "polynucleotide onro~;ng a polypeptide"
encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-co~; ng sequence.
The present invention further relates to variants of the her~;n~hove described polynucleotides which PncoAe for frasm~nts, analogs and derivatives of the polypeptide having the ~e~llce~ amino acid sequence of Figure 1 (SFQ ID NO:2) or the polypeptide ~nco~ by the cDNA of the deposited clone(s). The variants of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides.
Thus, the present invention includes polynucleotides ~ncoA; ng the same mature polypeptide as shown in Figures 1 (SBQ ID NO:2) or the same mature polypeptide ~nco~ by the cDNA of the deposited clone(s) as well as variants of such polynucleotides which variants PnCo~e for a fragment, derivative or analog of the polypeptide of Figure 1 (SFQ ID
NO:2) or the polypeptide encoded by the cDNA of the deposited W O 96~9521 PCT/U~3~ 7~g4 clone (8). Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As herP;n~hove indicated, the polynucleotides s y have a coding sequence which is a naturally occurring allelic variant of the coding se~uence shown in Figure 1 (SEQ ID
NO:1) or of the co~in~ sequence of the deposited clone(s).
As known in the art, an allelic variant is an alternate form of a polynucleotide sequenCe which s y have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
The present invention also includes polynucleotides, wherein the coding sequence for the s ture polypeptidesmay be fused in the same r~i n~ frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a ~Le~L~Lein and may have the leader sequence cleaved by the host cell to form the sture form of the polypeptide. The polynucleotide~ s y also ~n~o~ for a proprotein which is the s ture protein plus additional 5~
amino acid residues. A s ture protein having a prosequence is a p~ oLein and is an inactive form of the protein. Once the prosequence is cleaved an active s ture protein r~m~;nc.
Thus, for example, the polynucleotide~ of the present invention may ~nco~ for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence).
The polynucleotides of the present invention may also have the co~; ng sequence fused in frame to a s rker sequence which allows for purification of the polypeptide of the present invention. The marker sequence s y be a hexa-histidine tag supplied by a pQE-9 vector to provide for CA 022l72l6 l997-l0-02 W O 96~9521 PCT~US95/07294 purification of the mature polypeptide ~used to the marker in the case of a bacterial host, or, for example, the marker seguence may be a hemagglutinin (HA) tag when a . l; An host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 ~1984)).
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening se~uences (introns) between individual coding segments (exons).
Fra~m~nt~ of the full length Ck~-13 gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence 5im;l~ity to the gene or Sim;l A~ biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe may also be used to identify a cDNA clone corregp~n~;n~
to a full length transcript and a genomic clone or clones that CQntA; n the complete Ck~-13 gene including regulatory and promotor region~, exons, and introns. An example of a screen comprises isolating the co~; ng region of the Ck~-13 gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which m~m~8 of the library the probe hybridizes to.
The present invention further relates to polynucleotides which hybridize to the her~;nAhove-described se~uences if there is at least 70%, preferably at least 90~, and more preferably at least 95% ;~nt;ty between the seql~nc~c. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the here;nAhove-described polynucleotides. As herein used, CA 022l72l6 l997-l0-02 W O 96~9521 PCTAJS95/07294 the term n stringent conditions~ means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the se~uences. The polynucleotides which hybridize to the herP~nAhove described polynucleotides in a preferred embodiment ~nco~ polypeptides which either retain substAntiAlly the same biological function or activity as the mature polypeptide ~ncoA~A by the cDNA of Figure 1 (SEQ ID NO:l) or the deposited cDNA(s), i.e. function as a chemokine polypeptide.
Alternatively, the polynucleotides may be polynucleotides which have at least 20 bases, preferably 30 bases and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which have an identity thereto, as her~inAhove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SBQ ID NO:1, or for variants thereof, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
Thus, the present invention is directed to polynucleotides having at lea8t a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which frA~ ~ ts have at least 30 bases and preferably at least 50 bases and to polypeptides ~n~oAe~ by such polynucleotides.
The deposit(s) referred to herein will be m~intAineA
under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. 112.
The sequence of the polynucleotides contAi n~A in the deposited materials, as well as the amino acid sequence of the polypeptides ~nroA~A thereby, are in~v~vlated herein by _g_ CA 022l72l6 l997-l0-02 W O 96~9521 PCTrUS95/07294 reference and are controlling in the event of any con~lict with any description of se~l~nce~ herein. A license may be required to make, use or sell the depo8ited materials, and no such license is hereby granted.
The present invention further relates to polypeptides which have the ~ lc~ amino acid sequence of Figure 1 (SBQ
ID NO:2 ) or which have the amino acid sequence encoded by the deposited cDNA, as well as fra~m~nts, analogs and derivatives of such polypeptides.
The terms "fragment, n "derivative" and "analog" when referring to the polypeptide o_ Figure 1 (SEQ ID NO:2) or that encoded by the deposited CDNA, means a po}ypeptide which retain essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the ~~ otein portion to produce an active mature polypeptide. A derivative or fragment may include, for ~X~rle~ a splice variant which has less amino acid residues than the polypeptide of Figure 1 but which still retains biological activity characteristic of human ch~m~kine polypeptides.
The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably reco~;n~nt polypeptides.
The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO: 2) or that ~n~-o~ by the deposited cDNAs may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one ~nro~ by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a c~..~ound to increase the half-life of the polypeptide ~for example, polyethylene glycol), or (iv) one in which the additional amino acids are W O 96~9521 PCTnUS95/07294 fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fra~r~ntc, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to h~oy~.eity.
The term ~lisolated" means that the material is removed from its original envi~u~ G-~t (e.g., the natural envi-o. -~.lt if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living ~n;m~l iS not isolated, but the same polynucleotide or polypeptide, separated from sOme or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be i801ated in that such vector or composition is not part of its natural envil~,-.e~t.
The polypeptides of the present invention include the polypeptide of SEQ ID N0:2 (in particular the mature polypeptide) as well as polypeptide6 which have at least 70%
similarity (preferably at least 70% identity) to the polypeptide of S8Q ID N0:2 and more preferably at least 90%
similarity (more preferably at least 90% identity) to the polypeptide of SBQ ID N0:2 and still ~ re preferably at least 95% s;~;l~rity (still more preferably at least 95% identity) to the polypeptide of SBQ ID N0:2 and also include portions of such polypeptides which generally ront~;n at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides is determ; n~ by rnmr~ring the amino acid seque2nce and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

W O 96~9521 PCTAJS95~/~94 Fra~-m~nt~ or portions of the polypeptides of the present invention may be employed for producing the corresponA;n~
full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synth~;ze full-length polynucleotides of the present invention.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recomh;n~nt techniques.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as 8~LC~ iate for activating promoters, selecting transformants or amplifying the Ck~-13 genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for pro~--~; ng polypeptides by recomh;n~nt t~rhn;~ues. Thus, for ~Y~mrle~ the polynucleotide may be included in any one of a variety of expression vector~ for expressing a polypeptide. Such vectors include chromosomal, nonC~ somal and synthetic DNA se~-~nc~s, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmidsi vectors derived from comh;n~tions of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox viru~, and pseudorabies.

