CA2210471A1 - Human chemokine beta-11 and human chemokine alpha-1 - 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 the polynucleotides encoding the chemokine polypeptides and for detecting altered levels of the polypeptide in a host.

Description

CA 022l047l l997-08-08 Human Chemokine Beta~ nd Human Chemokine Alpha-1 This invention relates to newly identi~ied 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 polypeptides o$ the present invention are hllm~n ~h~mokine polypeptides, sometimes hereinafter referred to as hllm~n c~Pmnkine beta-ll (Ck~-11) and hllm~n ~hpmnkine alpha-l (Ck~-1). The invention also relates to inhibiting the action of such polypeptides.
~ hPm~kines~ also referred to as intercrine cytokine~, are a subfamily of structurally and functionally related cytokines. These molecule~ are 8-10 kd in size. In general, ~h~mnkines 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, ~h~mnkines 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 96/24668 PCT~US9~J~78D
"C-C" subfamily. Thus far, at least eight different members of this family have been identified in hllm~n~
The intercrine cytok~ n~s exhibit a wide variety of functions. A h~ rk feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many rhemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of hist~m;ne release, lysosomal enzyme and leukotriene release, increased adherence of target ~mm~lne cells to endothelial cells, ~nh~nced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain ch~mokines have been shown to exhibit other activities. For example, macrophage inflammatory protein 1 (MIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-3 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for m~l~nom~ cells.
In light of the diverse biological activities, it is not surprising that chPmokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound he~l~ng, hematopoietic regulation and ~mmlln~logical disorders such as allergy, asthma and arthritis.
Members of the "C-C" branch exert their effects on the following cells: eosinophils which destroy parasites to lessen parasitic infection and cause chronic inflammation in the airways of the respiratory system; macrophages which suppress tumor formation in vertebrates; and basophils which release hist~m;ne which plays a role in allergic inflammation. However, members of one branch may exert an effect on cells which are normally responsive to the other W096/24668 PCT~S95)01780 branch of chPm~kines and, therefore, no precise role can be attached to the member~ o~ the br~nch~
While members of the C-C branch act pre~omin~ntly on m~non11Clear cells and m~mh~rs of the C-X-C branch act pr~n~;n~ntly on neutrophils a distinct chemnAttractant property cannot be assigned to a chPm~kine based on this gui~-Pl~ne. Some rh~mnkineS from one family show characteristics of the other.
The polypeptides of the present invention have the conserved cysteine residues, namely Ck~-ll has "C-C" and Ck~-l has "C-X-C" regions, and they have high amino acid sequence homology to known rh~mokines and have, therefore, been putatively characterized as h11m~n ch~nkines.
In accordance with one aspect o~ the present invention, there are provided novel polypeptides which are h11m~n Ck~
and Ck~-l 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 therapeutically useful fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to Ck~-ll and Ck~-l sequences.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, cont~n~ng a Ck~-ll or Ck~-l nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said protein.

CA 022l047l l997-08-08 PCTnUS9~178 In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides for therapeutic purposes, for example, to treat solid tumors, chronic infe~tions, lellk~m;~, T-cell mediated auto-~
diseases, parasitic infections, psoriasis, asthma, allergy,to regulate hematopoiesis, to stimulate growth factor activity, to ~ nh; h~ t angiogenesis and to promote wound he~l ing.
In accordance 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-;mmllne diseases, atherosclerosis, chronic inflammatory and infectious diseases, hist~m;ne and IgE-mediated allergic reactions, prostagl~n~;n-independent fever, bone marrow failure, cancers, silicosis, sarcoidosis, rheumatoid arthritis, shock, hyper-eos;noph;l;c syndrome and fibrosis in the asthmatic lung.
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 Ck~-ll or Ck~-l nucleic acid sequences and 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 vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

W096/24668 PCT~S95J01780 The ~ollowing drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the inve~tion as encompassed by the claims.
Figure l displays the cDNA sequence and corresponding deduced amino acid sequence of Ck~-ll. The initial 17 amino acids represent the leader sequence such that the putative mature polypeptide comprises 81 amino acids. The stAn~d one-letter abbreviations for amino acids are used Sequencing was performed usina a 373 Automatec1 DNA sequencer (Applied Biosystems, Inc.). Sequencing accuracy is predicted to be greater than 97% accurate.
Figure 2 displays the cDNA sequence and corresponding deduced amino acid sequence of Ck~-l. The initial 22 amino acids represent the leader sequence such that the putative mature polypeptide comprises 87 amino acids. The stAn~rd one-letter abbreviations for amino acids are used.
Figure 3 displays the amino acid sequence homology between Ck~-ll (top) and the Rat RANTES polypeptide (bottom).
Figure 4 displays the amino acid sequence homology between Ck~-l and Ovis Aries interleukin-8 (bottom).
In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature polypeptides having the deduced amino acid sequences of Figures l (SEQ ID No. 2) and 2 (SEQ
ID No. 4) or for the mature polypeptides encoded by the cDNAs o~ the clones deposited as ATCC Deposit No. 75948 (Ck~-ll) and 75947 (Ck~-l) on November ll, 1994.
Polynucleotides encoding Ck~-ll may be isolated from numerous hl1m~n adult and fetal cDNA libraries, for example, a human fetal spleen cDNA library. Ck~-ll is a member of the C-C branch of chem~kines. It cont~in~ an open reading frame encoding a protein of 98 amino acid residues of which approximately the first 17 amino acids residues are the putative leader sequence such that the mature protein comprises 81 amino acids. The protein exhibits the highest W 096124~68 P~-l/u~fJvI780 degree of homology to the Rat R~NTBS polypeptide wltn identity and 47% s;m;l~ity over a stretch of 89 amino acids.
It is also important that the four spatially conserved cysteine residues in rhemnkines are found in the polypeptide~.
Polynucleotides encoding Ck~-1 may be isolated from numerou~ h~ n adult and fetal cDNA libraries, for example, h~ n tonsils cDNA library. Cka-1 is a member of the C-X-C
branch of rhPmnk;ne~ It Cr~nt~; n~; an open rP~1; n~ frame ~n~A~ ng a protein of 109 amino acid residues of which a~ro~lmately the fir8t 22 amino acids residues are the putative leader sequence such that the mature protein comprises 87 amino acid The protein ~h;h; ts the highest degree of homology to interleukin-8 from Sheep (Ovis Aries) with 31% ;~nt; ty and 80~ similarity over a stretch of 97 amino acids. It is also important that the four sp~t~lly conserved cysteine residues in chemok; nP~ are found in the polypeptides.
The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes CDN~, genomic DN~, and synthetic DNA. The DNA may be double-str~n~eA or single-stranded, and if single str~nA~A may be the coding strand or non-coding (anti-sense) strand. The coA;ng sequence which ~ncoAPs the mature polypeptides may be ; ~nt; cal to the coding se~l~nc~ shown in Figure~ 1 (SBQ ID
No. 1) and 2 (SEQ ID No. 3) or that of the deposited clones or may be a different coding sequence which coding sequence, as a result of the rP~llnA~cy or degeneracy of the genetic code, Pnco~c the same mature polypeptides as the DNA of Figures 1 (SEQ ID No. 1) and 2 (SEQ ID No. 3) or the deposited cDNA8.
The polynucleotides which Pn~o~ for the mature polypeptides of Figures 1 (SEQ ID No. 2) and 2 (SEQ ID No. 4) or for the mature polypeptides encoded by the deposited cDNAs may include: only the ~oA~ ng sequence for the mature W096/24668 PCT~S95101780 polypeptide; the coding sequence for the mature polypeptide and additional coding sequence ~uch a~ a leader or secretory ~equence or a ~L~Lotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding ~equence 5' and/or 3' of the coding sequence for the mature polypeptides.
Thus, the term "polynucleotide encoding a polypeptide"
~ncnmrA~seS a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which include~ additional coding and/or non-coding sequence.
The present invention further relates to variants of the her~n~hove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the ded~ced amino acid se~uences of Figures 1 ~SEQ ID No. 2) and 2 (SEQ ID No. 4) or the polypeptides encoded by the cDNAs of the deposited clones. The variant 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 encoding the same mature polypeptides as shown in Figures l (SEQ ID No. 2) and 2 (SEQ ID No. 4) or the same mature polypep~ides encoded by the cDNA of the deposited clones as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides of Figures l (SEQ ID No. 2) and 2 (SEQ ID No. 4) or the polypeptides encoded by the cDNA of the deposited clones. Such nucleotide variants include deletion variants, su~stitution variants and addition or insertion variants.
A~ here~n~hove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequences shown in Figures l (SEQ ID
No. l) and 2 (SEQ ID No. 3) or of the coding sequence of the deposited clones. As known in the art, an allelic variant is t PCTnUS95101780 an alternate form of a polynucleotide ~equence which may have a ~ubstitution, 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 mature polypeptides may be fused in the same reading ~rame to a polynucleotide 8equence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a ~ecretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prosequence is a ~ U~7 otein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein re~; n~ .
Thus, for example, the polynucleotides of the present invention may encode 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 coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a m~ n 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)).

