CA2242908A1 - Human g-protein chemokine receptor hsatu68 - Google Patents

Abstract

Human G-protein chemokine receptor polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptides for identifying antagonists and agonists to such polypeptides and methods of using the agonists and antagonists therapeutically to treat conditions related to the underexpression and overexpression of the G-protein chemokine receptor polypeptides, respectively. Also disclosed are diagnostic methods for detecting a mutation in the G-protein chemokine receptor nucleic acid sequences and detecting a level of the soluble form of the receptors in a sample derived from a host.

Description

W O 97/25340 PCT~US96/00499 _, ~MAN G-PROTEIN r~FM~K ~ N~ K~W~O~ HSAT~68 This invention relates to newly identi~ied polynuoleotides, polypeptides encoded by such polynucleotides, the use o~ such polynucleotides and polypeptide~, as well as the ~loduction of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a h~lm~n 7_ tr~n~:m~ e receptor which ha5 been putatively identi~ied as a rhPmok;~ receptor, 50metimes hereinafter re~erred to as "G-Protein ~h~mokine Receptor" or "~SATU68". The invention also relates to inhibiting the action o~ such polypeptides.
It is well established that many medically signi~icant biological processes are mediated by proteins participating in signal tr~n~llction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Le~kowitz, Nature, 3~1:35~-354 (1991)). Herein these proteins are re~erred to as proteins participating in pathways with G-proteins or PPG proteins. Some ~mples o~ these proteins include the GPC receptors, such as those ~or adrenergic agents and dor:lm;n~ (Kobilka, B.K., et al., PN~S, 84:46-50 (1987);
Kr~h;lk~, B.~C., et al., Science, 238:6~0-656 ~lg87); ~unzow, J.R., et al., Nature, 336:783-787 ~1988)), G-proteins themselves, e~ector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodie8terase, and actuator WO 97/25340 PCT~US96/00499 proteins, e.g., protein kina~e A and protein kinase C
(Simon, M.I., et al., Science, 2~2:802-8 ~1991)).
For example, in one ~orm of signal transduction, the e~ect o~ hormone binding is activation of an enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence o~ the nucleotide GTP, and GTP also in~ n~s hormone bi n~i ng . A G~protein connects the hormone receptors to adenylate cyclase. G-protein was shown to ~h~nge GTP ~or bound GDP when activated by hormone receptors. The GTP-carryiny ~orm then binds to an activated adenylate cyclase. Hydrolysis o~ GTP
to GDP, catalyzed by the G-protein itsel~, returns the G-protein to its basal, inactive ~orm. Thus, the G-protein serves a dual role, as an interm~ te that relays the signal ~rom receptor to e~ector, and as a clock that controls the duration of the signal.
The membrane protein gene super~amily o~ G-protein coupled receptors has been characterized as having seven putative tr~n~ ane ~ i n~ . The ~om~ i n~ are believed to represent tran~ e ~-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, s~ch as hormone, viral, growth ~actor and neuroreceptors.
G-protein coupled receptors have been characterized a~
including these seven conserved hydrophobic stretches o~
about 2Q to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein ~amily o~
coupled receptors includes dopamine receptors which bind to neuroleptic drugs used f or treating p~ychotic and neurolo~ical disorders. Other examples o~ mem~ers o~ this ~amily include calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, hist~m;nP, throm~in, k; ni n, ~ollicle stimulating hormone, opsins, endotheli~l di$~erentiation gene-1 receptor and rhodopsins, odorant, cytomegalovirus receptors, etc.
G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various CA 02242908 l998-07-lO

W O 97/25340 PCT~US96/001~
intracellular enzymes, ion ~h~nn~ls and transporters ~see, Johnson et al., Endoc., ~ev.,- 10:3~7-331 (1989)).
Different G-protein ~-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors have been identified as an important mechanism for the regulation of ~-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a m~mm~l ~ ~n host.
~ mnkines, also referred to as intercrine cytok~n~s~
are a subfamily of structurally and functionally related cytokines. These molecules are 8-10 kd in size. In general, chemokines ~h~ h~ t 2096 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~mokines 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, re~erred to as the l~C-C~ sub~amily. Thus $ar, at least nine dif f erent members of this family have been identified in hllm~n~
The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many chemokines have pro-inflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include st;mn-~tion of histamine release, lysosomal enzyme and ~ leukotriene release, increased adherence of target ~mmllne cells to endothelial cells, ~nh~nced h~ n~; ng of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst.
In addition to their involvement in inflammation, certain .

W O 97/25340 PCT~US96/00499 ~h~m~kines have been shown to exhibit other activitie~.
For example, macroph~ge 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-8 (IL-8) promotes proliferation of keratinocytes, and GR0 is an autocrine growth factor for m~l ~nom~ cell5, 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 h~l in~, hematopoietic regulation and ;mmlln~logical disorders such as allergy, asthma and arthritis.
In accordance with one aspect of the present invention, there are provided novel mature receptor polypeptides as well as biologically active and diagnostically or therapeutically useful fra~Pnts, analogs and derivatives thereof. The receptor polypeptides o~ the present invention are of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the receptor polypeptides of the present invention, including mRNAs, cDNAs, genomic DNA as well as antisense analogs thereo~ and ~iologically active and diagnostically or therapeutically useful fragments thereo~.
In accordance with another aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressed by the hn~-n cDNA ~nt~i n~ in ATCC Deposit No. 97334.
In accordance with a further aspect of the present invention, there are provided processes for producing such receptor polypeptides by reC~mhin~nt techniques comprising culturing recomh;n~nt prokaryotic and/or eukaryotic host ce-ls, cont~;n~ng nucleic acid sequences encoding the receptor polypeptides of the present invPntion, under conditions promoting expression of said polypeptides and subse~uent recovery of said polypeptides.

CA 02242908 l998-07-lO

W O 97/25340 PCT~US~C'0015~
In accordance with yet a ~urther aspect o~ the present invention, there are provided antibodies against such receptor polypeptides.
In accordance with another aspect o~ the present invention there are provided methods of screening for compounds which bind to and activate or inhibit activation o~ the receptor polypeptides o~ the present invention.
In accordance with still another embodiment o~ the present invention there are provided processes of ~nm~ni~tering compounds to a host which bind to and activate the receptor polypeptide o~ the present invention which are use~ul in stimulating haematopoiesis, wound hF~l tng~ coagulation, angiogenesis, to treat tumors, chronic in~ections, leukemia, T-cell mediated auto-immllne diseases, parasitic in~ections, psoriasis, and to stimu~ate growth ~actor activity.
In accordance with another aspect of the present invention there is provided a method o~ A~m;n;stering the receptor polypeptides of the present invention via gene therapy to treat conditions related to underexpression o~
the polypeptides or underexpression o~ a ligand ~or the receptor polypeptide.
In accordance with still another embodiment o~ the present invention there are provided processes o~
~mtnt~tering compounds to a host which bind to and inhibit activation o~ the receptor polypeptides o~ the present invention which are use~ul in the prevention and/or treatment o~ allergy, atherogenesis, ~n~rhylaxis, malignancy, chronic and acute in~lammation, histamine and IgE-mediated allergic reactions, prostagl~n~tn-indep~n~nt ~ever, bone marrow ~ailure, silicosis, sarcoidosis, rheumatoid arthritis, shock and hyper-eosinophilic syndrome.
In accordance with yet another aspect of the present invention, there are provided nucleic acid probes comprising nucleic acid molecules o~ su~icient length to speci~ically hybridize to the polynucleotide sequences o~
the present invention.