W O 96~9521 PCTAUS9S/07294 However, any other vector may be used as long as it is replicable and viable in the host.
The appropriate DNA sequence may be in~erted into the vector by a variety of procedure~. In general, the DNA
sequence is inserted into an appropriate restriction ~n~nnnclease site~s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an d~' ~' iate expre8sion control sequence(s) (promoter) to direct mRNA 8ynthesi8. As representative examples of such promoters, there may be ment~one~: LTR or SV40 promoter, the E. coli. lac or trp, the phage 1 A~A p~
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also rontA; n~ a ribosome h; n~; ng site for translation initiation and a transcription tenminator.
The vector may also include d~' ~' iate sequences ~or amplifying expression.
In addition, the expression vectors preferably cnntA~n one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in B. coli.
The vector contA;n;n~ the ~ iate DNA sequence as her~;nAhove described, as well a~ an a~ iate promoter or control 6equence, may be employed to transform an appropriate host to penmit the host to express the protein.
As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, strePtomYces, ~1 m~n~l la tYPhimurium; fungal cells, such as yeast; insect cells such as DrosophilA S2 and SPodo~tera Sf9;
~n;m~l cells such as CH0, COS or Bowes ~-lAnO--;
adenoviruses; plant cells, etc. The selection of an WO 96~9521 PCT~US95/07294 appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes reco~h~nAnt constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embo~;m~nt, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large num.~ers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQB70, pQE60, pQ~-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SR, pBSRS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); pTRC99a, pRR223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Bukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
Promoter regions can be selected from any desired gene using CAT (chlor~mrh~nicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, 1~mhtl~ PR~ PL and trp.
Eukaryotic promoters include CMV immediate early, HSV
thym;~n~ kinase, early and late SV40, LTRs from retrovirus, and mou~e metallothionein-I. Selection of the ~lo~liate vector and ~ Ler i8 well within the level of ordinary skill in the art.
In a further em.~o~; -nt, the present invention relates to host cells cont~;n;ng the above-described constructs. The host cell can be a higher eukaryotic cell, such as a m~ ian cell, or a lower eukaryotic cell, such as a yeast WO 96~9521 PCT~US95/07294 cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DBAB-Dextran mediated tran8fection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
The constructs in ho8t cells can be used in a conventional ~-nn~r to produce the gene products encoded by the recombinant se~l~nC~. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expre88ed in mammalian cells, yeast, bacteria, or other cell8 under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sd,.~look, et al., Molecular Cloning: A Laboratory MAnllAl, Second _dition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA ~nco~; ng the polypeptides of the present invention by higher eukaryotes is increased by inserting an ~nhAncer sequence into the vector. RnhAnC~r8 are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a ~l- -Ler to increase its transcription.
Bxamples include the SV40 ~nh~nr~r on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter ~nhAncer, the polyoma ~nhAncer on the late side of the replication origin, and adenovirus ~nhAncers.
Generally, re~_ ~nAnt expression vectors will include origins of replication and 8electable markers permitting transfonmation of the host cell, e.g., the ampicillin resistance gene of B. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct W O 96~9521 PCTAUS95/07294 transcription of a downstream structural sequence Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ~-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in ~-~riate phase with translation initiation and termination sequences, and preferably, a leader sequence c~p~hle of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence ~ncoA; n~
a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will ~_ , ise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include B. coli, Bacillus subtilis, S~l~nella t~h~ ~ium and various species within the genera Psell~om~n~, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but non 1; m~ ting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available pl ~r~ ~ c~..~Lising genetic elements of the well known cloning vector pBR322 ~ATCC
37017). Such __ -~cial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGBM1 (~l~ -_ Biotec, Madison, WI, USA). These pBR322 -W O 96~9521 PCT/U~J/~7~54 ~Ibackbone" sections are ~ h~n~A with an ~-o~riate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by d~o~riate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract re~; n~ for further purification.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
Various ~-mm~ 1 ian cell culture systems can also be employed to express re~omh~ n~n~ protein. Examples of m~ l ian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines c~p~hle of expressing a compatible vector, for example, the C127, 3T3, CH0, HeLa and BHK cell lines. ~ ian expre~sion vectors will comprise an origin o~ replication, a suitable promoter and ~nh~n~
and also any necessary ribosome h~ n~ ng sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA seql~n~eC derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic el~ - t~.
The polypeptides can be recovered and purified from reco~h~n~nt cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exch~nge chromatography, phosphocellulose chromatography, hydroph~h~c interaction chromatography, a~inity chromatography, hyd~oxylapatite chromatography and CA 022l72l6 l997-l0-02 W O 96~9521 PCT/u'~sJ~7~54 lectin chrom.atography. Protein re~olding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography ~PLC) can be employed for final purification steps.
The polypeptides of the present invention m.ay be a naturally purified product, or a product o~ chemical synthetic procedures, or produced by recom.binant techni~ues from a prokaryotic or eukaryotic host ~for example, by bacterial, yeast, higher plant, insect and m~mmalian cells in culture) . Dep~n~; ng upon the host employed in a recQmh~n~nt production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial methionine amino acid residue.
The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and ~;~gnostics to human disease.
The polypeptide of the present invention m.~ay be employed to i nh; h; t bone marrow stem cell colony formation as adjunct protective treatment during c~nc~r chemotherapy. The Ck~-13 polypeptide may inhibit the proliferation and differentiation of hematopoietic cells such as bone marrow stem cells. The inhibitor ef~ect on the population of committed ~ uye.itor cells, ~for example granulocytes, and macrophage/...~,.~ytes) may be employed therapeutically to ; nh; h; t proliferation of leukemic cells.
The polypeptides of the present invention may also be employed to ;nh;h;t epidermal keratinocyte proliferation for treatment of psoriasis, which is characterized by keratinocyte hyper-proliferation, since Langerhans cells in skin have been found to produce ~ -k; n~c, The polypeptides of the present invention may also be employed to treat solid tumors, for example, Rarposi sarcoma, by stimulating the invasion and activation of host defense cells, e.g., cytotoxic T cells and macrophages via ., WO 96~9521 PCTAUS95/07294 chemotaxis, and by ;nhi h; ting the angiogenesis o~ tumors.
They may also be employed to ~nhAnre host defenses against resistant chronic and acute infections, for example, mycobacterial infection8 via the attraction and activation of microbicidal leukocytes.
The the polypeptides of the present invention may also be employed to inh;hit T cell proliferation by the inhibition of IL-2 biosynthesis for the treatment of T-cell mediated auto-;mm~lne diseases and lymphocytic leuks~;~.
Ck~-13 may also be employed to st;m~ te wound healing and prevent scarring during wound h~l;ng, both via the recruitment of debris clearing and connective tissue promoting inflammatory cells and also via its control of excessive TGF~-mediated fibrosis. In this same m~nner~ Ck~-13 may also be employed to treat other fibrotic disorders, including liver cirrhosis, osteoarthritis and plllm~n~ry fibrosis.
The polypeptides of the present invention also increase the presence of eosin~rh;l~ which have the distinctive function of killing the larvae of parasites that invade tissues, as in schistosomiasis, trirh;no~is and ascariasis.
They may also be employed to regulate hematopoiesis, by regulating the activation and differentiation of various hematopoietic ~ye~itor cells, for example, to release mature leukocytes from the bone marrow following chemotherapy.
The polypeptide of the present invention may also be employed to target ~l-- nted cells, such as in the treatment of r~n~r, for apoptosis.
The polynucleotides and polypeptides Pnro~ by such polynucleotides may also be utilized for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors and for designing therapeutics and diagnostics for the treatment of human disease.