PCT~S95101780 The present invention further relates to polynucleotides which hybridize to the herPin~hove-described sequences if there is at least 50~ and pre~erably 70~
identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the her~n~hove-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there i8 at least 95~ and preferably at least 97~ identity between the sequences. The polynucleotides which hybridize to the her~tn~hove described polynucleotides in a preferred embo~m~nt encode polypeptides which retain substantially the same biological function or activity as the mature polypeptides ~nco~ by the cDNAs of Figures 1 (SEQ ID No. l) and 2 tSEQ ID No. 3) or the deposited cDNAs.
The deposit(s) referred to herein will be m~lnt~ined under the terms o~ the Budapest Treaty on the International Recognition of the Deposit of Micro-org~ni ~m~ 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 cont~n~ in the deposited materials, as well as the amino acid seguence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be reguired to make, use or sell the deposited materials, and no such license is hereby granted.
The present invention further relates to polypeptides which have the deduced amino acid seguences of Figures 1 (SEQ
ID No. 2 ) and 2 (SEQ ID No. 4) or which have the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptides.
The terms "fragment," "derivative~ and ~analog~ when referring to the polypeptides of Figures 1 (SEQ ID No. 2) and _g_ W O 96/2~668 ~CT~S95101780 2 (SEQ ID No. 4) or that encoded by the deposited cDNA, means polypeptides which retain ess~nt~lly the same biological function or activity as such polypeptides. Thus, an analog -includes a proprotein which can be activated by cleavage of the ~lv~Lotein portion to produce an active mature polypeptide.
The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably re~omhin~nt polypeptides.
The fragment, derivative or analog of the polypeptides of Figures l (SEQ ID No. 2) and 2 (SEQ ID No. 4) or that encoded by the deposited cDNAs may be (i) one in which one or more o~ the amino acid residues are substituted with a conserved or non-conserved amino acid residue (pre~erably a conserved amino acid residue) and such substituted amino acid residue may or may not ~e one encoded 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 ma~ure polypeptide i~ fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acid~ are 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 ~equence. Such fragment~, 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 fonm, and preferably are purified to homogeneity.
The term llisolated" means that the material is removed from its original envi~ollme~lt (e.g., the natural environment i~ it i~ naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living An~m~l is not isolated, but the same polynucleotide or -PCTnUS95~0~780 polypeptide, separated ~rom ~ome or all o~ the coexisting materi~ls 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 isolated in that such vector or composition is not part of its natural envi~o~ t.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors o~ 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 ~iral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate ~or activating promoters, selecting transformants or amplifying the Ck~
or Ck~-1 genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell ~elected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techni~ues. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, no~chromosomal and synthetic DNA se~uences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from C~Tnh~n~tions of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is replicable and viable in the host.
-CA 022l047l l997-08-08 W 096/24668 PCTnUS955~1780 The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction ~n~nnllClease 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 ~equence in the expression vector is operatively linked to an a~lopriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: ~TR or SV40 promoter, the E. coli. lac or trP, the phage 1;~mh~:3 PL
~ n~oter and other promoters known to control expression o~
genes in prokaryotic or eukaryotic cells or their ~iruses.
The e~q?ression vector also c~n~ n~ a ribosome h; nA~ng site for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for am.plifying expression.
In addition, the expression vectors preferably contain one or more selectable marker genes to pro~ide 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 ampic;ll; n resistance in E. coli.
The vector cont~; n~ ng the appropriate DNA sequence as here~n~hove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
AS representative examples of .~c~riate hosts, there may be mentioned: bacterial cells, such as E. coli, StrePtomyces, S~lmonella tYph~mllrium; fungal cells, such as yeast; insect cells such as DrosoPhila S2 and Spodoptera Sf9;
~n;m~l cells such as CHO, COS or Bowes m~l ~n~m~;
adenoviruses; plant cells, etc. The selection of an u~riate 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 recombinant 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 embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers 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: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, 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 (chloramphenicol 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, lambda PR, PL and trp.
Eukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a high eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host W O ~6/~4668 PCTnUS95101780 cell can be e~ected by calcium phosphate transi~ection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner~ M., Battey, I., Basic Methods in Molecular Biology, (1986)~.
The constructs in host cells can be used in a conventional m~nnPr to produce the gene products encoded by the recQmh~n~nt se~lPncP~. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide ~ynthesizers.
Mature proteins can be expressed in m~mm~l ian cells, yeast, bacteria, or other cells 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.
A~Lo~riate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory ~ml~ 1, Second Bdition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by re~erence.
Transcription of the DNA ~nco~; ng the polypeptides of the present invention by higher eukaryotes is increased by inserting an enh~ncer sequence into the vector. Rnh~ncers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription.
Examples include the SV40 Pnh~n~er on the late side of the replication origin bp 100 to 270, a cytomegalovirus early ~o.l~ter Pnh~ncer, the polyoma Pnh~ncer on the late side of the replication origin, and adenovirus Pnh~ncers.
Generally, recombinant expression vectors will include origins of replication and selectable marker~ permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic PCTnUS95101780 enzymes ~uch as 3-phosphoglycerate kinase ~PGK), a!-~actor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phafie with translation initiation and termination seguences, 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 Pn~O~P a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
Use~ul expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable m~rkers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide ampli$ication within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, SAlm~nella ty~himllrium and various species within the genera Psell~nm~n~, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors ~or bacterial use can comprise a selectable marker and bacterial origin of replication derived from ro~mPrcially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC
37017). Such cs~mPrcial vectors include, for example, pKK223-3 ~Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are cs~hinP~ with an appropriate promoter and the structural sequence to be expressed.

W ~ 96/24668 PCTrUS9~0178 Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate 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 retained for further puri~ication.
Microbial cells employed in expression o~ 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 m~mm~l ian cell culture systems can also be employed to express recombinant protein. Examples of m~mm~ n expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 ~1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CH0, HeLa and BHK cell lines. ~mm~ n expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed se~uences. DNA se~uences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The polypeptides can be recovered and purified from recombinant cell cultures by methods including ~mmoni um sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature W O 96/24668 PCTnUS95101780 protein. Finally, high per~ormance liquid chromatography (HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a natura]ly purified product, or a product o~ chemical synthetic procedures, or produced by recom~binant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and m~m~ n cells in culture) . Dep~n~; n~ upon the host employed in a rer~m~;n~nt production procedure, the polypeptides of the present invention may be glycosylated or ,m,~y be non-glycosylated.
Polypeptides o~ 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 diagnostic~ to hllm~n disease.
The hllm~n r~Pm~kine polypeptides m, y be employed to inhibit bone marrow stem cell colony formation as adjunct protective treatment during cancer chemotherapy and ~or leukemia.
The hnm~n rh~mokine polypeptides may also be employed to ~nh;h;t epidermal keratinocyte proliferation for treatment of psoriasis, which is characterized by keratinocyte hyper-proli~eration.
The hnm~n rhPmokine polypeptides may also be employed to treat solid tumors by stimulating the invasion and activation of host defense cells, e.g., cytotoxic T cells and macrophages and by inhibiting the angiogenesis of tumors.
They may also be employed to enh~nce host defenses against resistant chronic and acute infections, for example, mycobacterial infections via the attraction and activation of microbicidal leukocytes.
The hllm~n che~okine polypeptides may also be employed to inhibit T cell proliferation by the inhibition of IL-2 biosynthesis for the treatment of T-cell mediated auto-;mml-nP
diseases and lymphocytic leukem;~s.