W O 97/~5340 PCTAUS96/00499 In accordance with still another aspect o~ the present invention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid seguences encoding such polypeptides and ~or detecting an altered level of the soluble ~orm o~ the receptor polypeptides.
In accordance with yet a further aspect o~ the present invention, there are provided processes for utilizing ~uch receptor polypeptides, or polynucleotides encoding s~ch polypeptides, for in vitro purposes related to scienti~ic research, synthesis o~ DNA and manu~acture of DNA vectors.
These and other aspects o~ the present invention should be apparent to those skilled in the art ~rom the teachings herein.
The ~ollowing drawings are illustrative o~ embo~;m~nt~
of the invention and are not meant to limit the scope o~
the invention as encompassed by the claims.
Figure 1 shows the cDNA sequence and the corresponA;ng deduced amino acid sequence of the G-protein ~h~m~kine receptor o~ the present invention The standard one-letter abbreviation ~or amino acids is used. Seguencing was per~onmed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).
Figure 2 illu~trates an amino acid alisnm~nt of the G-protein ~hPm~kine receptor of the present invention (top) and the human interleukin-8 receptor (bottom) (SEQ ID NO:9) In accordance with an aspect o~ the present invention, there is provided an isolated nucleic acid (polynucleotide) which ~nco~ ~or the mature polypeptide having the deduced amino acid sequence o~ Figure 1 (SEQ ID NO:2).
In accordance with another aspect o~ the present invention there are provided isolated polynucleotides encoding a mature polypeptide expressed by the h~ n cDNA
con~A~neA in ATCC Deposit No. 97334, deposited with the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, USA, on November 6, 1995. The deposited material is a cDNA insert, encoding a polypeptide .

.

W O 97/25340 PCT~US~G~
- o~ the present invention, cloned into a pBluescript SK(-) vector (Stratagene, La Jolla, CA~, which will con~er ampic;lli n resistance upon trans~ormation.
The deposit(s) has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms ~or purposes of Patent Procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. These deposits are provided merely as convenience to those o~ skill in the art and are not an admission that a deposit is required under 35 U.S.C. 112.
The sequence o~ the polynucleotides cont~;ne~ in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by re~erence and are controlling in the event of any conflict with any description of sequences herein. A
license may be required to make, use or sell the deposited materials, and no such license i5 hereby granted.
The polynucleotide of this invention was discovered in a human genomic library derived ~rom human activated T
cells. It is structurally related to the G protein-coupled receptor ~amily. It cont~i n~ an open reading ~rame encoding a protein o~ 415 amino acid residues. The protein exhibits the hiyhest degree of homology at the amino acid level to a hllm~n interleukin-8 receptor with 39.31 %
identity and 58.405 ~ s~m; 1 ~ity.
The polynucleotide o~ the present invention may be in the ~orm o~ RNA or in the ~orm o~ DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-str~n~, and i~ single stranded may be the coding strand or non-coding ~anti-sense) strand.
The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID NO:1) which coding se~uence, as a result o~ the re~lln~ncy or degeneracy o~ the genetic code, encodes the same mature polypeptide as the DNA o~ Figure 1 (SEQ ID
NO:1).

WO 97/2534Q PCT/US961'0~ S~l The polynucleotide which encodes ~or the mature polypeptide of Figure 1 ~SEQ ID NO:~) may include: only the coding se~uence for the mature polypeptide; the coding seguence ~or the mature polypeptide and additional coding sequence such as a tr~n~mh~ane (TM) or intra-cellular ~n~A;n; the coding sequence for the mature polypeptide (and optionally additional coding se~uence) and non-coding sequence, such as introns or non-coding sequence 5~ and/or 3' o~ the coding sequence for the mature polypeptide.
The present invention further relates to variants of the her~n~hove descri~ed polynucleotides which ~nco~ ~or ~ragments, analogs and derivatives o~ the polypeptide having the deduced amino acid sequence of Figure 1 ~SEQ ID
NO:2) The variant of the polynucleotide may be a naturally occurring allelic variant o~ the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) as well as variants o~ such polynucleotides which variants encode for a ~ragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2). Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As her~;n~bove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding se~uence shown in Figure 1 (SEQ ID
NO:1). As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function o~ the encoded polypeptide.
The polynucleotides may al~o encode ~or a soluble ~orm of the G-protein chem~kine receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved ~rom the TM and intracellular ~nm~ ~ n of the full-length polypeptide o~ the present invention.
The polynucleotides of the present invention may also have the coding sequence fused in ~rame to a m~rker .

sequence which allows ~or puri~ication o~ the polypeptide of the present inven~ion. The marker se~uence m~y be a hexa-histidine tag supplied by a pQE vector (Qiagen) to provide ~or puri~ication o~ the mature polypeptide ~used to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (BA) tag when a m~mm~ lian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglll~in~n protein ~Wilson, I., et al., Cell, 37:767 (1984)).
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and ~ollowing the coding region ~leader and trailer) as well as intervening sequences (introns) between individual codiny segments (exons).
Fragments o~ the ~ull length gene o~ the present invention may be used as a hybridization probe ~or a cDNA
library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence sim;l~ity to the gene or similar biological activity. Probes of this type preferably have at least 15 bases, preferably 30 bases and most preferably, may contain, ~or example, 50 or more bases. The probe may also be used to identi~y a cDNA clone corresponding to a ~ull lenyth transcript and a genomic clone or clones that Cont~ n the complete gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene o$ the present invention are used to screen a library of h~ n cDNA, genomic DNA or mRNA to determine which memh~S of the library the probe hybridizes to.
The present invention ~urther relates to polynucleotides which hybridize to the hereinabove-described se~nces i~ there is at least 70~, preferably at least 90~, and more pre~erably at least 9~ identity between the sequences. The present invention particularly _g _ relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides.
As herein used, the term "stringent conditions~ means hybridization will occur only i~ there is at least 95~ and preferably at least 97% identity between the sequences.
The polynucleotides which hybridize to the her~in~hove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA o~ Figure 1 (SEQ ID NO:1).
Alternatively, the polynucleotide may have at least 15 bases, pre~erably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide o~ the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes ~or the polynucleotide of SEQ ID NO:1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences i~ there is at least 70~, pre~erably at least 90~, and more pre~erably at least 95~ identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides.
As herein used, the term "stringent conditions" means hybridization will occur only i~ there is at least 95~ and preferably at least 97~ identity between the sequences.
The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred Pmhn~;mPnt encode polypeptides which either retain ~ubst~nti~lly the same biological function or activity as the mature polypeptide encoded by the cDNA o~ Figure 1 (SEQ ID NO~
The present invention ~urther relates to a G-protein çhPmnkine receptor polypeptide which has the deduced amino acid se~uence of Figure 1 (SEQ ID NO:2), as well as fragments, analogs and derivatives of such polypeptide.