L

W O 96~9521 PCTAJS95/07294 The polypeptide may also be used to m~h;l; ze bone marrow stem cells to peripheral blood, which allows ea8y isolation of stem cells. The isolated stem cells may be employed for bone marrow colonization after high dose chemotherapy.
This-invention is also related to the use of the Ck~-13 gene as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the nucleic acid sequences ~nco~; n~ a the polypeptide of the present invention. Such diseases are related to under-expression of the human chPmokine polypeptides, for example, tumors and c~ncP~s.
Individuals carrying mutations in a gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obt~;nP~ from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose.
As an example, PCR primers complementary to the nucleic acid PnCo~;ng Ck~-13 can be used to identify and analyze Ck~-13 mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing ampli~ied DNA to radiolabeled Ck~-13 RNA or alternatively, radiolabeled Ck~-13 antisense DNA
se~-PncP~. Perfectly matched se~PncPs can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
Genetic testing based on DNA ~equence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA
fr~ments of different se~lPncPC may be disting~ hP~ on CA 022l72l6 l997-l0-02 W O 96~9521 P ~ ~US9S/07294 denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985)).
Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection of a 8pecific DNA sequence may be achieved by methods suCh as hybridization, RNase protection, chemical cleavage, direct DNA 8e~'~nC; n~ or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA se~lPnring~ mutations can also be detected by in situ analysis .
The present invention also relates to a diagnostic a say ~or detecting altered levels of the polypeptide of the present invention in various tissues since an over-expression of the polypeptide compared to normal control tissue samples may detect the presence of a disease or susceptibility to a disease, for example, a tumor. A88ays used to detect levels of a the polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and include radioi~-noA~says, competitive-bi n~i ng assays, Western Blot analysis, BLISA assays and nsAn~'richn assay. An RT-TC~ ae~say (Coligan, et al., Current Protocols in T lnology, 1(2), h~pter 6, (1991)) initially cv~ ises preparing an Ant;h~y specific to a Ck~-13 antigen, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal Antiho~y. To the reporter antibody is attArh~ a detectable reagent such as radioactivity, fluore~cence or, in thi~ example, a horseradish peroxidase enzyme. ~ sample is removed from a WO 96~9521 PCT~US95/07294 host and incubated on a solid su~ L, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein b;nA;ng sites on the dish are then covered by incubating with a non-specific protein like BSA. Next, the monoclonal antibodv is incubated in the dish during which time the monoclonal ant;hoA;es attach to any Ck~-13 protein attAcheA to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter Ant; hOAY
linked to horseradish pero~;~Ase is now placed in the dish resulting in h; n~li ng of the reporter Ant;hody to any monoclonal antibody bound to the Ck~-13 polypeptide.
UnattArh~A reporter antibody is then washed out. Peroxidase sub6trates are then added to the di~h and the amount of color developed in a given time period is a measurement of the amount of Ck~-13 protein present in a given volume of patient sample when ._ ~A~ed against a stAnA~rd curve.
A competition assay may be employed wherein antibodies specific to the Ck~-13 polypeptide are attached to a solid 5U~V1L and labeled Ck,~-13 polypeptide and a sample derived from the host are passed over the solid su~o L and the amount of label detected, for example by liquid sc;nt;llation chromatography, can be correlated to a quantity of Ck~-13 polypeptide in the sample.
A "sandwich" assay is s;m;lA~ to an ELISA assay. In a ~sandwich" a~say the Ck~-13 polypeptide i~ passed over a solid support and binds to Ant;hoAy attArh~A to a solid support. A second Ant;hoAy i8 then bound to the Ck~-13 polypeptide. A third antibody which is labeled and specific to the second Ant;ho~y is then passed over the solid 8u~0 L
and binds to the second antibody and an amount can then be qllAnt;fied~
This invention provides a method for i~nt;fication of the receptors for the polypeptide of the present invention.
The gene ~ncoA;ng the receptor can be identified by numerous methods known to those of skill in the art, for example, CA 022l72l6 l997-l0-02 ligand p~nning and FACS sorting ~Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the labeled polypeptides. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and retransfected using an iterative sub-pooling and rescreening process, eventually yielding a single clone(s) that ~nco~s the putative receptor.
As an alternative approach for receptor i~nt~fication, the labeled polypeptides can be photoaffinity linked with cell .lle"~e or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE
analysis and exposed to X-ray film. The labeled complex contA;n~ng the receptors of the polypeptides can be excised, resolved into peptide fra~ ntS, and subjected to protein microseq~l~nc;ng~ The amino acid sequence obtA;ne~ from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes ~nC~; ng the putative receptors.
This invention provides a method of screening compounds to identify agonists and antagonists to the polypeptide of the pre~ent invention. An agonist is a compound which bind~
to and activates a receptor to the polypeptide of the present invention, while antagonists bind to and ;nh; h; t such receptors or simply compete with Ck~-13 for such receptors.
Chemotaxis may be assayed by placing cells, which are rh~oAttracted by the polypeptide of the present invention, W O 96~9521 PCT~US95/07294 on top of a filter with pores of sufficient diameter to admit the cells (about 5 ~m). Solutions of potential agonists are placed in the bottom of the rhAmh~r with an appropriate control medium in the upper cg~r~rtment, and thus a concentration gradient of the agonist is measured by counting cells that migrate into or through the porous ~ dne over time.
When assaying for antagonists, the polypeptide of the present invention is placed in the bottom chAmh~r and the potF~nt; A 1 antagonist i8 added to determine if chemotaxis of the cells is prevented.
Alternatively, a m~mm~l;An cell or ' dne preparation expressing the receptors of the polypeptides would be incubated with a labeled polypeptide of the present invention, eg. radioactivity, in the presence of the compound. The ability of the c~..~o~d to block this interaction could then be measured.
13xamples of pot~'nt; Al antagonists to the polypeptide Of the present invention include Antiho~;es, or in some cases, oligonucleotides, which bind to the polypeptides. Another example of a potential antagonist is a negative ~nm; nAnt mutant of the polypeptide~. Negative ~omtnAnt mlltAnt~ are polypeptides which bind to the receptor of the wild-type polypeptide, but fail to retain biological activity.
Antisense constructs prepared using antisense technology are also pot~nt; Al antagonists. Anti~ense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on h~ nA; ng of a polynucleotide to DNA or RNA. For example, the 5' ro~; ng portion Of the polynucleotide sequence, which rnrsA~s for the mature polypeptides of the present invention, is used to design an antisense RNA
oligonucleotide of from about 10 to 40 base pairs in length.
A DNA oligonucleotide is designed to be comple~snt~ry to a region of the gene involved in transcription (triple- helix, W O 96~9521 PCTAUS95/07294 see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 ~1988); and Dervan et al., Science, 251:
1360 (1991)), thereby preventing transcription and the production of the polypeptide of the present invention. The antisense RNA oligonucleotide hybridizes to the m~NA ~n vivo and blocks translation of the mRNA molecule into the polypeptides (antisense - Okano, J. Neurochem., 56:560 ~1991); Oligodeoxynucleotides as Antisense Tnh;h;tors of Gene ~xpression~ CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that the antisen8e RNA or D'.~A may be expressed in vivo to i nh; hit production of the polypeptide of the present invention.
Another potential antagonist is a peptide derivative of the polypeptides which is a naturally or synthetically modified analog of the polypeptide that have lo~t biological function yet still recognizes and binds to the receptors of the polypeptide to thereby effectively block the receptors.
~xamples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules.
The antagonists may be employed to inh; h; t the chemotaxis and activation of macrophages and their precursors, and of neutrophils, h~Corh;ls, B lymphocytes and some T cell subsets, e.g., activated and CD8 cytotoxic T
cells and natural killer cells, in certain auto-; ~ and chronic inflammatory and infective diseasee. Bxamples of auto-immune diseases include multiple sclerosis, and insulin-dep~n~nt diabetes.
The antagonists may al~o be employed to treat infectiou~
diseases including silicosis, ~arcoidosis, idiopathic pl~lm~n~y fibrosis by prev~ntt ng the recruitment and activation of monnntlclear phagocytes. They may also be employed to treat idiopathic hyper-eos;nnrh;lic syndrome by preventing eos;norh~l production and migration. Endotoxic shock may also be treated by the antagonists by preventing ~.