W O 96/24668 PCTnUS95J0~78 Ck~-11 and Ck~-1 may al~o be employed to stimulate wound he~l iny, both via the recruitment of debris clearing and connective tissue promoting inflammatory cells and also via it~ control of excessive TGF~-mediated fibrosis. In this 8ame m~nnPr, Ck~-11 and Cka-1 may also be employed to treat other fibrotic disorders, including liver cirrhosis, osteoarthritis and plllm~n~y fibrosis.
The hllm~n ch~mokine polypeptides also increase the presence of eosinophils which have the distinctive function of killing the larvae of parasites that invade tissues, as in schistosomiasis, trichinosis and ascariasis.
They may also be employed to regulate hematopoiesis, by regulating the activation and differentiation of various hematopoietic progenitor cells, for example, to release mature leukocytes from the bone marrow ~ollowing chemotherapy.
The polynucleotides and polypeptides ~ncoAed by such polynucleotides may also be utilized for in vitro purposes related to scientific research, synthesi~ of DNA and manufacture of DNA vectors and for designing therapeutics and diagnostics for the treatment of human disease.
Fragments of the full length Ck~-11 or Ck~-1 genes 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 similarity to the gene or similar biological activity. Probes of this type generally have at least 20 bases. Preferably, however, the probes have at least bases and generally do not exceed 50 bases, although they may have a greater number of bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that CQnt~ i n the co~plete genes including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the genes by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled W096/Z4668 PCT~S95101780 oligonucleotides having a sequence complementary to that of the genes of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA ~o determine which members of the library the probe hybridizes to.
This invention i~ also related to the use of the Ck~
or Ck~-1 gene as part of a diagnostic assay for detectiny diseases or susceptibility to diseases related to the presence of mutations in the Ck~-11 or Ck~-1 nucleic acid seque~ces. Such diseases are related to under-expression of the hllm~n ~hemnkine polypeptides, for example, tumors and c~nc~s.
Individuals carrying mutations in the Ck~-11 or Ck~-1 gene may be detected at the DNA level by a variety of techniques. Nucleic acids ~or diagnosis may be obt~;n~ from a patient's cells, such as from blood, urine, saliva, tissue biop~y 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 purpo~e.
As an example, PCR primers complementary to the nucleic acid encoding Ck~-11 or Ck~-1 can be used to i~ntl fy and analyze Ck~-ll or Ck~-1 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 amplified DNA to radiolabeled Ck~-11 or Ck~-1 RNA or alternatively, radiolabeled Ck~-11 or Ck~-1 antisense DNA sequences.
Perfectly matched sequences can be distinguished from m~sm~tched duplexes by RNase A digestion or by differences in melting temperatures.
Genetic testing based on DNA sequence 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
-W096/24668 PCT~S95J0178 ~ragments of different sequences may be distinguished on denaturing ~ormamide gradient gels in which the mobilities of different DNA fra~m~nt~ are retarded in the gel at di~erent 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 re~ealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection of a speci~ic DNA sequence may be achie~ed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA se~lPnc~ ng 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~l~n~ing~ mutations can also be detected by in situ analysis.
The present invention also relates to a diagnostic assay for detecting altered level~ of Ck~-11 or Ck~-1 protein in various tissues since an over-expression of the proteins comp~red to normal control tissue samples may detect the presence of a disease or susceptibility to a disease, for example, a tumor. Assays used to detect levels of Ck~-11 or Ck~-1 protein in a sample derived ~rom a host are well-known to those of skill in the art and include radioimmllnoassays, competitive-h~ n~ ng assays, Western Blot analysis, ELISA
assays and "sandwich" assay. An ELISA assay (Coligan, et al., Current Protocols in Tmmllnology, 1(2), Chapter 6, (1991)) initially comprises preparing an antibody specific to the Ck~-11 or Ck~-1 antigen, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or, in this example, a horser~sh peroxidase W 096~668 PCTrUS9S~1780 enzyme. A sample is removed ~rom a host and incuDa~u ~
solid cu~o~L, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein h;nAin~ sites on the dish are then covered by incubating with a non-specific protein like BSA. Next, the monoclonal Ant;hoAy is incubated in the di~h during which time the monoclonal Ant; hoA; es attach to any Ck~-11 or Ck~-1 proteins attArhP~ to the poly~L~-~--e dish. All unbound m~noclonal Ant;hoAy is ~7-~hP~
out with buffer. The L~uLLer An~h~Ay l;nkP~ to horser~Ad;~h ~r~ A~e is now placed in the dish resulting in binding of the ~vLLer Ant;hoAy to any monoclonal Ant;hoAy holln~l to Ck~-11 or Cka-1. UnattA~hpA reporter AntihoAy is then W-A~heA
out. Per~Y;~A~e substrates are then AAAP~ to the di~h and the amount of color developed in a given time period is a measurement of the amount of Ck~-11 or Ck~-1 protein pre~ent in a given volume of patient sample when cQ~rA~ed against a stAn~A~d curve.
A competition assay may be employed wherein An~; hodies speci~ic to Ck~-11 or Cka-1 are attArh~A to a solid support and labeled Ck~-11 or Ck~-1 and a sample derived from the host are passed over the solid 8U~Ol L and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of Ck~-11 or Ck~-1 in the sample.
A ~ s~n~' ich" assay is similar to an ELISA assay. In a "sandwich" assay Ck~-11 or Cka-1 i~ pas~ed over a solid s~oLL and bind8 to Ant~hoAy attAchP~ to a 801id support.
A second antibody is then bound to the Ck~-11 or Cka-1. A
third An~; ho~y which is labeled and ~pecific to the second antibody is then passed over the solid support and binds to the second ~n~; ho~y and an amount can then be ~lAnt; fied.
This invention provides a method for identification of the receptors for the hn~~n chp-m~k;np polypeptides. The gene F~nco~l;n~J the receptor can be i~f~nt; fied by numerous methods known to those of skill in the art, for example, ligand W 096/24668 PCTnUS95Jal78 pAnn~n~ and FACS sorting (Coliyan, et al., Current Protocols in I-m--mun.l 1(2), Chapter 5, ~1991)). Preferably, expression cloniny 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 proces~, eventually yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell ~ .~Ldne or extract preparation~ that express the receptor molecule. Cross-linked material is resol~ed by PAGE
analysis and exposed to X-ray film. The labeled complex cont~n;ng the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microse~l~nc~ng~ The amino acid sequence obtained from microse~l~c;ng would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptor~.
This invention provides a method of screening compounds to identify agonists and antagonists to the human ch~mokine polypeptides of the present invention. An agonist is a compound which has S;m; 1 ~r biological functions of the polypeptides, while antagonists block such functions.
Chemotaxis may be assayed by placing cells, which are chemoattracted by either of the polypeptides of the present invention, 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 o~ the chamber with an appropriate control medium in the upper compartment, and thus a concentration gradient of the agonist is measured by colln~ i ng cells that migrate into or through the porous membrane over time.
When assaying ~or antagonists, the hllm~n ~h~mokine polypeptides of the present invention are placed in the bottom chamber and the potential antagonist is ~ to determine if chemotaxi~ of the cells is prevented.
Alternatively, a m~mm~ n cell or n,~ dne preparation expressing the receptors of the polypeptides would be incubated with a labeled human chemskine polypeptide, eg.
radioactivity, in the presence of the compound. The ability of the compound to block this interaction could then be measured. When assaying for agonists in this fashion, the hllm~n ~h~m~kines would be absent and the ability of the agonist itself to interact with the receptor could be measured.
Examples of potential Ck~-11 and Ck~-1 antagonists include antibodies, or in some cases, oligonucleotides, which bind to the polypeptides. Another example of a potential antagonist is a negative ~ n~nt mutant of the polypeptides.
Negative ~n~i n~nt m~l~Ants 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 potential antagonists. Antisense technology can be used to control yene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encoAes for the mature polypeptides of the present invention, is used to design an antisense RNA
oligo~ucleotide of from about 10 to 40 base pairs in length.

PCT~S95/0~780 A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple- helix, 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 ~eve~lting transcription and the produc~ion of the hllm~n ch~mokine polypeptides. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptides (antisense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vi~o to ; nh; h; t production of the hllm~n ch~okine polypeptides.
Another potential human ~h~m~kine antagonist is a peptide derivative of the polypeptides which are naturally or synthetically modified analogs of the polypeptides that have lost biological function yet still recognize and bind to the receptors of the polypeptides to thereby effectively block the receptors. Examples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules.
The antagonists may be employed to inhibit the chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T cell subsets, e.g., activated and CD8 cytotoxic T
cells and natural killer cells, in certain auto-;mmllnP and chronic inflam~atory and infective diseases. Examples of auto-immllne diseases include multiple sclerosis, and insulin-dependent diabetes.
The antagonists may also be employed to treat infectious diseases including silicosis, sarcoidosis, idiopathic plllm~n~ry fibrosis by preventing the recruitment and activation of mononllrlear phagocytes. They may also be employed to treat idiopathic hyper-eosinophilic syndrome by W O 96/~4668 PCT~S9~101780 preventing eosinophil production and migration, Endotoxic shock may also be treated by the antagonists by preventiny the migration of ~acrophages and their production of the hllm~n rh~mok; n~ polypeptides of the present invention.
The antagonists may also be employed for treating atherosclero~i8, by preventing monocyte infiltration in the artery wall, The antagonists may also be employed to treat histamine-m~ ted allergic reactions and im-m-lmological disorders including late phase allergic reactions, chronic urticaria, and atopic dermatitis by i nh~ h; ting ch~mnkine-induced mast cell and basophil degranulation and release of hist~m; n~, IgE-me~;~ted allergic reactions such as allergic asthma, rhinitis, and eczem.a m.ay also be treated.
The antagonists m~y also be employed to treat chronic and acute in~lam~tion by pre~enting the attraction of monocytes to a wound area. They may also be employed to regulate normal plllmnn~ry macrophage populations, since chronic and acute inflammatory plllm~n~ry diseases are associ.ated with sequestration of msnsnllrlear phagocytes in the lung.
Antagonists m.~y 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 inflammatory arthropathies.
The antagonists may be employed to interfere with the deleterious cascades attributed prim,arily to IL-l and TNF, which prevents the biosynthesis of other inflammatory cytokines. In this way, the antagonists may be employed to prevent inflammation. The antagonists may also be employed to inhibit prostagl~n~ n-independent fever induced by chl~m~kines.