W O 97/25340 PCT~US96/00499 The terms ~ragment,~ "derivative" and ~analog~ when referring to the pol~peptide of Fi~ure 1 ~SEQ ID NO:2) !
means a polypeptide which either retains substantially the same biological function or activity as such polypeptide, i.e. functions as a G-protein ~.h~mokine receptor, or retains the ability to bind the ligand for the receptor, for example, a soluble form of the receptor. An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a rec~mh;n~nt polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a rero~hin~nt polypeptide.
The fragment, derivative or analoy of the polypeptide of Figure 1 tSEQ ID NO:2) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conser~ed amino acid residue) and such substituted amino acid residue may or may not be 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 mature polypeptide is fused with another compound, such as a compound to increase the hal~-life o~
the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide or (v) one in which a fragment of the polypeptide is soluble, i.e. not ,.,e"~ldne bound, yet still binds ligands to the 1.._..~ dne bound receptor. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the t~h;ngs herein.
The polypeptides and polynucleotides of the present - invention are preferably provided in an isolated form, and preferably are purified to hu~L~yelleity.
Thus, the present invention is directed to polynucleotides having at least a 70~ identity, preferably at least 90% and more preferably at least a 95~ identity to W O 97/25340 PCTnJS96/0~499 a polynucleotide which ~nco~s the polypeptide o~ SEQ ID
N0:2 and polynucleotides complementary thereto as well as portions thereo~, which portions have at least 15 consecutive bases, pre~erably 3~ consecutive ba~es and more pre~erably at least 50 consecutive bases and to polypeptides encoded by such polynucleotides.
As known in the art "s;m;l~ity~ between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence o~ a second polypeptide.
Fragments or portions o~ the polypeptides o~ the present invention may be employed ~or producing the corresponding ~ull-length polypeptide by peptide synthesis, there~ore, the ~ragments may be employed as intenmediates for producing the full-length polypeptides. Fragments or portions o~ the polynucleotides o~ the present invention may be used to synthesize ~ull-length polynucleotides of the present invention.
The term "gene" means the segment o~ DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer~ as well as intervening seguences (introns) between individual coding segments (exons).
The term "isolated" means that the material is l~ulo~d ~rom its original envi.~-l".e~t (e.g., the natural envi~olllLIellt if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living ~n~m~l iS not isolated, but the same polynucleotide or polypeptide, separated ~rom some or all o~ the coexisting materials in the natural system, is isolated. Such polynucleotides could ~e part o~ a vector and/or such polynucleotides or polypeptides could be part o~ a composition, and still ~e isolated in that such vector or composition is not part o~ its natural envi~ -t.
The present invention also relates to vectors which include polynucleotides o~ the present invention, host cells which are genetically engineered with vectors o~ the W O 97/25340 PCTrUS96/00499 invention and the production o~ polypeptides o~ the invention by recombinant techniques.
Host cells are yenetically engineered (transduced or trans~ormed or trans~ected) with the vectors of this invention which may be, ~or example, a cloning vector or an expression vector. The vector may be, ~or example, in the ~orm of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modi~ied as appropriate ~or activating promoters, selecting trans~ormants or amplifying the genes o~ the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected ~or expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides o~ the present invention may be employed for producing polypeptides by rec~mh;n~t techniques. Thus, ~or example, the polynucleotide may be included in any one o~ a variety o~ expression vectors ~or expressing a polypeptide. Such vectors include chromosomal, non~hromosomal and synthetic DNA seqlPn~, e.g., derivatives o~ SV40; ~acterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived ~rom combinations o~ plasmids and phaye DNA, viral DNA such as vaccinia, adenovirus, ~owl pox virus, and pseudora~ies.
However, any other vector may be used as lon~ as it is replicable and viable in the host.
The d~LO~ iate DNA sequence may be inserted into the vector by a variety o~ procedures. In general, the DNA
sequence is inserted into an d~LV~riate restriction ~n~nllClease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope o~
those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As ~ representative examples o~ such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage ~ Amh~l~ PL promoter and other promoters known to WO 97~5340 PCT~US9GtO01~
control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include a~riate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait ~or selection of trans~ormed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampi-cillin resistance in E. coli.
The vector cont~i n~ ny the d~Lo~riate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to trans~orm an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as ~. coli, StreptomYces, S~m~nella tYPhimurium; fungal cells, such as yeast; insect cells such as Droso~hila and S~odoptera Sf9;
~n~m~l cells such as CHO, COS or Bowes m~l~n~m~;
a~enovirus; plant cel~s, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs ~u,.,~isiny one or more o~ the se~uences 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 em~o~mp~t~ 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, WO 97/25340 ~CTAJS~G~
pKK223-3, pKK233-3, pDR540, pRIT5 ~Pharmacia). Eukaryotic:
pWLNEO, pSV2CAT, pO~44, pXT1, pSG (Stratagene) p~VK3, pBPV, pMSG, pSVL (Pharmacia) However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
Promoter regions can be selected from any desired gene using CAT (chlor~r~n~col transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular n~m~ bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda ~?R~ PL and trp. ~ukaryotic promoters include CMV ~mm~ te early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection o~ the appropriate vector and promoter is well within the level o~
ordinary skill in the art.
In a ~urther embodiment, the present invention relates to host cells contA~n~ng the above-described constructs.
The host cell can be a higher eukaryotic cell, such as a m~m~lian 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 cell can be e~fected by calcium phosphate trans~ection, DEAE-Dextran mediated trans~ection, 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~nn~ to produce the gene product encoded by the recombinant se~uence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in m~m-lian cells, yeast, ~acteria, or other cells under the control of d~riate promoters. ~ell-~ree translation systems can also be employed to produce such proteins using RNAs derived ~rom the DNA constructs o~ the present invention.
Appropriate cloning and expression vectors ~or use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory ~nll~l, Second W O 97/25340 PCT~US96/00499 Edition, Cold Spring Harbor, N.Y., (1989), the disclo~ure of which is hereby i~corporated by reference.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an ~nh~ncer seguence 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 including the SV40 ~nh~n~er on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter ~nhAncer, the polyoma ~nh~n~er on the late side of the replication origin, and adenovirus ~nh~ncers ~ enerally, recombinant expression vectors will includP
origins of replication and selectable markers 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 enzymes such as 3-phosphoglycerate kinase ~PGK), ~-factor, acid phosphatase, or heat shock proteins, among others.
The heterologous structural se~uence is assembled in a~l~riate phase with translation initiation and termination se~l~nce~, and prefera~ly, a leader sequence capable o~ directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous se~uence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stAh;l~zation or simplified purification of expressed recombinant product.
Useful 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 markers and an origin of replicAt~ on to ensure maintenance of the vector and to, if desirable, provide amplification within the host Suitable prokaryotic hosts ~or trans~ormation include E. coli, Bacillus subtilis, Salmonella tY~h~mllrium and various species within the genera Psell~omnn~-~, Streptomyces, and Staphylococcus, although others may also be employed as a matter o~ choice.
As a representative but nonl~m; ting example, useEul expression vectors ~or bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements o~ the well known cloning vector pB~322 (ATCC 37017). Such commercial vectors include, ~or example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "ba~-khon~" sections are c-~mhin~d with an a~:L~riate promoter and the structural se~uence to be expressed.
Following trans~ormation o~ a suitable host strain and growth o~ the host strain to an d~1o~liate cell density, the selected promoter is induced by appropriate means (e.g., temperature shi~t or chemical induction) and cells are cultured ~or an additional period.
Cells are typical~y harvested by centri~ugation, disrupted by physical or chemical means, and the resulting crude extract retained ~or ~urther puri~ication.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including ~reeze-thaw cycling, sonication, mechanical disruption, or u~e o~
cell lysing agents, such methods are well know to those ~killed in the art.
Various m~mm~l ian cell culture systems can also be employed to express recombinant protein. Examples of m~ n expression ~ystems include the COS-7 lines o~
monkey kidney ~ibroblasts, described by Gluzman, Cell, 23:175 ~1981), and other cell lines capable of expressing a compatible vector, ~or example, the C127, 3T3, CH0, HeLa and BHK cell lines. ~ ian expression vector~ will compri~e an origin o~ replication, a suitable promoter and enh~n~er, and also any necessary ribosome h~ n~ ng sites, polyadenylation site, spliCe donor and acceptor sites, WO 97/25340 PCTAUS96/00499 transcriptional termination sequences, and 5~ ~l ~nk~ ng nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic el~m~nt~.
The ~-protein ch~m~kine receptor polypeptides can be recovered and puri~ied from recombinant cell cultures by methods including ~mmon~ um sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydropho~ic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Protein re~olding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography ~HPhC) can be employed ~or final puri~ication steps.
The polypeptides of the present invention may be a naturally puri~ied product, or a product o~ chemical synthetic procedures, or produced by recnmh;n~nt techniques ~rom a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and m~mmAlian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials ~or discovery of tr~tm~nts and diagnostics to hllm~n disease.
The ~-protein ch~mok~ ne receptors of the present invention may be employed in a process ~or screening for c~--~ounds which activate (agonists) or inhibit activation (antagonists) of the receptor polypeptide o~ the present invention .
In general, such screening procedures involve providing appropriate cells which express the receptor polypeptide of the present invention on the surface thereof. Such cells include cells from m~m~ yeast, W O 97/25340 PCT~US96/00499 drosophila or E. Coli. In particular, a polynucleotide encoding the recepto~ o~ the present invention is employed to trans~ect cells to thereby express the G-protein rh~mokine receptor. The expressed receptor is then contacted with a test compound to observe hi nr~ing, s~im~ tion or inhibition o~ a functional response.
One such screening procedure involves the use o~
melanophores which are trans~ected to express the G-protein chemokine receptor of the present invention. Such a screening technique is described in PCT WO 92/Q1810 published February 6, 1992.
Thus, ~or example, such assay may be employed ~or screening ~or a compound which inhibits activation o~ the receptor polypeptide o~ the present invention by contacting the melanophore cells which encode the receptor with both the receptor ligand and a compound to be screened.
Tnhi h; tion o~ the signal generated by the ligand indicates that a compound is a pot~nt;~l antagonist for the receptor, i.e., i nh; hi ts activation o~ the receptor.
The screen may be employed ~or determining a compound which activates the receptor by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor.
Other screening techni~ues include the use o~ cells which express the G-protein rh~m~kine receptor (~or example, trans~ected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, volume 246, pages 181-296 (October 1989). For example, compounds may be contacted with a cell which expresses the receptor polypeptide o~ the present invention and a second messenger response, e.g.
signal transduction or pH changes, may ~e measured to determine whether the potential compound activates or - ;nhi~its the receptor.
Another such screening technique involves introducing RNA encoding the G-protein ~h~mnki n~ receptor into X~nopus oocytes to transiently express the receptor. The receptor oocytes may then be contacted with the receptor ligand and W O 97~5340 PCTrUS96/00499 a compound to be screened, followed by detection o~
inhibition or activation o~ a calcium signal in the case of screening ~or compounds which are thought to inhibit activation o~ the receptor.
Another screening technique involves expressing the G-protein rh~mnk; n~ receptor in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there may be mentioned endoth~l ~Al cells, smooth muscle cells, embryonic kidney cells, etc. The screening may be accomplished as hereinabove described ~y detecting activation o~ the receptor or i nhi h~ tion of activation o~ the receptor ~rom the phospholipase second signal.
Another method invol~es screening ~or compounds which ; nh; h; t activation o~ the receptor polypeptide o~ the present invention by determining the inhibition o~ h,n~ing of a labeled ligand to cells which have the receptor on the sur~ace thereo~. Such a method comprises trans~ecting a eukaryotic cell with DNA encoding the G-protein ch~mnkine receptor of the present invention such that the cell expresses the receptor on its sur~ace and cont~cting the cell with a compound in the presence o~ a laheled ~orm of a known ligand. The ligand can be labeled, e.g., hy radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity o~
the receptors. I~ the compound binds to the receptor as determ;n~ by a reduction o~ labeled ligand which binds to the receptors, the h; n~;ng of~ la~eled ligand to the receptor is ;nh; h; ted.
An Ant;hs~y may antagonize a G-protein chemok;n~
receptor o~ the present invention, or in some cases an oligopeptide, which bind to the ~-protein ~hemokine receptor but does not elicit a second messenger response such that the activity of the G-protein ~h~mnk;ne receptors is ~level~ted. Anti~odies include anti-idiotypic antibodies which recognize uni~ue determ;n~nts generally associated with the antigen-h; n~; ng site o~ an antibody. Potential antagonist compounds also include proteins which are , CA 02242908 l998-07-lO