W O 96~9521 PCTrUS9SJ'~/~94 the migration of macrophages A nd their production o~ the polypeptide of the present invention.
The antagonists may also be employed for treating atherosclerosis, by preVPnti n~ monocyte infiltration in the artery wall.
The antagonists may also be employed to treat hiStAm; n~_ mediated allergic reactions and ;m~-7nological disorders including late phase allergic reactions, chronic urticaria, and atopic dermatitis by i nh; h; ting ch~mokine-induced mast cell and basophil degranulation and release of his7-Am; n~, Ig~-mediated allergic reactions such as allergic asthma, rhinitis, and eczema may also be treated.
The antagonists may also be employed to treat chronic and acute inflammation by preventiny the attraction of monocytes to a wound area. They may also be employed to regulate normal plll Ary macrophage populations, since chronic and acute inflammatory plllmonAry diseases are associated with sequestration of m~nonllclear phagocytes in the lung.
Antagonists may also be employed to treat rheumatoid arthritis by preventing the attraction of monocytes into synovial fluid in the joints of patients. Monocyte influx and activation plays a significant role in the pathogenesis of both degenerative and inflawmatory arthropathies.
The antagonists 7nay be employed to interfere with the deleterious cascades attributed primarily to IL-1 and TNF
which p e~el-Ls the biosynthesis of other inflammatory cytokines. In this way, the antagonists may be e7~nployed to prevent inflammation. The antagonists may also be employed to inhihit prostagl~n~;n-independent fever induced by ch~mokines .
The antagonists may also be employed to treat cases o~
bone marrow failure, for example, aplastic ~nP~i~ and myelodyspla~tic syndrome.

CA 022l72l6 l997-l0-02 W O 96~9521 PCT/U',5/'~7~94 The antagonists may also be employed to treat asthma and allergy by preventing eosinophil accumulation in the lung.
The antagonists may also be employed to treat subepithelial basement membrane fibrosis which is a pr~in~nt feature of the asthmatic lung.
The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
The polypeptide of the present invention and agonists and antagonists may be employed in rc~mh;nAtion with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation 8hould suit the mode of A~m; n; stration.
The invention also provides a pharmaceutical pack or kit comprising one or more ~nntA;n~rs filled with one or more of the ingredients of the pharmaceutical cv ~o~itions of the invention. Associated with suCh contA;ner(s) can be a notice in the form prescribed by a gov~nm~ntal agency regulating the madnufacture, use or sale of pharmaceuticals or biological products, which notice reflect~ d~o~al by the agency of manufacture, use or sale for human A~m;n; stration. In addition, the polypeptides and agonists and antagonists may be employed in conjunction with other therapeutic compounds.
The pharmaceutical ~-J~ ition~ may be A~m; ni ~tered in a convenient ~-nn~ such as by the topical, intravenous, intraperitoneAl, intramuscular, intratumor, subcutaneous, intrAnA~Al or intradenmal route~. The pharmaceutical compositions are A~m;n;stered in an amount which is effective for treating and/or prophylaxis of the specific indication.
In general, the polypeptides will be A~m;n;~tered in an amount of at least about 10 ~g/kg body weight and in most W O 96~9521 PCTAUS95/07294 cases they will be ~mini~tered in an amount not in excess o~
about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 ~g/kg to about 1 mg/kg body weight daily, taking into account the routes of A~mi ni stration, symptoms, etc.
The polypeptide of the present invention, and agonists or antagonists which are polypeptides, may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy."
Thus, ~or example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) ~nCO~;ng a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA ~nCOAi ng a polypeptide of the present invention.
S;m;la~ly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for pro~l~c~ng a retroviral particle c~nt~ining RNA
encoding the polypeptide of the present inv~nt~on may be ~m; n;stered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the te~chings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after comb;n~tion with a suitable delivery vehicle.
Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine ~eukemia Virus, spleen necrosis W O 96~9521 PCTAUS95/07294 virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immllno~eficienCy virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus In one ~m~g~; ~ t, the retroviral plasmid vector is derived from Moloney Murine Leukemia viru~.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ~-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and Bl9 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings cont ,A ~neA herein.
The nucleic acid seguence ~nCOA; ng the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late ~ ~er; or hetorologous promoters, such as the cyt~ ovirus (CMV) promoter; the respiratory syncytial virus (RSV) ~l~ ~Ler; ;n~l~c;hle promoters, such as the MMT promoter, the metallothionein promoter; heat shock -ters; the A 1 h lm; n ~ r ~ ~ Ler; the ApoAI ~ r ~ ~ Ler; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs her~;nAhove described); the ~-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the genes ~nro~;ng the polypeptides.