W096/24668 PC~S95~0~780 The antagonists may also be employed to treat cases o~
bone marrow failure, for example, aplastic AnPm~ and myelodysplastic syndrome.
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 subepith~
basement membrane fibrosis which is a prom~n~nt feature o~
the asthmatic lung.
The antagonist~ may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereina~ter described.
The human ch~okine polypeptides and agonists and antagoni~ts may be employed in romh;nAtion with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically ef~ective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to SAl ~ne, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of n~ stration .
The invention also provides a pharmaceutical pack or kit comprisiny one or more contA~Pr~ ~illed with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such contA~ner(s) can be a notice in the form prescribed by a governmental agency regulating the ma~ufacture, use or sale of phar~aceuticals or biological products, which notice re~lects d~lo~al by the agency of manu~acture, use or sale for human A~m; ni stration. In addition, the polypeptides and agonists and antagonists may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositions may be A~m1 n~ stered in a convenient m~nner such as by the topical, intravenous, intraperitoneal, intramuscular, intratumor, subcutaneous, intr~n~l or intradermal routes. The pharmaceutical compo~ition~ are ~min;stered in an amount which is e~ective PCT~S9~ 780 for treating and/or prophylaxis of the specific indicatlon.
In general, the polypeptides will be ~*m; n~ stered in an amount of at least about 10 ~g/kg body weight and in most cases they will be ~m~n~tered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage i~ from about lQ ~g/kg to about 1 mg/kg body weight daily, k; n~ into account the routes of ~-; n; ~tration, syn~toms, etc.
The hl~-n chemokine polypeptide~, and agonists or antagonists which are polypeptide~, may be employed in accordance with the present invention by e~re~sion of such polypeptide~ iR YiVo, which i~ often referred to a~ "gene therapy n Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) en~oA;~g a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
Such method8 are well-known in the art. For example, cells may be enyineered by procedures known in the art by use of a retroviral particle cQntA;n;ng RNA ~nr~;ng a polypeptide of the present invention.
S;m;l~ly, cells may be engineered in vivo for expre~sion of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle contA;n;ng RNA
encoding the polypeptide of the present invention may be ;n;~tered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for A~m;n~tering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the ~A~h;ngs 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 comh~nAtion with a ~uitable delivery vehicle.

W O 96/24668 PCTnUS95101780 The sequences o~ the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromo~ome marking reagents based on actual se~uence data (repeat polymorphi~ms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important ~irst step in correlating those se9uences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) ~rom the cDNA.
Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process.
These primers are then used.for PCR screening of somatic cell hybrids cont~ining individual human chromosomes. Only those hybrids contA;n~ng the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
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 clones in an analogous m~nn~, Other mapping strategies that can similarly be used to map to its chromosome include in situ 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
clones to a met~h~e chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases;
however, clones larger than that have a higher likelihood of W096/24668 PCT~S95101780 hi n~i ng to a unique chromosomal location with su~ficient signal intensity for simple detection. FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review o~ thi~
technique, see Verma et al., ~llm~n Ch~u~l~osomes: a ~nll~l of Basic Techniques, Pel~d,.,o-l 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. McKusick, Men~elian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of.physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffec~ed individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely ~o 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 of between 50 and 500 potential causative genes. (This assumes l megabase mapping resolution and one gene per 20 kb).
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an imm~lnogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes ch;m~ic, single chain, and hllm~nized antibodies, as well as Fab fragments, or the PCTnUS95/01780 product o~ an Fab expression library. various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obt~inP~ by direct injection of the polypeptides into an ~n;m~l or by ~m;ni stering the polypeptides to an ~nim~l, preferably a nonhllm~n . The antibody so obtained will then bind the polypeptides itself. In this m~nn~r, even a sequence encoding only a fragment o~ the polypeptides can be used to generate antibodies h; n~l ng the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any tech~ique which provides ~nt ~ ho~; es produced by continuous cell line cultures can be used. Examples include the hybrido technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Tmmllnology Today 4:72), and the EBV-hybridoma technique to produce hum.~n monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and r~nc~
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 immnnogenic polypeptide products of this invention. Also, transgenic mice may be used to express h~lm~nized antibodies to immllnogenic 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 is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

CA 022l047l l997-08-08 PCT~S95101780 In order to ~acilitate underst~n~ng of the ~ollowing examples certain frequently occurring methods and/or terms will ~e described.
"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmid~ herein are either ~omm~rcially available, publicly aV~A;lAhle on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent pla~mid~ to those described are known in the art and will be apparent to the ordinarily ~killed arti~an.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequence~ in the DNA. The various re~triction enzyme~ u~ed herein are comm~rcially avail_ble and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily ~killed arti~an. For analytical purposes, typically l ~g of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 ~l of bu~er ~olution. For the purpo~e o~ i~olating DNA
fragments for plasmid construction, typically 5 to 50 ~g of DNA are digested with 20 to 250 unit~ of enzyme in a larger volume. Appropriate buffer~ and substrate amounts for particular restriction enzyme~ are ~pecified by the manufacturer. Incubation times of about l hour at 37~C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophore~ed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel de~cribed by Goeddel, D.
et al ., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single strAn~e~
polydeoxynucleotide or two compl~m~ntAry polydeoxynucleotide strands which may be chemically synthe~ized. ~uch synthetic W096124668 PC~S95101780 oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without ~ g a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated .
"~igation" refers to the process of ~orming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation ,m,~y be accomplished using known buffers and conditions with lO units to T4 DNA ligase ("ligase") per 0.5 ~g of approximately equimolar amounts of the DNA fragments to be ligated.
Unle~ otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Exam~le l Bacterial Ex~ression and Purification of Ck~
The DNA ~equence encoding for Ck~-ll, ATCC # 75948, is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the processed Ck~-ll nucleic acid sequence (minus the putative signal peptide sequence). Additional nucleotides corresponding to the Ck~-ll gene are added to the 5' and 3' end seqllPnr~-~respectively. The 5' oligonucleotide primer has the sequence 5' CCCGCATGCCAA~ L~GTGGCACCA 3' contains a SphI restriction enzyme site (bold) followed by 18 nucleotides of Ck~-ll coding sequence (underlined) starting from the second nucleotide o~ the sequences rn~in~ 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 rem~ining two bases correspond to the second and third base of the first codon (residue 18) of the putative mature protein. The 3' sequence 5' CCCGGATCCCAATGCTTGACTCGGACT 3' ront~in~ complem~nt~ry PCT/USg5/01780 seq~l~n~ C to a BamHl site (bold) and is followed by 18 nucleotides of gene specific se~n~fi preceding the termination codon. The restriction enzyme _ites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. Chat~worth, C~). pQ~-9 ~nco~
~nt;h~otic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable ~ro...oter operator (P/O), a ribo80me h; n~; n~ site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 i8 then digested with SphI
and R, ~'1 . The ~rl;fied ~e~l~nc~Q are ligated into pQE-9 and are inserted in frame with the se~uence ~nroA;~ for the histidine tag and the RBS. The ligation mixture is then used to transform the E. coli ~train M15/rep 4 (Qiagen, Inc.) by the procedure described in S~l,~ ook, ~. et al., Molecular Cloning: A Laboratory ~nll~l, Cold Spring Laboratory Press, (1989). M15/rep4 cont~;nQ multiple copies of the pl~Pr;A
pREP4, which expresse8 the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are i~nt;fied by their ability to grow on ~B plate~ and ampic;ll;n/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis. Clones cont~n~ng the desired constructs are grown overnight (0/N) in liquid culture in LB m~; A supplemented with both Amp (100 ug/ml) and Ran ~25 ug/ml). The 0/N culture i8 used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are yl~.~ to an optical density 600 (O.D.~) of between 0.4 and 0.6. IPTG ("I~o~o~l-B-D-thiogalacto pyranoside~) is then ~e~ to a final conc~ntration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/0 l~ ng to increased gene expre sion. Cells are y ~.~ an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pelle~ is solubilized in the chaotropic agent 6 Molar Guanidine HCl pH 5Ø ~fter clarification, solubilized Ck~-11 is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight W O 96/24668 PCTAUS9~101780 h;n-l~n~ by proteins ~ont;:l;n~ng the 6-His tag (Hochuli, E. et al., J. a~"~tography 411:177-184 (1984)). Ck~-11 ( ~98~
pure) is eluted from the column in 6M guanidine HCl. Protein renaturation out of GnHCl can be accomplished by several protocols ~Jaenicke, R. and Rudolph, R., Protein Structure -A Practical Approach, IRL Press, New York (1990)).
Initially, step dialysis is utilized to remove the GnHCL.
Alternatively, the purified protein isolated ~rom the Ni-chelate column can be bound to a second column over which a decreasing l~ne~r GnHCL gradient is run. The protein is allowed to renature while bound to the column and is subsequently eluted with a buffer con~;n~ng 250 mM
Imidazole, 150 mM NaC1, 25 mM Tris-HCl pH 7.5 and 10%
Glycerol. Finally, soluble protein is dialyzed against a storage buffer cont~;ntng 5 mM ~m~on~um Bicarbonate.