W O 97/2~340 PCT~US~ 19~
closely related to the ligand of the G-protein ch~m~kine receptors, i.e. a fragment of the li-gand, which have lost biological function and when binding to the G-protein chem~kine receptor elicit no response.
An antisense construct prepared through the use o~
antisense technology, m~y be used to control gene expression through triple-helix ~ormation or antisense DNA
or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5I coding portion of the polynucleotide sequence, which encodes for the m~ture polypeptides of the present invention, i8 used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be compl~m~nt~y 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, 241:4~6 ~1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of G-protein rh~m~kine receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of mRNA molecules into G-protein coupled receptor (antisense - Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene ~xpres~ion, 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 tn vivo to i nht ht t production of G-protein ch~m~ktne receptor.
A small molecule which binds to the G-protein ch~okine receptor, making it inaccessible to ligands such that normal biological activity is prevented, ~or example small peptides or non-peptide antagonists, m~y also be used to ; nh; h~ t activation of the receptor polypeptide of the present invention.
A soluble form of the G-protein ch~mok~n~ receptor, e.g. a ~ragment o~ the receptors, may be used to ; nh; h; t activation of the receptor by b; n~; ng to the ligand to a polypeptide of the present invention and preventing the W O 97~5340 PCT~US96/00499 ligand ~rom interacting with membrane bound G-protein ch~m~kine receptors.
The compounds which bind to and activate the G-protein ~hemnkine receptors o~ the present invention may be employed to stimulate haematopoiesis, wound healing, coagulation, angiogenesis, to treat tumors, chronic in~ections, leukemia, T-cell mediated auto-;mmnn~ diseases, parasitic in~ections, psoriasis, and to stimulate growth ~actor activity.
The compounds which bind to and inhibit the G-protein chemokine receptors o~ the present invention may be employed to treat alleryy, atherogenesis, ~nAphylaxis, maliynancy, chronic and acute in~lammation, histAm~ne and IgE-mediated allergic reactions, prostagl ~n~;n -independent ~ever, bone marrow ~ailure, silicosis, sarcoidosis, rheumatoid arthritis, shock and hyper-eosinophilic syndrome.
The compounds m~y be employed in combination with a suitable pharmaceutical carrier Such compositions comprise a therapeutically e~ective amount o~ the compound and a pharmaceutically acceptable carrier or excipient.
Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The ~ormulation should suit the mode o~ A~m; n t~tration.
The invention also provides a pharmaceutical pack or kit comprising one or more cont~in~s ~illed with one or more o~ the ingredients o~ the pharmaceutical composition~
o~ the invention. Associated with such cont~;n~s) can be a notice in the ~orm prescribed by a governmental agency regulating the manu~acture, use or sale o~ pharmaceutical~
or biological products, which notice re~lects approval ~y the agency o~ manu~acture, use or sale ~or human ;n;~tration. In addition, the compounds of the present invention may be employed in conjunction with other therapeutic compounds.
The ~hArmAceutical compositions may be A~m~ n ~stered in a convenient mAnn~ such as by the topical, intravenous, W O 97/2~340 PCTAUS96/00499 intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are ~mi n~ stered in an amount which is e~ective ~or treating and/or prophylaxis o~ the speci~ic indication. In general, the pharmaceutical compositions will be ~mi n; stered in an amount o~ at least about 10 ~g/kg body weight and in most cases they will be ~mi n; stered in an amount not in excess o~ about 8 mg/Kg body weight per day. In most cases, the dosage is ~rom about 10 ~g/kg to about 1 mg/kg body weight daily, taking into account the routes of ~m; n i ~tration, symptoms, etc.
The soluble G-protein ch~mnkine receptor polypeptides and antagonists or agonists which are polypeptides, may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is o~ten re~erred to as "gene therapy."
Thus, ~or example, cells ~rom a patient may be enyineered with a polynucleotide (DNA or RNA) encoding a polypeptide OE vivo, with the engineered cells then beiny provided to a patient to be treated with the polypeptide.
Such methods are wel~-known in the art For example, cells may be engineered by procedures known in the art by use o~
a retroviral particle cont~in;ng RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo ~or expression o~ a polypeptide in vivo by, ~or example, procedures known in the art. As known in the art, a producer cell ~or producing a retroviral particle cont~;ning RNA encoding the polypeptide of the present invention may be ~min~stered to a patient ~or engineering cells in vivo and expression o~ the polypeptide in vivo.
These and other methods ~or ~ministering a polypeptide o~
the present invention by such method should be apparent to those skilled in the art from the teachings o~ the present invention. For example, the expression vehicle ~or - engineering cells may be other than a retrovirus, ~or example, an adenovirus which may be used to engineer cells in vivo a~ter cQ~hin~tion with a suitable delivery vehicle.