W O 96~9521 PCTAUS95/07294 The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17 ~2, ~CRE, ~CRIP, GP+F-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Thera~, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by re~erence in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use o~
liposomes, and CaP0~ precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then ~m;n;~tered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) ~nco~ ng the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or i~ vivo. The transduced eukaryotic cells will express the nucleic acid seguence(s) ~nCo~ing the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic car~;nor-- cells, as well as hematopoietic stem cells, hepatocytes, ~ibroblasts, myoblasts, keratinocytes, endothelial cells, and bronch;Al epith~l;~l cells.
The sequences of the present invention are also valuable ~or chromosome i ~Qnt; fication. The sequence is ~pecifically targeted to and can hybridize with a particular location on an individual human ch-~ ~ ~. Moreover, there is a current need for i~nt;fying particular sites on the chromosome. Few Cl~ ~ v~ ome m~k; n~ reagents based on actual ~equence data (repeat polymorphisms) are presently aV~ hl e ~or marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step W O 96~9521 PCTAUS95/07294 in correlating those sequences with genes associated with disease.
Briefly, seguences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
Computer analysis of the 3' untranslated region is used to rapidly select primers that do not 8pan more than one exon in the genomic DNA, thus complicatin~ the amplification process.
These primers are then used for PCR screening of somatic cell hybrids cont~n~ng individual human chromosomes. Only those hybrids contA~n~ng the human gene correspnn~;ng to the primer will yield an amplified fragment.
PCR mapping of somatiC cell hybrid8 is a rapid procedure for assigning a particular DNA to a particular cl~ ~some.
Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clone(s) in an analogous ~nne~. Other mapping strategies that can similarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA
clone(s) to a met~ph~e ch~ ~mal spread can be used to provide a precise c~o."o~omal location in one step. This technique can be used with cDNA as short as 500 or 600 bases.
For a review of this technique, see Verma et al., ~nm~n Chromosomes: a ~nn~l of Basic Techniques, Pely~ o~, Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. Mc~usick, M~n~lian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relatinnch~ r between genes and diseases that have been mapped to the same W O 96~9521 PCTAUS95/07294 chromosomal region are then identi~ied through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the di~ferences in the cDNA or genomic sequence between a~ected and unaffected individuals. If a mutation is observed in some or all of the a~fected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one o~ between 50 and 500 potential causative genes. (Thi~
assumes 1 m~g~hAce mapping resolution and one gene per 20 kb).
The polypeptides, their ~ragments or other derivatives, or analogs thereof, or cells expressing them can be used as an ; ~ ~n~en to produce ~ntihoA;es thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes rh;m~iC, single chain, and hnm~n;zed Ant;hodies, 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 o~ such ant;hoA;es and ~ragm~nt~.
~ nt;hoA;es generated ~-;n~t the polypeptides corresponA;ng to a sequence o~ the present invention can be obt~;neA by direct injection of the polypeptides into an ~n;m-l or by a ministering the polypeptides to an ~nim~l, pre~erably a n~n~ lan. The antibody so obt~;n~A will then bind the polypeptides itself. In this m~nn~, even a sequence encoding only a ~ragment of the polypeptides can be used to generate ~nt;hoA;es h;n~;ng the whole native polypeptides. Such ~ntiho~;es can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of m~norlQn~l antihodies, any technique which provides ant;hn~;es pr~All~e~ by cont;nlloll~ cell line CA 022l72l6 l997-l0-02 W O 96~9521 PCTAUS95/07294 cultures can be used. Bxamples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:4g5-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Tmmllnology Today 4:72), and the BBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to i lno~eniC polypeptide product~
of this invention. Also, tran8genic mice may be used to express hll~-nized antibodies to i~mlnogeniC polypeptide products 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 i8 not limited to such examples. All parts or amounts, unle8s otherwise specified, are by weight.
In order to facilitate understAn~ing of the following examples certain frequently occurring methods and/or terms will be described.
"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or nllmh~s The starting plasmids herein are either ro-~cially available, publicly avA; l ~hl e on an unrestricted basis, or can be constructed from available plasmids in accord with pllhl i ~h~
procedures. In addition, equivalent plA~mi~c to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially aVA i l A~l e and their reaction conditions, cofactors and other requir~ntc were used as would be known to the ordinarily skilled artisan. For W O 96~9521 PCTAUS95/07294 analytical purposes, typically 1 ~g o~ plasmid or DN~
fragment is used with a~out 2 units o~ enzyme in about 20 ~1 of buffer solution. For the purpose of isolating DNA
fra3r~nt~ for plasmid construction, typically 5 to S0 ~g of DNA are digested with 20 to 250 units o~ enzyme in a larger volume. Appropriate buf~ers and substrate amounts for particular restriction enzymes are specified by the manu~acturer. Tn~ tion times o~ about 1 hour at 37 C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired ~ragment.
Size separation of the cleaved fra~m~nt~ is performed using 8 percent polyacrylamide gel described by Goeddel, D.
et al ., Nucleic Acids Res., 8:4057 ~1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. 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 eFhoFphorylated .
nLigation" refers to the process o~ ~orming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accompli~h~ using known buffers and conditions with 10 units to T4 DNA ligase (nligasen1 per 0.5 ~g of approximately equimolar amounts of the DNA fr~l nts to be ligated.
Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Fb, A., Virology, 52:456-457 (1973).