Example 2 Bacterial ExDression and Purification of Ck~-1 The DNA ~equence encoding for Ck~-1, ATCC # 75947, is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the processed Ck~-l nucleic acid se~uence (minus the putative signal peptide sequence). Additional nucleotides corresponding to Ck~-l are added to the 5' and 3' end se~l~nce~ respectively.
The 5' oligonucleotide primer has the sequence 5~
CCCGCATG~-l-l~-l~AGGTCTATTACACA 3' contains a SphI restriction enzyme site (bold) followed by 21 nucleotides of Ck~-1 coding sequence starting from the second nucleotide of the sequences coding 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 rem~;n~ng two bases correspond to the second and third base of the ~irst codon (residue 23) of the putative mature protein. As a consequence, the first base in this codon is changed from G
to C comr~ring with the original sequences, resulting in a W 09612466~
P ~ nUS9~01780 Val to Leu ~ub~titution in the recorl~n~nt protein. The 3' sequence 5' ~C~A-~C'~;~A~ AAAC 3~ contAin~
compl~ - t~y seqllPnr~fi to a BamH1 site (bold) and is followed by 19 nucleotides of gene speci~ic ~equences preceding the termination codon. The restriction enzyme site~ corre~pond to the restriction enzyme ~ite~ on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, CA). pQE-9 Pn~o~e~ A~t;h~otic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-re~llAt~hle promoter operator (P/0), a ribo~ome h;nn;n~ site (RBS), a 6-His tag and restriction enzyme ~ite~. pQ~-9 i~ then digested with SphI and R~l, The amplified ~eqll~nce~ are ligated into pQE-9 and are inserted in frame with the se~uence PnrO~i for the hi~tidine tag and the RBS. The ligation mixture is then used to transform the ~. coli M15/rep 4 (Qiagen, Inc.) by the procedure de~cribed in Sambrook, J. et al., Molecular Cloning: A Laboratory M~n~ , Cold Spring Laboratory Press, (lg89). M15/rep4 cont~;nC multiple copie~ of the p~
pREP4, which expresses the lacI repressor and also confers k~nA~ycin resi~tance (Kanr). Transformant~ are identified by their ability to grow on LB plate~ and ampicillin/kanamycin resi~tant colonies are selected. Plasmid DNA is isolated and confinmed by re~triction analysis. Clone~ contA;n;ng the desired con~truct~ are grown overnight (0/N) in liquid culture in LB mP~; A supplemented with both Amp (100 ug/ml) and ~an (25 ug/ml). The 0/N culture i~ used to inoculate a large culture at a ratio of 1:100 to 1:250. The cell~ are grown to an optical density 600 (O.D.~) of between 0.4 and 0.6. IPTG ("I~o~yl-B-D-thiogalacto pyrano~ide") is then AA~P~ to a final concentration of 1 mM. IPTG ;n~llrP~ by inactivating the lacI repre~sor, clearing the P/0 leading to increa~ed gene expre~~ion. Cells are y. o;~n an extra 3 to 4 hour~. Cell~ are then harvested by centrifugation. The cell pellet i~ ~olubilized in the chaotropic agent 6 Molar Guanidine HCl pH 5Ø After clarification, ~olubilized Ck~-1 W 096t24668 PCT~US9S~27 is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for ti~ht h; n~ ng by proteins cont~in~ng the 6-His tag (Hochuli, E. et al., ~.
C~ "~tography 411:177-184 (1984)). Ck~ 98~ pure) is eluted ~rom the column in 6M guanidine HCl. Protein renaturation out of GnHCl can be accomplished by several protocol~ ~Jaenicke, R. and Rudolph, R., Protein Structure -A Practical Approach, IRL Press, New York (1990)).
Initially, step dialysi8 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 linear GnHCL gradient is run. The protein is allowed to renature while bound to the column and is subsequently eluted with a bu~fer cont~;n~ng 250 mM
Imidazole, 150 m.M NaCl, 25 mM Tris-HCl pH 7.5 and 10%
Glycerol. ~inally, soluble protein is dialyzed against a storage bu~fer contA~n~ng 5 mM Pmmontum Bicarbonate.

Exam~le 3 Expression of Recom~inant Ck~-11 in COS cells The expression of plasmid, Ck~-11 HA is derived ~rom a vector pcDNAI/Amp (Invitrogen) cont~n~ng: 1) SV40 origin of replication, 2) ampic~ll tn resistance gene, 3) B.coli replication origin, 4) CMV promoter followed by a polylinker region, a Sv40 intron and polyadenylation site. A DNA
fragment encoding the entire Ck~-ll precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recomh~n~nt protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the in~luenza 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 recombinant protein with an antibody that recognizes the HA epitope.

W096/24668 PCT~S95/01780 The plasmid construction strategy is described as follows:
The DNA sequence encoding ~or Ck,B-11, ATCC # 75948, i~;
constructed by PCR using two primers: the 5' primer si AAAAAGCTTGCCATGGCCCTGCTACTG 3~ ~ntA; n~ a HindIII site followed by 18 nucleotides of Ck~-11 coding sequence starting from the minus 3 position relative to initiation codon; the 3~ seguence 5'CGCT~T~ TTAAGCGTAGT~ ACGTCGTATG&GTATAGGTTA
ACTGCTGCGAC 3' contains compl~m~nt~y sequences to an XbaI
site, translation stop codon, HA tag and the last 18 nucleotides o~ the Ck~-11 coding sequence (not including the stop codon). Therefore, the PCR product contains a HindIII
site, Ck~-11 coding se~uence followed by HA tag fused in ~rame, a tran~lation termination stop codon next to the HA
tag, and an XbaI site. The PCR amplified DNA ~ragment and the vector, pcDNAI/Amp, are digested with HindIII and XbaI
restriction enzyme and ligated. The ligation mixture is transformed into 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 e~m;ne~ by restriction analysis for the presence of the correct fragment. For expre~sion of the recom~inant Ck~-11, COS
cells are transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory ~n~ , Cold Spring Laboratory Press, (1989)). The expression of the Ck~-11 HA
protein is detected by radiolabelling and ~mmllnoprecipitation method (B. Harlow, D. Lane, Antibodies: A Laboratory ~n~
Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled 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 W O 96/24668 PCTlUS9~178D

mP~ are precipitated with a HA specific monoclonal ~nt;hoAy, Proteins precipitated are analyzed 3)y SDS-PAGE.