.

W O 97/25340 PCT~US~6~55 Retroviruses ~rom which the retroviral pla~mid vectors hereina~ove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Viru~, avian leukosis virus, gibbon ape leukemia viru~, human ,mmllnode~iciency virus, adenovirus, Myeloproli~erative Sarcoma Virus, and m~mmA~y tumor viru~.
In one embo~;m~nt, the retroviral plasmid vector is derived ~rom Moloney Murine Leukemia Virus.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the hl~mAn cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eu~aryotic cellular promoters including, but not limited to, the histone, pol III, and ~-actin promoter~). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine ki~ase (TK) promoters, and Bl9 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
The nucleic acid se~uence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; ~nAllc;hle promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin ~r ~--~oters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modi~ied retroviral ~TRs her~nAhove described); the ~-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the genes encoding the polypeptides.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples o~ packaging cells which may be trans~ected include, but are not limited to, the PE501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H2, ~CRE, ~CRIP, GP+E-86, GP+envAm12, and DAN
cell lines as described in Miller, Human Gene Therapy, Vol.
1, pgs. 5-14 (199~), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells throuyh any means known in the art. Such means include, but are not limited to, electroporation, the use o~ liposomes, and CaP04 precipitation. In one alternative, the retroviral plasmid vector m y be encapsulated into a liposome, or coupled to a lipid, and then ~m~n~stered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid se~uence~s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limlted to, embryonic stem cells, embryonic carc;nnm~ cells, as well as hematopoietic stem cells, hepatocytes, ~ibroblasts, myoblasts, keratinocytes, endoth~ l cells, and bron~h~l epith~li~l cells.
The present invention also provides a method ~or determin; ng whether a ligand not known to be cAp~hle o~
h;n~l;ng to a G-protein rh-~mnkine receptor can bind to such receptor which comprises contacting a m~ n cell which expresses a G-protein ~h~mnkine receptor with the ligand under conditions permitting binding o~ ligands to the G-protein rhem~kine receptor, detecting the presence o~ a ligand which binds to the receptor and thereby determ;n;ng whether the ligand binds to the G-protein rh~m~kine ~ receptor. The systems hereinabove described ~or determ;n;ng agonists and/or antagonists m~y also be .

W O 97~5340 PCT~US96/00499 employed ~or determt ni ng ligands which bind to the receptor. - -This invention also provides a method of detectingexpression of a G-protein ~h~m~k~ne receptor polypeptide of the present invention on the sur~ace o~ a cell by detecting the presence of mRNA coding for the receptor which comprises obt~;n~ng total mRNA ~rom the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 10 nucleotides capable of speci~ically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence o~ mRNA hybridized to the probe, and thereby detecting the expression o~ the receptor by the cell.
The present invention also provides a method ~or identifying receptors related to the receptor polypeptides of the present invention. These related receptors may be i~nt; fied by homology to a G-protein chemokine receptor polypeptide o~ the present invention, by low stringency cross hybridization, or by identi~ying receptors that interact with related natural or synthetic ligands and or elicit s~mt 1~ behaviors after genetic or pharmacological blockade o~ the ~-hl~m~k~ ne receptor polypeptides of the present invention.
The present invention also contemplates the use of the gene of the present invention as a diagnostic, ~or example, some diseases result from inherited defective gene . These genes can be detected by comparing the se~l~nce~ of the de~ective gene with that of a normal one. Subsequent~y, one can ve~ify that a 'imutant" gene is associated with a~normal receptor activity. In addition, one can insert mnt~nt receptor genes into a suitable vector for expression in a ~unctional assay system (e.g., colorimetric assay, expression on MacConkey plates, compl~m~nt~tion exper~nt~, in a receptor deficient strain of ~EK293 cell~) as yet another means to verify or identi~y mutations. Once ~Imutant~ genes have been identified, one . .

W O 97/25340 PCT~US~6/C~~9~
can then screen population ~or carriers o~ the "mllt~nt"
receptor gene.
Individuals carrying mutations in the gene o~ the present invention may be detected at the DNA level by a variety o~ techniques. Nucleic acids used ~or diagnosis may be obtained ~rom a patient~s cells, including but not limited to such as ~rom blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly ~or 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 ~or the same purpose. As an example, PCR primers compl~m~nt~y to the nucleic acid o~ the instant invention can be used to identi~y and analyze mutations in the gene of the present invention. For example, deletions and insertions can be detected by a change in size o~ the ampli~ied product in comparison to the nonmal genotype. Point mutations can be identi~ied by hybridizing ampli~ied DNA to radio labeled RNA o~ the invention or alternatively, radio labeled antisense DNA sequences of the invention. Perfectly matched se~l~nc~s can be distinguished ~rom m; ~m~tched duplexes by RNase A digestion or by di~erences in melting temperatures. Such a diagnostic would be particularly use~ul ~or prenatal or even n~on~tal testinq.
Sequence differences between the re~erence gene and ~lm~ ntS~ may be revealed by the direct DNA se~lPn~ ng method. In addition, cloned DNA segments may be used as probes to detect speci~ic DNA se~m~nt~. The sensitivity o~
this method is greatly Pnh~n~A when combined with PCR.
For example, a sequence primer is used with double str~n~
PCR product or a single str~n~e~ template molecule generated by a modi~ied PCR. The sequence determination is per~ormed by conventional procedures with radio labeled nucleotide or by an automatic se~l~nc~ng procedure with ~luorescent-tags.
Genetic testing based on DNA sequence di~erences may be achieved by detection o~ alterations in the electrophoretic mobility o~ DNA ~ragments in gels with or -W O 97/25340 PCT~US96/00499 without denaturing agents. Sequences changes at speciEic locations may also -be revealed by nucleus protection assays, such RNase and S1 protection or the chemical cleavage method ~e.g. Cotton, et al., PNAS, USA, 85:4397-4401 1985).
In addition, some diseases are a result o~, or are characterized by changes in gene expression which can be detected by changes in the mRNA. Alternatively, the genes o~ the present invention can be used as a re~erence to identify individuals expressing a decrease of ~unctions associated with receptors o~ this type.
The present invention also relates to a diagnostic assay ~or detecting altered levels of soluble ~orm o~ the G-protein ~h~m~k; n~ receptor polypeptides o~ the present invention in various tissues. Assays used to detect le~els o~ the soluble receptor polypeptides in a ~ample derived ~rom a host are well known to those o~ skill in the art and include radio;mmnno~ssays, competitive-b~ nAing assays, Western blot analy~is and preferably as ELISA as~ay.
An ELISA assay initially comprises preparing an antibody speci~ic to antigens o~ the G-protein ~h~mokine receptor polypeptides, pre~erably a monoclonal anti~ody.
In addition a reporter ~nt; hody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, ~luorescence or in this example a horseradish peroxidase enzyme. A sample is now le,~oved ~rom a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the ~ample. Any ~ree protein hi n~; ng sites on the dish are then covered by incubating with a non-speci~ic protein such as bovine serum albumin. Next, the monoclonal ~nt; h~dy is incu-hated in the dish during which time the monoclonal antibodies attach to any G-protein ~hem~kine receptor proteins attached to the polystyrene dish. All unbound monoclonal ~nt;hody is washed out with bu~er. The reporter antibody l; nk~ to horseradish peroxidase is now placed in the dish resulting in h; n~ ng of the reporter ~nt; h~Ay to any monoclonal ~nt; hody bound to G-protein W O 97/2S340 PCT~US96100499 rhF.m~k; n~ receptor proteins. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount o~ G-protein ch~mokine receptor proteins present in a given volume o~
patient sample when compared against a st~n~A~d curve.
The se~uences 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 par~icular sites on the chromosome. Few chromosome mar~ing reagents based on actual se~uence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, se~l~nc~ can be mapped to chromosomes by preparing PCR primers (pre~erably 1~-25 bp) from the cDNA.
Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the ampli~ication process. These pri~ers are then used ~or PCR screening o~ somatic cell hybrids cont~tn~ng individual hnm~n chromosomes. Only those hybrids cont~n;ng the hllm~n gene corresponding to the primer will yield an amplified ~ragment.
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 r~nels o~ ~ra~ments from specific chromosomes or pools of large genomic clones in an analogous m~nn~r, Other mapping strategies that can similarly be used to map to it~
cl~. r~ include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome speci~ic-cDNA
libraries.