W O 96~9521 PCTAUS95/07294 ExamDle 1 Bacterial Bxpression and Purification of Ck~-13 The DNA sequence encoding for Ck~-13, ATCC # 97113, is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the processed Ck~-13 nucleic acid 8equence (minus the putative signal peptide sequence). Additional nucleotides corresponA;ng to the Ck~-13 gene are added to the 5' and 3' end sequences respectively. The 5' oligonucleotide primer has the sequence 5~ CCCGCATGCCCAACATGGAAGACAG 3' (S8Q ID N0:3) cont~n~ a SphI
restriction enzyme site (bold) followed by 16 nucleotides of Ck~-13 coding sequence starting from the second nucleotide of the sequence roA; ng for the mature protein. The ATG codon is included in the SphI site. In the next codon following the ATG, the first base is from the SphI site and the r~m~ntng two bases correspond to the 8econd and third base of the first codon (residue 29) of the putative mature protein. The 3~ sequence 5' AAAGGA~ -lw CTCAGCTTATTGAG 3' (SBQ ID N0:4) cont~i nc Compl~m~nt~ry Seqll~nc~c to a BamH1 site (bold) and is followed by 18 nucleotides of gene specific sequences preceding the termination codon. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQB-9 (Qiagen, Inc. Chatsworth, CA). pQB-9 ~nCOA~ antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/0), a ribosome h~nA~ng site (RBS), a 6-His tag and restriction enzyme sites. pQB-9 is then digested with SphI and BamH1. The amplified sequences are ligated into pQE-9 and are inserted in frame with the sequence encoding for the hist; Ai n~ tag and the RBS. The ligation mixture is then used to transform the B. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sa~look, J. et al., Molecular Cloning: A Laboratory ~nn~l, Cold Spring Laboratory Press, (1989). M15/rep4 cone~ nc mu~tiple copies of the plasmid pRBP4, which expresses the lacI repressor and CA 022l72l6 l997-l0-02 W O 96~9521 PCT~US9S/07294 also confers kanamycin resistance (Kanr) Transformants are i~nt;fied by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected.
Plasmid DNA is isolated and confirmed by restriction analysis. Clone(s) r~nt~;n;ng the desired constructs are grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml).
The 0/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.~) of between 0.4 and 0.6. IPTG
(nIsopropyl-B-D-thiogalaCtO pyranoside") i8 then added to a final concentration of 1 mM. IPTG ;n~llce~ by inactivating the lacI repressor, clearing the P/0 leading to increased gene expression. Cells are grown an extra 3 to 4 hours.
Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl pH 5Ø After clarification, solubilized Ck~-13 is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight h; nrl;ng by proteins cont~;n~ng the 6-His tag (~orhlll~, E. et al., J.
Chromatography 411:177-184 (1984)). Ck~-13 ( ~98~ pure) is eluted from the column in 6M guanidine HCl. Protein renaturation out of GnHCl can be accompl; ~h~l by several protocols (Jaenicke, R. and Rudolph, R., Protein Structure -A Practical Approach, IRL Pres~, New York (1990)).
Initially, step dialysis is utilized to remove the GnHCL.
Alternatively, the purified protein isolated from the Ni-chelate column can be bound to a second column over which a decreasing 1;n~ GnHCL gradient is run. The protein is allowed to renature while bound to the column and is subsequently eluted with a buffer cont~;ning 250 mM
Imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.S and 10~
Glycerol. Finally, soluble protein is dialyzed against a storage buffer rnnt~;n;ng 5 mM r ;um Bicarbonate.

W O 96/39521 P ~/U~S/~/~51 ~xamPle 2 Ex~ression of Recombinant Ck~-13 in COS cells The expression of plasmid, Ck~-13 HA is derived from a vector pcDNAI/Amp (Invitrogen) cont~;n;ng: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA
fragment ~ncoA;ng the entire Ck~-13 precursor and a HA tag fused in frame to its 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 correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to the target protein allows easy detection of the rec~h;n~nt protein with an ~nt;hoAy that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence encoding for Ck~-13, ATCC # 97113, is constructed by PCR using two primers: the 5' primer 5' AAA
AAGCTTAACATAGGCTCGCCTACAGACT 3' (SEQ ID NO:5) contains a HindIII site ~Jllowed by 18 nucleotides of Ck~-13 ~oA~ng sequence starting from the minus 3 position relative to initiation codon; the 3' sequence 5' CGCTrT'~TTAAGCGT
A~l~-l~4A~1~lA1~lAl-lw ~-l~AGCTTATTGAGAAT 3' (SEQ ID NO:6) cont~;nR c~mplementary seq~nc~ to an XbaI site, translation stop codon ~underlined), HA tag and the last 21 nucleotides of the Ck~-13 coA~g sequence (not including the stop codon).
Therefore, the PCR product cont~;nR a HindIII site, Ck~-13 coA;ng sequence followed by HA tag fused in frame, a translation tenmination stop codon next to the HA tag, and an XbaI site. The PCR amplified DNA fragment and the vector, pcDNA3/Amp, are digested with HindIII and XbaI restriction enzyme and ligated. The ligation mixture is transfonmed into WO 96~9521 PCT~US95/07294 E. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and ~Y~m;ne~ by restriction analysis for the presence of the correct fragment. For expression of the recombinant Ck~-13 polypeptide, COS cells are transfected with the expression vector by D}3AE-DEXTRAN
method (J. S~,-~-ook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory M~n~ , Cold Spring Laboratory Press, (1989)). The expression of the Ck~-13 HA protein is detected by radiolabelling and ; ~ ~nnr~ecipitation method (~. Harlow, D. Lane, Antibodies: A Laboratory M~nll~l, Cold Spring Harbor Laboratory Press, (1988)). Cells are l~h~lled 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, 50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)).
Both cell lysate and culture media are precipitated with a HA
specific monoclonal ~ntihO~y Proteins precipitated are analyzed by SDS-PAGB.

Exam~le 3 Cloninq and expression of CkB-13 usinq the baculovirus expression svstem The DNA sequence ~nco~;n~ the full length Ck~-13 protein, ATCC # 97113, is amplified using PCR oligonucleotide primers corr~r~n~ng to the 5' and 3' seqll~nr~c of the gene:
The 5' primer has the sequence 5' AAAG&ATCCG~
GCCTACAGACT 3' (SBQ ID NO:7) and cont~;n~ a BamHI restriction enzyme site (in bold) followed by 6 nucleotides res~mhl;ng an efficient signal for the initiation of translation in eukaryotic cells (Rozak, M., J. Mol. Biol., 196:947-950 (1987) and the first 18 nucleotides of the Ck~-13 gene (the initiation codon for translation "ATG" is underlined).
The 3' primer has the sequence 5' AAAGGTACCTCATTGG

CA 022l72l6 l997-l0-02 W O 96~9521 PCTAUS95/07294 CTCAGCTTATT 3' (SEQ ID NO:8) and cont~;n~ the cleavage site for the restriction ~n~Qn~ ease Asp718 and 18 nucleotides compl~m~nt~y to the 3' non-translated sequence of the Ck~-13 gene. The am.plified se~l~nc~ are isolated from a 1% agarose gel using a commercially available kit ("Geneclean,~ BIO 101 Inc., ~a Jolla, Ca.). The ~ragment is then digested with the ~n~9nllcleaSes BamHI and Asp718 and then purified again on a 1~ agarose gel. This fragment is designated F2.
The vector pRG1 (modification of pVL941 vector, discussed below~ is used for the expression of the Ck~-13 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.B. 1987, A m~nll~l of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experim~nt~l Station Bulletin NO:1555).
This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction ~n~onll~l eases BamHI and Asp718. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant viruses the beta-galactosidase gene from B.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin se~l~nc~fi are flanked at both sides by viral sequences for the cell-mediated homologou~ rer~mhin~tion of cotransfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
The plasmid is digested with the restriction enzymes BamHI and Asp718 and then ~phoqphorylated using calf intestinal phosphatase by procedures known in the art. The DNA is then isolated from a 1% agarose gel using the commercially available kit (nGeneclean" BI0 101 Inc., La Jolla, Ca.). This vector DNA is designated V2.