ExamPle 4 ExDression of Recombinant Ck~-1 in COS cells The expression of plasmid, Cka-1 HA is derived ~rom a vector pcDNAI/Amp (Invitrogen) cont~'n;ng: 1) SV40 origin of replication, 2) ampi~ n resistance gene, 3) E.coli replication origin, 4) CMV promoter ~ollowed by a polylinker region, a SV40 intron and polyadenylation site. A DNA
fragment encoding the entire Ck~-1 precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the rec~mh;n~nt 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 re~mh;n~nt protein with an ~nt; ho~y that recognizes the HA epitope.
The plasmid construction strategy is described as follow~:
The DNA sequence encoding ~or Ck~-1, ATCC # 75947, is constructed by PCR using two primers: the 5' primer 5' AAAAAGCTTAGAATGAAGTTCATCTCG 3' contains a HindIII site followed by 18 nucleotides of Cka-l coding sequence starting from the minus 3 position relative to the initiation codon;
the 3' sequence 5' CGCTCTAGATTAAGCGTAGTCTGGGACGTCGTAl~lAG
GG~ -l-l 3' contains complPmpnt~ry sequences to an XbaI
site, translation stop codon, HA tag and the last 18 nucleotides of the Ck~-1 coding sequence (not including the stop codon). Therefore, the PCR product contains a HindIII
site, Cka-1 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA
tag, and an XbaI site. The PCR amplified DNA fragment and PCT/us9s~al78a the vector, pcDNAI/Amp, are digested with HindIII and an XbaI
restriction enzyme and liyated. The ligation mixture i~
tran~ormed into E. coli strain SURE (Stratagene ~loning Systems, ~a Jolla, CA) the trans~onmed culture is plated on ampic;ll;n ~e~;~ plates and re~istant colonies are selected.
Plasmid DN~ is isolated from transformants and P~m; nP~ by restriction analysi~ for the presence of the correct fragment. For expres~ion of the reComLhin~nt Ck~-1, COS cells are transfected with the expression vector by DEAE-DEXTRAN
method (~. Sambrook, E. Fritsch, T. Maniati~, Mol~r~ r Cloning: A Laboratory ~nll~l, Cold Spring Laboratory Press, (1989)). The expression of the Ck~-1 HA protein is detected by radiol~hPll;n~ and i~m~lnoprecipitation method (E. Harlow, D. Lane, ~nt;hofl;es: A Laboratory ~ml~l, Cold Spring Harbor Laboratory Press, (1988)). Cells are 1 ~h~l 1 ed for 8 hours with 35S-cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent ~RIPA
bu~er (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
~pecific monoclonal antibody. Proteins precipitated are analyzed by SDS-PAGE.
Example 5 Cloninq and expression of Ck~-11 usinq the baculovirus expression sYstem The DNA sequence encoding the full length Ck~-11 protein, ATCC # 75948, is a~p1ified using PCR oligonucleotide primers corresponA~n~ to the 5' and 3' seqll~n~ps of the gene:
The 5' primer has the sequence 5' cGr~r5~ccGccATcATG
GCCCTGCTACTGGCCCT 3' and c~nt~i ns a BamHI restriction enzyme site (in bold) followed by 6 nucleotides resPmhl ~ ng an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M., ~. Mol. Biol., 196:947-950 (1987) which is just behind the first 20 nucleotides of the W 096/24668 PCTrUS9001780 Ck~-11 gene (the initiation codon for translation "ATG" is underlined).
The 3' primer has the sequence 5~
CGGCGGTACCTGGCTGCACGGTCCATAGG 3' and cont~in~ the cleavage ~;ite i~or the restriction ention-7~lease A8p781 aIld 19 nucleotides complement~ry to the 3' non-translated sequence of the Ck~-11 gene. The amplified sequences are isolated ~rom a 1~ agarose gel using a co~m~rcially available kit ("Geneclean," BIO 101 Inc., La ~olla, Ca.). The fragment is then digested with the ~n~on~lcleases BamHI and Asp781 and then purified again on a 1~ agarose gel. This ~ragment is designated F2.
The vector pRG1 (modification of pVL941 vector, discussed below) is used ~or the expression of the Ck~
protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A m~nll~l of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Exper~mPnt~l Station Bulletin No. 1555).
This expression vector cont~; n.C the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction ~n~onll~leaseS BamHI and Asp781. The polyadenylation site of the simian virus (SV)40 is used ~or efficient polyadenylation. For an easy selection of recombinant viruses the beta-galactosidase gene ~rom E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination 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 Asp781 and then dephosphorylated using calf PCTlUSg~0]78D
intestinal phosphatase by procedures known in the art. The DNA is then isolated from a 1~ agarose gel using the co~ercially available kit ("Genecleanll BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase. E.coli HB101 cell~ are then transformed and bacteria i~nt;fied that contained the plasmid (pBac-Ck~-11) with the CK~-11 gene using the enzymes BamHI and Asp781. The sequence of the cloned ~ragment is confirmed by DNA se~l~ncing.
5 ~g of the plasmid pBac-CK~-11 is cotransfected with 1.0 ~g of a ~omm~rcially aV~ hl e l;ne~rized baculo~rirus (IlBaculoGold~ baculovirus DNA", Pharmingen, San Diego, CA.) u~ing the lipofection method (~elgner 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~-11 are mixed in a sterile well of a mlcrotiter plate cont~;n;n~ 50 ~l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards lO ~l Lipofectin plus 90 ~1 Grace's medium are ~PA, m~ ~eA and incubated for 15 minutes at room temperature. Then the transfection mixture is added ~lo~ise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with lml Grace's medium without serum. The plate is rocked back and ~orth to mix the newly ~ solution. The plate is then incubated for 5 hours at 27~C. After 5 hours the transfection solution is lc.~.o~ed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is ~ . The plate is put back into an incubator and cultivation continued at 27~C for four days.
After four days the supernatant i~ collected and a plaque assay performed s;m;l~r as described by Summers and Smith (supra). As a modification an agarose gel with IlBlue Gal" tLife Technologies Inc., Gaithersburg) i8 used which allows an easy isolation of blue stained plaques. (A

W O 96t24668 PCT~USgSJ~7 detailed description of a "plaque assay~ can also be ~ound in the u~er's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10) .
Four days after the serial dilution, the viruses are ~e~ to the cells and blue st~ ne~ plaques are picked with the tip of an ~ppendorf pipette. The agar cont~n1ny the recomhin~nt viruses is then re8uspended in an Eppendorf tube cont~;n~ng 200 ~l of Grace's medium. The agar is l~-.,~ved by a brief centrifugation and the ~upernatant c~nt~; n; ng the reCo~mh;n~nt baculovirus is used to infect Sf9 cells seeded in 35 mm ~hes~ Four days later the supernatants of these culture ~ hF~F: 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 infected with the recomh;n~nt baculovirus V-CK~-11 at a multiplicity of infection (MOI) of 2. Six hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburq). 42 hours later 5 ~Ci of 35S-methionine and 5 ~Ci 35S cysteine (Amersham) are ~ . The cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

Example 6 Cloninq and exPression of Ck~-1 usinq the baculovirus exPre~ion system The DNA sequence encoding the full length Ck~-1 protein, ATCC # 75947, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5' GCC~TCCGCCATC
ATGAAGTTCATCTCGACATC 3' and r~nt~;n~ a BamHI restriction enzyme ite (in bold) followed by 6 nucleotides re~emhl;ng an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol., 196:947-950 PCTrU~9~D~78D
(1987) which is just hPhin~ the first 20 nucleotides of the Cka-1 gene (the initiation codon ~or translation ~ATG~ is underli n~
The 3' primer has the sequence 5' CGCGGGTACCGG
GTGG~AA 3' and cont~n~ the cleavage site for the restriction Pn~nllrlease ASp781 (in bold) and 17 nucleotides complementary to the 3' non-translated sequence of the Ck~-1 gene. The ampli~ied sequences are isolated from a 1% agarose gel using a cqmmPrcially av~ hle kit ("Geneclean," BI0 101 Inc., La Jolla, Ca.). The fragment is then digested with the en~Qnllcleases BamHI and Asp781 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~-1 protein using the baculovirus expression gystem (for review see: Summers, M.D. and Smith, G.E 1987, A m~nll~l of methods for baculovirus vector~ and insect cell culture procedures, Texas Agricultural Exper~mPntAl Station Bulletin No. 1555).
This expres~ion vector contA~n~ the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction ~n~nnllcleases BamHI and Asp781. 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 E.coli is inserted in the same orientation as the polyhedrin ~ L-oter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-m~ ted homologous recomh~n~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 Asp781 and then dephosphorylated using calf PCT~Ss~1780 intestinal phosphatase by procedures known in the art. The DNA i~ then isolated from a 1% agarose gel using the commercially av~ ble kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated v2.
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 contained the plasmld (pBac-Ck~-1) with the Ck~-1 gene using the enzymes BAmHI and Asp781. The sequence of the cloned fragment is confirmed by DNA se~lPncing.
5 ~g of the plasmid pBac-Ck~-1 is cotransfected with 1.0 g of a comm~rcially av~ hl e 1ine~ized baculovirus ("BaculoGold~ baculovirus DNA", Phanmingen, San Diego, CA.) using the lipofection method ~Felgner et al. Proc. Natl.
Acad. SCi. USA, 84:7413-7417 (1987)).
l~g of BaculoGold~ ~irus DNA and 5 ~g of the plasmid pBac-Ck~-1 are mixed in a sterile well of a microtiter plate ~nnt~tntng 50 ~l of serum free Grace~s medium ~hife Technologies Inc., Gaithersburg, MD). Afterwards 10 ~l ~ipofectin plus 90 ~l Grace's medium are ~e~ ~e~ and incu~ated for 15 minutes at room temperature. Then the trans~ection mixture is added ~ .ise to the Sf9 insect cell~ (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with lml Grace's medium without serum. 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 trans~ection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10~ fetal calf serum is ~e~. The plate is put back into an incubator and cultivation continued at 27~C for four day~.
After four days the supernatant is collected and a pla~ue assay performed stm; 1 ~r as described by Summers and Smith (supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) i~ used which allows an easy isolation of blue st~i ned plaques. (A

W096~668 PCT~S95101~80 detailed description of a "plaque assay~' can also be ~ound in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10) .
Four days after the serial dilution, the viruses are ~e~ to the cells and blue st~; n~ pla~ues are picked with the tip of an Eppendorf pipette. The agar cont~; ni ng the rec~mb;n~nt viruses is then resuspended in an Eppendorf tube contA;n;ng 200 ~l of Grace's medium. The agar is .e.l.o~ed by a brief centrifugation and the supernatant cnnt~ining the recomh;n~nt baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture ~ h~$: 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 infected with the recombinant baculovirus V-Cka-l at a multiplicity of infection (MOI) of 2. Six hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg, MD). 42 hours later 5 ~Ci of 35S-methionine and 5 ~Ci 35S cysteine (Amersham) are ~ . The cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.
Numerous modifications and variations of the present invention are possible in light of the above teachings and, there~ore, within the scope of the appended cl~im~, the invention m~y be practiced otherwise than as particularly described.