' -29-W O 97/25340 PC~US~G/C~199 Fluorescence in situ hy~ridization (FIS~) o~ a cDNA
clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases.
For a review of this technique, see Verma et al., ~l~m~n Chromosomes: a ~nll~ 1 of Basic Techniques, PeLydll~oll Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the seque~ce on the chromosome can be correlated with genetic map data.
Such data are found, for example, in V. McKusick, M~n~lian Inheritance in Man (aV~ hl e on line through ~ohns 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 di~ferences in the cDNA or genomic sequence between a~ected and una~ected individuals. I~ a mutation is observed in some or all of the a~ected individuals but not in any normal individuals, then the mutation is likely to be the causative agent o~ the disease.
With current resolution of physical ~apping 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 senes. (This assumes 1 meyabase mapping resolution and one gene per 20 kb).
The polypeptides, their ~ra~m~nts or other derivatives, or analogs thereo~, or cells expressing them can be used as an i~m~lnogen to produce anti~odies thereto.
These antibodies can be, for example, polyclonal or monoclonal ~n~;hodies The present invention also includes ~him~ric, gingle chain, and hllm~nized antibodies, as well as Fab ~La~J~ ts~ or the product o~ an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragment~.

W O 97/2~340 PCT~US9G/00~
Antibodies generated again~t the polypeptides corresponding to a sequence o~ the present invention can be obtained by direct injection o~ the polypeptides into an ~n~m~l or by ~m1 n; stering the polypeptides to an ~nim~l, preferably a nonhl~m~n. The antibody so obtained will then bind the polypeptides itsel~. In this m~nn~r, even a sequence encoding only a ~ragment o~ the polypeptides can be used to generate ~nt;~odies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide ~rom tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the hllm~n B-cell hybridoma technique ~Kozbor et al., 1983, Tmmllnology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies ~Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production o~ single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain ant;bc)A; es to immllnogenic polypeptide products o~ this invention. Also, transgenic mice may be used to express hllm~n1zed antibodies to ;~mllnogenic polypeptide products o~ this invention.
The above-described antibodies may be employed to i~olate the polypeptide o~ the present invention by atta~hmPnt o~ the antibody to a solid SU~OLL and per~orming a~inity chromatography by passing the polypeptide desired to be puri~ied over the column and recovering the puri~ied polypeptide.
The present invention will be ~urther described with re~erence to the ~ollowing examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise speci~ied, are by weight.

WO 97/25340 PC~fUS96/00499 In order to ~acilitate underst~n~ng o~ the ~ollowing examples certain ~re~ently occurring methods and/or terms will be described.
~ 'Plasmidsl' are designated by a lower case p preceded and/or ~ollowed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed ~rom available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" o~ DNA refers to catalytic cleavage o~ the DNA with a restriction enzyme that acts only at certain ~equences in the DNA. The various restriction enzymes used herein are c~mm~rcially available and their reaction conditions, co~actors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 ~g o~ plasmid or DNA
~ragment is used with a~out 2 units o~ enzyme in about 20 ~1 o~ bu~er solution. For the purpose of isolating DNA
~ragments for plasmid construction, typically 5 to 50 ~g of DNA are digested with 20 to 250 units o~ enzyme in a larger volume. Appropriate bu~ers and substrate amounts ~or particular restriction enzymes are speci~ied by the manu~acturer. Incubation times o~ about 1 hour at 37 C are ordinarily used, but may vary in accordance with the supplier's instructions. A~ter digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired ~ragment.
Size separation o~ the cleaved ~r~gm~n~c is per~ormed using 8 percent polyacrylamide gel described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 ~1980).
"Oligonucleotides" re~ers to either a single str~n~
polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a ~ragment that has not been dephosphorylated.
'ILigation~ re~ers 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 may be accomplished using known bu~fers and conditions with 10 units to T4 DNA ligase (nligase") per 0.5 ~g o~ approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, trans~ormation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 ~1973).

Exam. ple Bacterial ExPression and Purification o~ HSATU68 The DNA sequence encoding for HSATU68, ATCC # 97334 is initially amplified using PCR oligonucleotide primers corresponding to the 5' and se~l~nr~s of the proces~ed HSATU68 protein (minus the signal peptide sequence) and the vector sequences 3' to the HSATU68 gene. Additional nucleotides correspon~ng to HSATU68 were added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5' CGGGA~ ~CATGGAGTTGAGGAAGTAC 3~
~SEQ ID NO:3) contains a BamHI restriction enzyme site ~ollowed by 18 nucleotides of HSATU68 coding se~uence starting ~rom the presumed terminal amino acid of the protein. The 3' sequence 5' GGCGGATCCCGCTCACAAGCCCGAGTAGGA
3' (SEQ ID NO:4) rnnt~n~ compl~m~nt~ry sequences to a BamHI site and is ~ollowed by 18 nucleotides o~ HSATU68 coding sequence. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQ~-9 (Qiagen, Inc., Chatsworth, CA, 91311). pQE-9 encodes antibiotic resistance (~mpr), a bacterial origin of ~ replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome htn~;ng site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 was then digested with BamHI. The ampli~ied seguences were ligated into pQE-9 and were inserted in ~rame with the sequence encoding for the WO 97/2534Q PCT~US96100499 histidine tag and the RBS. The ligation mix~ure was then used to transfonm E.-coli ~train M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory ~nl7~-, Cold Spring Laboratory Pre~s, (1989). M15Jrep4 cont~tn~ multiple copies o~ the plasmid pREP4, which expresses the lacI
repressor and also con~ers kanamycin resistance (Kanr).
Transformants are identified by their ability to grow on ~B
plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones cont~;n~ng the desired constructs were grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio o~ 1:100 to 1:250. The cells were grown to an optical density 600 (o~D~6~) o~ between 0.4 and 0.6.
IPTG (nIsopropyl-B-D-thiogalacto pyranoside") was then added to a ~inal concentration o~ 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression. Cell~ were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation.
The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HCl. A~ter clari~ication, solubilized HSATU68 was puri~ied ~rom this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins cont~ntng the 6-His tag (Hochuli, E. et al., ~. Chromatography 411:177-184 (1984)).
HSATU68 was e}uted from the column in 6 molar guanidine HCl pH 5.0 and ~or the purpose o~ renaturation adjusted to 3 molar ~l~n;~;ne HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized).
A~ter tncl~h~tion in this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.