W O 96~9521 PCT/U'~5/~/~54 Fragment F2 and the dephosphorylated plasmid v2 are ligated with T4 DNA ligase. E.coli B 101 cells are then transformed and bacteria identified that cont~in~ the plasmid (pBac-Ck~-13) with the Ck~-13 gene using the enzymes BamHI and Asp718. The sequence of the cloned fragment is confirmed by DNA se~l~nc;ng.
5 ~g of the plasmid pBac-Ck~-13 is cotransfected with 1.0 ~g of a commercially available lin~rized baculovirus ("BaculoGold~ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)).
l~g of BaculoGold~ virus DNA and 5 ~g of the plasmid pBac-Ck~-13 are m.ixed in a sterile well of a microtiter plate cont~tning 50 ~l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~l Lipofectin plus 90 ~l Grace~s medium are added, mi Y~ and incubated for 15 minutes at room temperature. Then the transfection mixture is added ~l~..ise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with lml Grace's medium without ~erum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27~C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace~s insect medium supplemented with 10~ fetal calf serum is added. The plate is put back into an incubator and cultivation continll~A at 27~C for four days.
After four day6 the supernatant is collected and a plaque assay performed similar as described by Summers and Smith (supra). A~ a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue st~in~A plaques. (A
detailed description of a "plaque assay" can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10) .

CA 022l72l6 l997-l0-02 W O 96~9521 PCTAUS95/07294 Four days after the 3erial dilution, the viruses are added to the cells and blue stained plaques are picked with the tip of an Eppendorf pipette. The agar rontA;ning the recombinant viru8es is then resuspended in an Eppendorf tube contAin~ng 200 ~l of Grace's medium. The agar is removed by a brief centrifugation and the supernatant ~OntA; n; ng the rec~nAnt baculovirus i8 used to infect Sf9 cells seeded in 35 mm ~i Sh~:. Four days later the supernatants of these culture dishes are harvested and then stored at 4~C.
Sf9 cells are grown in Grace's medium supplemented with 10~ heat-inactivated FBS. The cells are in~ected with the reC~mh~nAnt baculovirus V-Ck~-13 at a multiplicity of infection (MOI) of 2. Six hours later the medium is ~_...oved and replaced with SF900 II medium minus methionine and cysteine (Life TechnolOgieS Inc., Gaithersburg). 42 hours later 5 ~Ci of 35S-meth;on;ne and 5 ~Ci 35S cysteine (Amersham) are added. The cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGB and autoradiography.

ExamPle 4 Ex~ression via Gene Therapy Fibroblasts are obtA;n~ from a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small ~hllnk~ of the tissue are placed on a wet surface of a tissue culture flask, a~.o~imately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% ~3S, penicillin and streptomycin, is added.
This is then incubated at 37~C for ~Lo~imately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in WO 96~9521 PCTrUS9~/07294 culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3~ end sequences respectively. The 5' primer cont~ning an EcoRI site and the 3~ primer further includes a HindIII site.
Equal quantities of the Moloney murine sarcoma virus l;n~r backbone and the amplified EcoRI and HindIII ~ragment are added together, in the presence o~ T4 DNA ligase. The resulting mixture is ~nt~;ne~ under conditions ~-~riate for ligation of the two fragments. The ligation mixture is used to transform bacteria B 101, which are then plated onto agar-~nnt~;n~n~ kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10~ cal~ serum (CS), penicillin and streptomycin. The MSV vector ront~;n;ng the gene is then added to the media and the packaging cells are transduced with the vector. The p~k~ging cells now produce infectious viral particles rQnt~;n;n~ the gene (the packaging cells are now referred to as producer cells).
Fresh ~ is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, cnnt~;n;ng the infectious viral particles, is filtered tl~o~yh a millipore filter to .eu~e det~ch~ pro~lc~r cells and t_is media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fiDroblasts and quickly replaced CA 022l72l6 l997-l0-02 WO 96~9521 PCT~US~ 7~1 with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.
Numerous modifications and variations of the present invention are possible in light of the above t~ch;ngs and, therefore, within the scope of the appended rl~;mc, the invention may be practiced otherwise than as particularly described.

CA 022l72l6 l997-l0-02 W O 96~9521 PCTrUS95/07294 SEQu~ LISTING

(1) r~RNR~AL INFORMATION:
(-i) APPLICANT: LI, ET AL.

(ii) TITLE OF lNv~sNllON: Human C'h' -~cine Beta-13 (iii) NUMBBR OF SBQ~S: 8 (iv) ~O~RR-~PONDBN OE Ann~R~s:

(A) ~nn~RC-~BE CARBLLA, BYRNE, BAIN, GILFILLAN, OE CCHI, STBWART & OLSTBIN
(B) STREET: 6 BECRBR FARM ROAD
(C) CITY: ~OSRT-~ND
(D) STATB: NBW JBRSBY
(B) ~OUN-1KY: USA
(F) ZIP: 07068 (V) COM~U 1~K RRADABLE FORM:
(A) MBDIVM TYPB: 3.5 INCH DISK~-l-(B) COh~ul~K: IBM PS/2 (C) OPBRATING SYSTBM: MS-DOS
(D) SOFTWARE: ~ORD PBRFBCT 5.1 (vi) ~U~RNT APPLICATION DATA:
(A) APPLICATION NU.MBBR:
(B) FILING DATB: Concurrently (C) CLASSIFICATION:
~vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER:
(B) FILING DATB:

CA 022l72l6 l997-l0-02 W O 96~9521 PCT~US95/07294 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION NUMBER: 36,134 (C) REFER_NOE/DOCKET N~3ER: 325800-~ix) TELECOMMUNICATION INFORMATION:
(A) TEL~Oh~: 201-994-1700 (B) TELEFAX: 201-994-1744 (2) lN~O~ ~TION FOR SEQ ID NO:1:

(i) SEQu~N~ CHARACTERISTICS
(A) L_NGTH: 282 BAS_ PAIRS
(B) TYPE: NUCLEIC ACID
(C) STR~N~RnNR.S.S: SINGL8 (D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

(xi) SBQu~ DESCRIPTION: SEQ ID NO:1:

A~G~-~CC TACAGACTGC A~-1C~L~-~ C~-~ CC ~C-.-G~ GGCG~60 GCAACTGAGG CAGKCCC~A CGGCGCCAAC ATGGA~GACA GC -~-~ CCGTGATTAC 120 CC-~-~ACC ~ ~CCC---- GCGC-~-~-G AA~C TTCT ACTGGACCTC AGACTCCTGC 180 CCGAGGCCTG GC-~-~-~--~ GCTAACCTTC AGGGATA~GG AGATCTGTGC CGATCCCAGA 240 GTGCC~.aGG TGAAGATGAT TCTr~T~A~- CTGAGCCAAT GA282 (2) lN~O.~. ~ATION FOR SEQ ID NO:2:
(i) S~UUKN~ CH~RACTERISTICS
(A) L_N~'l~: 93 AMINO ACIDS
(B) TYPB: AMINO ACID
(C) STR~NI lKI ~, ~.CS
(D) TOPOLOGY: LINBAR