W0~6/24668 PCT~S95)017X~

SEQUENCE LISTING

(1) r7RNRR~L INFORMATION:
(i) APPLICANT: LI, ET AL.

(ii) TITLE OF lNV~NllON: ~llm~n rhpm~kine Be~a-ll and ~llm~n ~h~m~kine Alpha-l (iii) NUMBER OF SE~u~N~S: 16 (iv) CORRESPON~ Ann~R~s:

(A) ~nn~ SEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OhSTEIN
(B) ~'lK~-l: 6 BECKER FARM ROAD
(C) CITY: ROS~r-~ND
(D) STATE: h-EW JERSEY
(E) ~OUN'1'~Y: USA
(F) ZIP: 07068 (v) COM~uI~K READABLB FORM:
(A) MEDI~M TYPE: 3.5 INCH DISKETTE
(B) COM~U'1~K: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.l (vi) CURR~NT APPLICATION DATA:
(A) APPhICATION NUMBER:
(B) FILING DATE: Con~ tly (C) CLASSIFICATION:
~vii) PRIOR APPLICATION DATA
(A) APPhICATION NUMBER:
(B) FILING DATE:

=

W 096/24668 PCTrUS95/01780 d ~ iled description of a "plaque assay~ can also be found in the ~ er's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10~
Four ~ays after the serial dilution, the viruses are ~ to the~cells and blue stA;ne~ pla~ues are picked with the tip of an Eppendorf pipette. The agar contA;n~ng the reromhinAnt vi~uses is then resuspended in an Eppendorf tube cont~;n;ng 200 ~ of Grace's medium. The agar is removed by a brief centrifu~ation and the supernatant cont~ n; ng the reco~h;n~nt baculo~irus is used to infect Sf9 cells seeded in 35 mm dishes. Fou~ days later the supernatants of these culture ~sh~s are h~rvested and then stored at 4~C.
Sf9 cells are grown in Grace's medium supplemented with 10~ heat-inactivated F~S. The cells are infected with the recomhin~nt baculovirus~; V-Ck~-1 at a multiplicity of infection (MOI) of 2. Sixt~hours later the medium is removed and replaced with SF900 ~ medium minus methionine and cysteine (Life Technologies ~Inc., Gaithersburg, MD). 42 hours later 5 ~Ci o~ 35S-meth~on; n~ and 5 ~Ci 35S cysteine (Amersham) are ~P~. The cells~-are further incubated for 16 hours before they are harvested~by centrifugation and the labelled proteins visualized by SDS~PAGE and autoradiography.
Numerous modifications and var~iations of the present invention are possible in light of th~e above teachings and, therefore, within the scope of the ~ppended claims, the invention may be practiced otherwise ~an as particularly described CA 022l047l l997-08-08 WO96/24668 PCT~S95101~80 (Viii) A1-1OKN~/AGENT INFORMATION
(A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION N ~3ER: 36,134 (C) R~N~/DOCKET NUMBER 325800-272 (iX) TELECOMMUNICATION INFORMATION:
(A) TEL~N~ 201-994-1700 (B) TEL~FAX: 201-994-1744 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQu~;N~:~; C~Z~Ri~rTERISTICS
(A) L~N~1n 297 BASB PAIRS
(B) TYPE: NUCLEIC ACID
(C) ST~Nn~n~S: SINGLE
(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE CDNA

(Xi) SE~N~ DESCRIPTION: SEQ ID NO:1:

ATGGCCCTGC TACTGGCCC~ CAGCCTGCTG L-llC'l~ CCC~AGC CCCAACTCTG 60 AGTGGCACCA ATGAAGCTGA AGACTGCTGC L~ ~-l~l~A CCCAGAAACC CAl-CCL-lL;LG 120 TACATCGTGA GGAACTTCCA CTACL-l-l--lC ATCAAGGATG G~TGCAGGGT GCCTGCTGTA 180 G'l-l~ACCA CACTGAGGGG CCGCCAGCTC TGTGCACCCC C~GACCAGCC CTGGGTAGA~ 240 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQU~N~ CHARACTERISTICS
(A) LISNC~1~: 98 AMINO ACIDS
(B) TYP~3: AMINO ACID
(C) ST~PN~ S:
(D) TOPOLOGY: T.TNRZI~

(ii) MOLECULE TYPE PROTEIN

CA 022l047l l997-08-08 W 096/24668 PCT~US95/01780 (xi) SEQu~N~ DESCRIPTION: SEQ ID NO:2:

Met Ala Leu Leu Leu Ala Leu Ser Leu Leu Val Leu Trp Thr Ser Pro Ala Pro Thr Leu Ser Gly Thr Asn Glu Ala Glu Asp Cys Cys Leu Ser Val Thr Gln Lys Pro Ile Pro Gly Tyr Ile Val Arg Asn Phe HiS Tyr Leu Leu Ile Lys Asp Gly Cys Arg Val Pro Ala Val . 35 40 Val Phe Thr Thr Leu Arg Gly Arg Gln Leu Cys Ala Pro Pro Asp Gln Pro Trp Val Glu Arg Ile Ile Gln Arg Leu Gln Arg Thr Ser Ala Lys Met Lys Arg Arg Ser Ser ~2) lN~'O~ SATION FOR SEQ ID NO:3:

(i) SEQU~N~ CHAR~CTERISTICS
(A) LENGTH: 333 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRPNnT~n~.~S: SINGLE
(D) TOPOLOGY: T.TNR~R

(ii) MOLECULE TYPE: cDNA

(xi) SEyu~N~ DESCRIPTION: SEQ ID NO:3:

AAG~l~l-l~l GGAGGTCTAT TAACACAAGC TTGAGGTGTA GAl~lG'lC~A AGAAGAAGCT 120 CA~l~-Ll-lAT CCCTAGACGC TTCATTGATC GAATTCAAAT CTTGGCCCCG TGGGAATGGT 180 TGTCC~AGAA AAGA~ATCAT A~l~-l~AAG AAGAACAAGT CAAl-l~l-~lG TGTGGACCCT 240 CAAGCTGAAT GGGTACAAAG A~TGATGGAA GTATTGAGAA AAAGAAGTTC TTCAACTCTA 300 CCAGTTCCAG TGl-l-lAAGAG AAAGATTCCC TGA 333 W096/24668 PCT~S95101780 (2) INFORMATION FOR SE:Q ID NO: 4:
(i) SEQu~ CHARACTERISTICS
(A) L~;N~1~: 10 9 AMINO ACIDS
(B) T~YPE: AMINO ACID
(C) STR~N~ S,~:
(D) TOPOLOGY T.T~l;~Z~

(ii) MOLECULE TYPE: PROTEIN

(xi) SE~u~L~ DESCRIPTION: SEQ ID NO:4:

Met ~yS Phe Ile Ser Thr Ser Leu heu Leu Met Leu ~eu Val Ser -20 -15 -l0 Ser Leu Ser Pro Val Gln Gly Val Leu Glu Val Tyr Tyr Thr Ser -5 l 5 Leu Arg ~ys Arg Cys Val Gln Glu Ser Ser Val Phe Ile Pro Arg Arg Phe Ile ASp Arg Ile Gln Ile ~eu Pro Arg Gly Asn Gly CyS
Pro Arg Lys Glu Ile Ile Val Trp Lys Lys Asn Lys Ser Ile Val Cys Val Asp Pro Gln Ala Glu Trp Ile Gln Arg Met Met Glu Val Leu Arg ~ys Arg Ser Ser Ser Thr Leu Pro Val Pro Val Phe Lys Arg Lys Ile Pro (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS
(A) L~l~: 27 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANv~vN~SS: SINGLE
(D) TOPOLOGY: LINEAR