~xam~le 2 ~xDression o~ Recombinant HSATU68 in COS cells The expression o~ plasmid, HSATU68 HA is derived ~rom a vector pcDNAI/Amp (Invitrogen) con~;n~n~ 1) SV40 W 097/25340 PCT~US96/00499 oriyin o~ replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter ~ollowed by a polylinker region, a SV40 intron and polyadenylation site.
A DNA ~ragment encoding the entire HSATU68 precursor and a HA tag ~used in ~rame to its 3' end was cloned into the polylinker region o~ the vector, there~ore, the recom~inant 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.
1erner, 1984, Cell 37, 767). The in~usion of HA tag to the target protein allows easy detection of the recombinant protein with an ~nt ~hody that recognizes the HA epitope.
The plasmid construction strategy is described as ~ollows:
The DNA sequence encoding ~or HSATU68, ATCC # 97334, was constructed by PCR using two primers: the 5' primer 5' GTCC
AAGCTTGCCACCATGGAGTT~7~GTAC 3' (SBQ ID NO:5) and contains a HindIII site ~ollowed by 18 nucleotides of HSATU68 coding sequence starting ~rom the initiation codon ~u n d e r l i n e d) ; t he 3 ' s e q u e n c e 5 ' CTGCTCGAGTCAAGCGTA~ ~ GA~ ~lAl~ w l~AGCACA
AGCCCGAGTAGGA 3' (SEQ ID NO:6) cont~n~ compl~men~y sequences to an XhoI site, translation stop codon, HA tag and the last 15 nucleotides o~ the HSATU68 coding sequence (not including the stop codon). There~ore, the PCR product cont~n~ a HindIII site HSATU68 coding sequence ~ollowed by HA tag fused in frame, a tran~lation termination stop codon next to the HA tag, and an XhoI site. The PCR ampli~ied DNA ~ragment and the vector, pcDNAI/Amp, were digested with HindIII and XhoI restriction enzyme and ligated. The ligation mixture was trans~ormed into E. coli strain SURE
(Stratagene Cloning Systems, La Jolla, C~ 92037) the trans~ormed culture was plated on ampicillin m~; ~ plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and ~mi n~ by restriction analysis ~or the presence Of the correct fragment. For W O 9712~340 PCTrUS96/00499 expression o~ the recombinant HSATU68, COS cell~ were transfected with the expression vector by DEAE-DEXTRAN
method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory ~Anll~l, Cold Spring Laboratory Press, (1989)). The expression o~ the HSATU68 HA protein was detected by radiolabelling and ~mm~lnoprecipitation method (E. Harlow, D. Lane, ~nt~hodies: A Laboratory ~nll~l, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with 35S-cysteine two days post trans~ection. Culture media were then collected and cells were lysed with detergent (RIPA buf~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 were precipitated with a HA speci~ic monoclonal antibody. Proteins precipitated were analyzed on 15~ SDS-PAGE gels.

E~ample 3 Cloninq and exPression o~ HSATU68 usinq the baculovirus exPression system The DNA sequence encoding the ~ull length HSATU68 protein, ATCC # 97334, was ampli~ied using PCR
oligonucleotide primers corresponding to the 5~ and 3 se~uences o~ the gene:
The 5~ primer has the sequence 5' CGGGATCCCTCCC
ATGGAGTTGAGGAAGTAC 3' (SEQ ID NO:7) and cont~;n~ a BamHI
restriction enzyme site ~ollowed by 5 nucleotides resemhl1 ng an e~icient signal ~or the initiation o~
translation in eukaryotic cells (J. Mol. Biol. 1987, 196, 947-g50, Kozak, M.), and just behind the ~irst 6 nucleotides o~ the HSATU68 gene (the initiation codon ~or translation is "ATG"). The 3' primer has the sequence 5' CGGGATCCCGCTCACAAGCCCGAGTAGGA 3' (SEQ ID NO:8) and contains the cleavage site ~or the restriction Pn~nl~clease BamHI
and 18 nucleotides c~mplem~nt~ry to the 3' non-tran~lated se~uence o~ the HSATU68 gene. The ampli~ied se~uences were isolated ~rom a 1~ agarose gel using a commercially available kit ('IGeneclean,'' BIO 101 Inc., La Jolla, Ca.).

CA 02242908 l998-07-lO

W O 97/25340 PCT~US~ 159 The ~ragment was then digested with the ~n~nllclease BamHI
and purified as described above.- This fragment is designated F2.
The vector pRG1 (modification of pVL941 vector, discussed below) is used for the expression of the HSATU68 protein using the baculovirus expression system ~for review see: Summers, M.D. and Smith, G.E. 1987, A mAnn~l of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Exper~mPntAl Station Bulletin No. 1555). This expression vector cont~in~ the strong polyhedrin promoter of the Autographa cali~ornica nuclear polyhedrosis virus (AcMNPV) ~ollowed by the recognition sites for the restriction ~n~onllclease BamHI.
The polyadenylation site of the simian virus SV40 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 promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at ~oth sides by viral seguences for the cell-mediated homologous recombination of co-transfected wild-type viral DN~. Many other baculovirus vectors could be used in place of pR&1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
The plasmid was digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1~ agarose gel as described above.
This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E. coli B 101 cells were then trans~ormed and bacteria ~nt; ~ied that cont~ n~ the plasmid (pBacHSATU68) with the HSATU68 gene using the enzyme BamHI. The sequence of the cloned fragment was confirmed by DNA se~l~ncin~.
- 5 ~g of the plasmid pR~r~TU68 were co-transfected with 1.O ~g of a ~n~mP~cially av~ hle linp~ized bacu30virus ("BaculoGold~ baculovirus DNA", Pharmingen, San CA 02242908 l998-07-lO

WO 97/25340 PCT~US9G/00199 Diego, CA.) using the lipofection method ~Felgner et al.
Proc. Natl. Acad. Sci. USA, 84:7413-7417 ~1987)).
l~g of BaculoGold~ virus DNA and 5 ~g of the plasmid pBacHSATU68 were mixed in a sterile well of a microtiter plate contA;n;ng 50 ~l o~ serum free Grace's medium (Li~e Technologies Inc., Gaithersburg, MD) Afterwards 10 ~l Lipofectin plus 90 ~l Grace's medium were added, mi~ and incubated _or 15 minutes at room temperature. Then the transfection mixture was added drop wise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace' medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate was then incubated for 5 hours at 27~C. A~ter 5 hours the trans~ection solution was Lel-l~ved from the plate and 1 ml o~ Grace's insect medium supplemented with 10~
~etal cal~ serum was added. The plate was put back into an incubator and cultivation continued at 27~C ~or ~our days.
After four days the supernatant was collected and a pla~ue assay performed sim;l~ as described by Summers and Smith ~supra). As a modification an agarose gel with "Blue Gal~ (Life Technologies Inc., Gaithersburg) was used which allows an easy isolation of blue st~ n~ plaques. (A
detailed description o~ a "pla~ue assay~ can also be ~ound in the user's guide ~or insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).
Four days after the serial dilution, the viruses were added to the cells and blue st~;ne~ pla~ues were picked with the tip of an ~ppendor~ pipette. The agar contAinin~
the recombinant viruses was then resuspended in an - ~ppendorf tube c~ntAtnin~ 200 ~l of Grace's medium. The agar was ~ ~ved by a brief centrifugation and the supernatant contA~nin~ the recomhin~nt baculoviruses was used to infect Sfg cells seeded in 35 mm dishes. Four days later the supernatants of these culture ~1;~:h~ : were harvested and then stored at 4~C.
Sf9 cells were grown in Grace's medium supplemented with 10~ heat-inactivated FBS. The cells were infected CA 02242908 l998-07-lO