(ii) MOL_CULE TYPE: PROTEIN

WO 96~9521 PCT~US95/07294 (xi) SEQu~ DESCRIPTION: SEQ ID NO:2:

Met Ala Arg Leu Gln Thr Ala Leu Leu Val Val Leu Val Leu Leu Ala Val Ala Leu Gln Ala Thr Glu Ala Gly Pro Tyr Gly Ala Asn Met Glu Asp Ser Val Cys Cys Arg Asp Tyr Val Arg Tyr Arg Leu Pro Leu Arg Val Val Lys His Phe Tyr Trp Thr Ser Asp Ser Cys Pro Arg Pro Gly Val Val Leu Leu Thr Phe Arg Asp Lys Glu Ile Cys Ala Asp Pro Arg Val Pro Trp Val Lys Met Ile Leu Asn Lys Leu Ser Gln (2) lN~O~TION FOR SBQ ID NO:3:

(i) SBQUBNCB CHARAC-l-~RISTICS
(A) LBNGTH: 25 BASE PAIRS
(B) TYPE: NUCLBIC ACID
(C) STRAN~K~K~S: SINGLE
(D) TOPOLOGY: T.TNR~

(ii) MOLBCULB TYPB: Oligonucleotide (xi) S~u~ DBSCRIPTION: SBQ ID NO:3:

(2) INFORMATION FOR SBQ ID NO:4:

(i) SEQu~ CHARACTERISTICS
~A) LBN~Th: 27 BASE PAIRS

W O 96~9521 PCTAUS95/07294 (B) TYPE: NUCLEIC ACID
(C) STR~Nl~Kl )~ KCs: SINGLE
(D) TOPOLOGY: LINBAR

(ii) MOLECULE TYPE: Oligonucleotide (Xi) :i~iyU~!;N-~S DBSCRIPTION: SBQ ID NO:4:

(2) INFORMATION FOR SBQ ID NO:5:

(i) SBQu~N~ CHARACTERISTICS
(A) LENGTH: 31 BASB PAIRS
(B) TYPB: NUCLBIC ACID
(C) ST~PN~K~ KSS: SINGLB
(D) TOPOLOGY: LINBAR

(ii) MOLBCULB TYPB: Oligonucleotide ~xi) SEQuh~ DBSCRIPTION: SBQ ID NO:5:

(2) lN~O~ ~TION FOR SEQ ID NO:6:

(i) SBQu~.~ CHARACTBRISTICS
(A) LBNGTH: 60 BASB PAIRS
(B) TYPB: NUCLBIC ACID
(C) STR~N~ )Kl ~._KS.~: SINGLB
(D) TOPOLOGY T.T ~ ~1~

(ii) MOLBCULE TYPB: Oligonucleotide (Xi) SEQU~K DBSCRIPTION: SBQ ID NO:6:

W O 96~9521 PCTAUS95/07294 C~ lA~AT TAAGCGTAGT CTGGGACGTC GTATGGGTAT 1 w ~-l~AGCT TATTGAGAAT 60 (2) INFORMATION FOR SBQ ID NO:7:

(i) SEQu~_ CHARACTBRISTICS
(A) LENGTH: 33 BASE PAIRS
(B) TYPE: NUCLBIC ACID
(C) STR~Nv_vw~SS: SINGLB
(D) TOPOLOGY: LINBAR

(ii) MOLBCULB TYPB: Oligonucleotide (xi) SBQUENCE DBSCRIPTION: SEQ ID NO:7:

AAAGGATCCG ~ TGGC TCGCCTACAG ACT 83 (2) lN~O.~TION FOR SBQ ID NO:8:

(i) ~_yu_N~K CHARACTBRISTICS
(A) LBNGTH: 26 BASE PAIRS
(B) TYPB: NUCLBIC ACID
(C) STRPN~K~NK~S: SINGLB
(D) TOPOLOGY: LINBAR

(ii) MOLBCULB TYPB: Oligonucleotide (xi) S_YU_N~_ DBSCRIPTION: SBQ ID NO:8:

Claims (20)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide comprising amino acid -28 to amino acid 65 as set forth in SEQ ID NO: 2;
(b) a polynucleotide encoding the polypeptide comprising amino acid 1 to amino acid 65 as set forth in SEQ ID NO: 2 (c) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a) or (b); and (d) a polynucleotide fragment of the polynucleotide of (a), (b) or (c).

2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3. The polynucleotide of Claim 2 which encodes the polypeptide comprising amino acid -28 to 65 of SEQ ID NO: 2.

4. The polynucleotide of Claim 2 which encodes the polypeptide comprising amino acid 1 to 65 of SEQ ID NO: 2 .

5. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide which encodes a polypeptide having the amino acid sequence expressed by the DNA
contained in ATCC Deposit No. 97113;
(b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a polynucleotide fragment of the polynucleotide of (a) or (b).

6. A vector containing the DNA of Claim 2.

7. A host cell genetically engineered with the vector of Claim 6.

8. A process for producing a polypeptide comprising:
expressing from the host cell of Claim 7 the polypeptide encoded by said DNA.

9. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 6.

10. A polypeptide selected from the group consisting of (i) a polypeptide having the deduced amino acid sequence of SEQ ID NO:2 and fragments, analogs and derivatives thereof; and (ii) a polypeptide encoded by the cDNA of ATCC
Deposit No. 97li3, and fragments, analogs and derivatives of said polypeptide.

11. A compound which mimics the activity of the polypeptide of claim 10.

12. A compound which antagonizes the activity of the polypeptide of claim 10.

13. An antibody against the polypeptide of claim 10 comprising a member selected from the group consisting of monoclonal and polyclonal antibodies.

14. A method for the treatment of a patient having need of Ck.beta.-13 comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 10.

15. The method of Claim 13 wherein said therapeutically effective amount of the polypeptide is administered by providing to the patient DNA encoding said polypeptide and expressing said polypeptide in vivo.

16. A method for the treatment of a patient having need to inhibit a Ck.beta.-13 polypeptide comprising:
administering to the patient a therapeutically effective amount of the compound of Claim 12.

17. A process for diagnosing a disease or a susceptibility to a disease related to an under-expression of the polypeptide of claim 10 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide.

18. A diagnostic process comprising:
analyzing for the presence of the polypeptide of claim 10 in a sample derived from a host.

19. A process for identifying a compound active as an agonist to the polypeptide of claim 10 comprising:
(a) combining a compound to be screened and a reaction mixture containing cells under conditions where the cells normally migrate in response to the polypeptide of claim 13; and determining the extent of migration of the cells to identify if the compound is effective as an agonist.

20. The process of claim 18 wherein a Ck.beta.-13 polypeptide is added to the combination of step (a), and the determination of the extent of migration identifies a compound effective as an antagonist.