W 096/24668 PCTrUS95/017X0 (ii) MOLECUhE TYPE: Oligonucleotide (xi) SEQu~ DESCRIPTION: SEQ ID NO:5:

CCCGCATGCC A~ ~-l~AGT GGCACCA 27 (2) INFORMATION FOR SEQ ID NO:6:

(i) SE~u~N~ CHAR~CTERISTICS
(A) hENGTH: 27 BASB PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRZ~)~nNR.ss: SINGLE
(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SE~u~N~ DESCRIPTION: SEQ ID NO:6:

TCcc AATGCTTGAC TCGGACT 27 (2) INFORMATION FOR SEQ ID NO:7:

(i) SE~;?u~sN~ RP,~'TERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STR~NDEDNESS: SINGLE
(D) TOPOLOGY: T.TNli~R

(ii) MOLECULE TYPE': Oligonucleotide (xi) S~YU~N~ DESCRIPTION: SEQ ID NO:7:

CCCGCATGCC ~ l~GA~GT CTATTACACA 30 (2) lN~ ~TION FOR SEQ ID NO:8:

W 096/24668 PCTrUS95101780 (i) SEyu~Nc~ CHARACTERISTICS
(A) LENGTH: 28 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STR~Nn~n~SS: SINGLE
(D) TOPOLOGY: T.TNRZ~

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEc~u~N~ DESCRIPTION: SEQ ID NO:8.

c~ ~TCCG GGAAlc-l-l-lc TCTTAAAC 28 (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 BASE PAIRS
~B) TYPE: NUCLEIC ACID
(C) STR~Nn~nNR-~S: SINGLE
(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SE~u~Nc~ DESCRIPTION: SEQ ID NO:9:

AAAAAGCrTG CCATGGCCCT GCTACTG 27 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 57 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: T~Tl~Z~R

(ii) MOLECULE TYPE: Oligonucleotide W 096/24668 PCTrUS95/01780 (Xi) SEQU~r~ ; DESCRIPTION SEQ ID NO:10:

CG~-l~-lAGAT TAAGCGTAGT CTGGGACGTC GTATGGGTAT AGGTTAACTG CTGCGAC 57 ~2) 1N~Okl-lATION FOR SEQ ID NO:11 (i) SEQU~N~ CHARACTERISTICS
(A) LENGTH: 27 BASE PAIRS
(B) TYPE NUCLEIC ACID
(C) STRPN~ J~-SS SINGLE
(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQu~N~ DESCRIPTION SEQ ID NO 11 (2) 1N~O~ ~TION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS
(A) L~N~1~: 54 BASE PAIRS
(B) TYPE NUCLEIC ACID
(C) STR~NV~V~SS SINGLE
(D) TOPOLOGY T.TNRZ~l~

(ii) MOLECULE TYPE: Oligonucleotide (Xi) SEQUENCE DESCRIPTION SEQ ID NO: 12:

CGCTCTAGAT TAAGCGTAGT CTGGGACGTC GTATGGGTAG GGAAl~-l-l-lC TCTT 54 (2) INFORMATION FOR SEQ ID NO :13:

(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 36 BASE PAIRS

W O g6/24668 PCTnUS95/01780 ~B) TYPE: NnUCLEIC ACID
(C) STRANDEDNESS: SINGLB
~D) TOPOLOGY: LINFAR

(ii) MOLECULE TYPE: O1igOnUC1eOtide (Xi) SEQU~N~ DESCRIPTION: SEQ ID NO:13:

(2) INFORMATION FOR SEQ ID NO:14:

(i) SE~U~N~ CHARACTERISTICS
(A) L~N~1~: 29 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STR~NV~N~SS: SINGLE
~D) TOPOLOGY: T.TNRAR

(ii) MOLECULE TYPE: O1igOnUC1eOtide (Xi) SEQU~N~ DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO:15:

(1) SEQUENCE CHARACTERISTICS
(A) LENGTH: 35 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRP~I~ N~SS SINGLE
(D) TOPOLOGY: T.TNR~R

(ii) MOLECULE TYPE: O1igOnUC1eOtide (Xi) SE~U~N~ DESCRIPTION: SEQ ID NO:15:

W 096/24668 PCTrUS95/01780 ~2) ~ INFORMATION FOR SEQ ID NO:16:

(i) SEQu~C~ CHARACTERISTICS
(A) LENGTH: 27 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRAN~ s: SINGLE
(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQu~N~ DESCRIPTION: SEQ ID NO:16: ~-

Claims (31)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide having the deduced amino acid sequence of SEQ ID No. 2 and fragments, analogs or derivatives of said polypeptide;
(b) a polynucleotide encoding a polypeptide having the deduced amino acid sequence of SEQ ID No. 4 and fragments, analogs or derivatives of said polypeptide;
(c) a polynucleotide encoding a polypeptide having the amino acid sequence encoded by the cDNA
contained in ATCC Deposit No. 75948 and fragments, analogs or derivatives of said polypeptide; and (d) a polynucleotide encoding a polypeptide having the amino acid sequence encoded by the cDNA
contained in ATCC Deposit No. 75947 and fragments, analogs or derivatives of said polypeptide.

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

3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA.

4. The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.

5. The polynucleotide of Claim 2 wherein said polynucleotide encodes a chemokine polypeptide selected from the group consisting of a polypeptide having the deduced amino acid sequence of SEQ ID No. 2 and a polypeptide having the deduced amino acid sequence of SEQ
ID No. 4.

6. The polynucleotide of Claim 2 wherein said polynucleotide encodes a chemokine polypeptide selected from the group consisting of a polypeptide encoded by the cDNA of ATCC Deposit No. 75948 and a polypeptide encoded by the cDNA of ATCC Deposit No. 75947.

7. The polynucleotide of Claim 1 having the coding sequence of SEQ ID No. 1.

8. The polynucleotide of Claim 1 having the coding sequence of SEQ ID No. 3.

9. A vector containing the DNA of Claim 2.

10. A host cell genetically engineered with the vector of Claim 9.

11. A process for producing a polypeptide comprising:
expressing from the host cell of Claim 10 the polypeptide encoded by said DNA.

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

13. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having Ck.beta.-11 activity.

14. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having Ck.alpha.-1 activity.

15. 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; (ii) a polypeptide having the deduced amino acid sequence of SEQ ID No. 4 and fragments, analogs and derivatives thereof; (iii) a polypeptide encoded by the cDNA of ATCC Deposit No. 75948 and fragments, analogs and derivatives thereof; and (iv) a polypeptide encoded by the cDNA of ATCC Deposit No. 75947 and fragments, analogs and derivatives thereof.

16. The polypeptide of Claim 15 wherein the polypeptide has the deduced amino acid sequence of SEQ ID
No. 2.

17. The polypeptide of Claim 15 wherein the polypeptide has the deduced amino acid sequence of SEQ ID
No. 4.

18. Antibodies against the polypeptides of claim 15.

19. Antagonists against the polypeptides of claim 15.

20. A method for the treatment of a patient having need of a Ck.alpha.-1 polypeptide comprising: administering to the patient a therapeutically effective amount of the polypeptide (ii) or (iv) of claim 15.

21. A method for the treatment of a patient having need of a Ck.beta.-11 polypeptide comprising: administering to the patient a therapeutically effective amount of the polypeptide (i) or (iii) of claim 15.

22. The method of claim 20 wherein the therapeutically effective amount of the polypeptide is employed to inhibit bone marrow colony formation.

23. The method of claim 21 wherein the therapeutically effective amount of the polypeptide is employed to inhibit bone marrow colony formation.

24. A method for the treatment of a patient having need to inhibit Ck.alpha.-1 polypeptide comprising:
administering to the patient a therapeutically effective amount of an antagonist against polypeptide (ii) or (iv) of claim 19.

25. A method for the treatment of a patient having need to inhibit Ck.beta.-11 polypeptide comprising:
administering to the patient a therapeutically effective amount of an antagonist against polypeptide (i) or (iii) of Claim 19.

26. The method of Claim 20 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.

27. The method of Claim 21 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.

28. A process for diagnosing a disease or a susceptibility to a disease related to an under-expression in a host of the polypeptide of claim 15 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide in a sample derived from a host.

29. A diagnostic process comprising:
analyzing for the presence of the polypeptide of claim 15 in a sample derived from a host.

30. A process for identifying a compound active as an agonist to the polypeptide of claim 15 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 15; and determining the extent of migration of the cells to identify if the compound is effective as an agonist.

31. The process of claim 30 for identifying compounds active as antagonists to the polypeptide of claim 15 wherein Ck.beta.-11 or Ck.alpha.-1 is added to the combination of step (a).