W O 97/25340 PCT~US~G/0019~
with the recombinant baculo~irus v-HSATU68 at a multiplicity o~ in~ection (MOI) of 2-. Six hours later the medium was removed and replaced with SF900 II medium minus methionine and cysteine ~Li~e Technologies Inc., Gaithersburg). 42 hours later 5 ~Ci o~ 35S-methionine and 5 ~Ci 35S cysteine ~Amersham) were added. The cells were ~urther incubated ~or 16 hours before they were harvested by centri~ugation and the labelled proteins visualized by SDS-PAG~ and autoradiography.

~xam~le 4 Expression via Gene TheraPy Fibroblasts are obt~; neA from a subiect by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks o~
the tissue are placed on a wet sur~ace o~ a tissue culture ~lask, approximately ten pieces are placed in each flask.
The ~lask is turned upside down, closed tight an~ le~t at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks o~ tissue remain ~ixed to the bottom o~ the ~lask and ~resh media (e.g., Ham's F12 m~; ~ , with 10% FBS, penicillin and streptomycin, is added. This is then incubated at 37~C ~or approximately one week. At this time, ~resh m~tA is added and subsequently changed every several days. A~ter an additional two weeks in culture, a monolayer o~ ~ibroblasts emerge. The monolayer is trypsinized and scaled into larger ~lasks.
pMV-7 (Kir~chmeier, P.T. et al, DNA, 7:219-25 (1988) ~ nke~ by the long terminal repeats o~ the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with cal~ intestinal phosphatase. The l~neAr vector is ~ractionated on agarose gel and puri~ied, using glass beads.
The cDNA encoding a polypeptide o~ the present invention is ampli~ied using PCR primers which correspond to the 5' and 3' end se9uences respectively. The 5' primer contA;n~ an EcoRI site and the 3' primer cnntA~n~ a HindIII

site. ~qual quantities o~ the Moloney murine sarcoma virus 1 ,ne~ backbone and the ~coRI and-HindIII ~ragment are added toyether, in the presence o~ T4 DNA ligase. The resulting mixture is maintA~ne~ under conditions appropriate ~or ligation of the two ~r~m~nt~ The ligation mixture i5 used to trans~orm bacteria HB101, which are then plated onto agar-ront~tning kanamycin ~or the purpose o~ con~irming that the vector had the gene o~
interest properly inserted.
The amphotropic pA317 or GP~aml2 packaging cells are grown in tissue culture to con~luent density in Dulbecco's Modi~ied ~agles Medium (DMEM) with 10% cal~ serum (CS), penicillin and streptomycin. The MSV vector c~n~;n~ng the gene is then ~e~ to the media and the packaging cells are transduced with the vector. The packaging cells now produce in~ectious viral particles cont~ning the gene (the packaging cells are now re~erred to as producer cells).
Fresh m~ iS ~lAetl to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate o~ con~luent producer cells. The spent media, ~ont~n~ng the in~ectious viral particles, is ~iltered through a millipore ~ilter to remove detached producer cells and this media is then used to in~ect ~ibroblast cells. Media is le..lo~ed ~rom a sub-con~luent plate o~ fibroblasts and quickly replaced with the media ~rom the producer cells.
This media is removed and replaced with fresh media. If the titer o~ virus is high, then virtually all ~ibroblasts will be in~ected and no selection is required. I~ the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, ~uch as neo or his.
- The engineered ~ibroblasts are then injected into the host. The ~ibroblasts now produce the protein product.
Numerous modi~ications and variations o~ the present invention are possible in light o~ the above t~h~ngs and, therefore, within the scope o~ the appended claims, the invention may be practiced otherwise than as particularly described.

Claims (28)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a polynucleotide having at least a 70% identity to a member selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide comprising an amino acid sequence as set forth in Figure 1:
b) a polynucleotide which is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising at least 30 bases of the polynucleotide of (a) or (b).

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 comprising nucleotide 1 to 1866 set forth in Figure 1.

6 The polynucleotide of Claim 2 comprising nucleotide 173 to 1477 set forth in Figure 1.

7. The polynucleotide of Claim 2 wherein said polynucleotide encodes a polypeptide comprising an amino acid sequence as set forth in Figure 1.

8. An isolated polynucleotide comprising a polynucleotide having at least a 70% identity to a member selected from the group consisting of:
(a) a polynucleotide encoding the same mature polypeptide expressed by the human cDNA contained in ATCC
Deposit No. 97334;
(b) a polynucleotide which is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising at least 30 bases of the polynucleotide of (a) or (b).

9. A vector comprising the DNA of Claim 2.

10. A host cell comprising 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 comprising:
transforming or transfecting the cells with the vector of Claim 9 to thereby express a polypeptide encoded by the human cDNA contained in said vector.

13. A polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) a polypeptide which is at least 70% identical to the amino acid sequence of Figure 1; and (b) a polypeptide comprising at least 30 amino acid residues of the polypeptide of (a).

14. An antibody against the polypeptide of claim 13.

15. An agonist to the polypeptide of claim 13.

16. An antagonist to the polypeptide of claim 13.

17. A method for the treatment of a patient having need to activate a G-protein chemokine receptor comprising:
administering to the patient a therapeutically effective amount of the compound of claim 15.

18. A method for the treatment of a patient having need to inhibit a G-protein chemokine receptor comprising:
administering to the patient a therapeutically effective amount of the compound of claim 16.

19. The method of claim 17 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to the patient DNA
encoding said agonist and expressing said agonist in vivo.

20. The method of claim 18 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to the patient DNA
encoding said antagonist and expressing said antagonist in vivo.

21. A method for identifying compounds which bind to and activate the polypeptide of claim 13 comprising:
contacting a cell expressing on the surface thereof said polypeptide, said polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor polypeptide, with a compound under conditions sufficient to permit binding of the compound to the polypeptide; and identifying if the compound is an effective agonist by detecting the signal produced by said second component.

22. A method for identifying compounds which bind to and inhibit activation the polypeptide of claim 13 comprising:
contacting a cell expressing on a surface thereof said polypeptide, said polypeptide being associated with a second component which provides a detectable signal in response to the binding of a compound thereto, with a compound to be screened under conditions to permit binding to the polypeptide; and determining whether the compound inhibits activation of by detecting the absence of a signal generated from the interaction of said compound with the polypeptide.

23. A process for diagnosing a disease or a susceptibility to a disease related to an under-expression of the polypeptide of claim 13 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide.

24. A process for diagnosing a disease or a susceptibility to a disease related to an over-expression of the polypeptide of claim 13 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide.

25. A process for diagnosing a disease or a susceptibility to a disease related to an under-activity of the polypeptide of claim 13 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide.

26. A process for diagnosing a disease or a susceptibility to a disease related to an over-activity of the polypeptide of claim 13 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide.

27. The polypeptide of Claim 13 wherein the polypeptide is a soluble fragment of the polypeptide and is capable of binding a ligand for the receptor.

28. A diagnostic process comprising:
analyzing for the presence of the polypeptide of claim 27 in a sample derived from a host.