CA2216912A1 - Human g-protein chemokine receptor hdgnr10 - Google Patents
Human g-protein chemokine receptor hdgnr10 Download PDFInfo
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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
O 96~9437 PCT/U',Sl'~7173 ~nMAN G-PROTEIN CEE~O~l~ RE~.~R ~n~ o This invention relates to newly identified polynucleotides, polypeptides encoded by such polynu.leotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a human 7-transmembrane receptor which has been putatively identified as a chemokine receptor, sometimes hereinafter referred to as "G-Protein Chemokine Receptor" or n~lx.N~1o". The invention also relates to inhibiting the action of such polypeptides.
It is well established that many medically significant biological processes are mediated by proteins participating in signaL transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 (1991)). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG
proteins. Some examples of these ?roteins include the GPC
receptors, such as tho~e for adrenergic agents and ~o~ine (Kobilka, B.K., et al., PN~S, 84:46-50 (1987); Robilka, 9.K., et al., Science 238:650-656 (1987); 9unzow, J.R., et al., Nature, 336:783-787 (1g88)), G-proteins themselve~, ef~ector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, ar.d actuator proteins, e.g., protein W096~9437 PCT~S95/07173 kinase A and protein kinase C ~Simon, M.I., et al., Science, 252:802-8 (l99l)).
For example, in one form of signal transduction, the effect of hormone hln~;ng iS activation of an enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP, and GTP also influences hormone hin~l;ng. A G-protein connects the hormone receptors to adenylate cyclase. G-protein was shown to ~Ych~nge GTP for bound GDP when activated by hon~one receptors. The GTP-carrying form then binds to an activated adenylate cyclase. Hydrolysis of GTP
to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
The ...~,~Lane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane a-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
G-protein coupled receptors have been characterized as including these seven conserved hydroph~bic stretches of about 20 -to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein family of coupled receptors includes doramine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, hist~mine, thrombin, kinin, follicle stimulating honmone, opsins, endothelial differentiation gene-l receptor and rhodop~ins, odorant, cytomegalovirus receptors, etc.
Wos6/3s437 PCT~S95/07173 G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion ch~nnels and transporters (see, Johnson et al., Endoc., Rev., 10:317-331 (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 G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host.
Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are 8-10 kd in size. In general, chemokines ~Yh;h; t 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine~
residues that form two disulfide bondg. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta.
In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the "C-X-C"
subfamily. In the beta subfamily, the two cysteines are in an adjacent po~ition and are, therefore, referred to as the "C-C~ subfamily. Thus far, at least nine different members of this family have been identified in humans.
The intercrine cytokines P}-h;h; t a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophil8, T lymphocytes, ha~oph;ls and fibrobla~ts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. TheQe activities include stimulation of histamlne release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cells, enhanced binding of complement proteins, induced expression W O 96~9437 PCT/U~5/'~7173 of granulocyte adhesion molecules and complement recéptors, and respiratory bur6t. In addition to their involvement in inflammation, certain chemokines 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-8 ~IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cell~.
In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and di6eage condition6, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.
In accordance with one a6pect of the present invention, there are provided novel mature receptor polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The receptor polypeptides of the present invention are of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules PncoAing the receptor polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA a6 well a6 antisense- analog6 thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with a further aspect of the present invention, there are provided processes for producing such receptor polypeptides by recombinant technique6 compri6ing culturing recombinant prokaryotic and/or eukaryotic ho6t cells, cont~ining nucleic acid sequences encoAing the receptor polypeptides of the present invention, under conditions promoting expre6sion of said polypeptides and subsequent recovery of said polypeptide6.
w096~9437 PCT/U535l~7173 In accordance with yet a further aspect of the present invention, there are provided antibcdies against such receptor polypeptides.
In accordance with another aspect of the present invention there are provided methods of screening for compounds which bind to and activate or inhibit activation of - the receptor polypeptides of the present invention.
In accordance with still another embodiment of the present invention there are provided processes of ~ministering compounds to a host which bind to and activate the receptor polypeptide of the present invention which are useful in stimulating haematopoiesis, wound healing, coagulation, angiogenesi~, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-immune diseases, parasitic infections, psoriasis, and to stimulate growth factor activity.
In accordance with another aspect of the present invention there is provided a method of administering the receptor polypeptides of the present invention via gene therapy to treat conditions related to undeLex~ession of the polypeptides or underexpression of a ligand for the receptor polypeptide.
In accordance with still another embodiment of the present invention there are provided processes of ~mi ni stering co~ro~nA~ to a host which bind to and inhibit activation of the receptor polypeptides of the present invention which are useful in the prevention and/or treatment of allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and Ig8-mediated allergic reactions, prostagl ~nAi n - independent fever, bone marrow failure, silicosis, sarcoidosis, rheumatoid arthritis, shock and hyper-eosin~rhilic syndrome.
In accordance with yet another aspect of,the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically W096~9437 PCT~S95/07173 structurallv related to the G protein-coupled receptor family. It cont~inc an open re~ing frame ~nco~in~ a protein of 352 amino acid residues. The protein exhibits the highest degree of homology to a human MCP-l receptor with 70.1 %
identity and 82.9 % similarity over a 347 amino acid stretch.
The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coAin~ strand or non-co~ing (anti-sense) strand. The coAing sequence which ~n~oAes the mature polypeptide may be identical to the ro~i ng sequence shown in Figure l (SBQ ID
NO:l) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the re~ln~ncy or degeneracy of the genetic code, encodes the-same mature polypeptide as the DNA of Figure l (SBQ ID NO~
or the deposited cDNA.
The polynucleotide which PncoAP~ for the mature polypeptide of Figure l or for the mature polypeptide PncoAeA
by the deposited cDNA may include: only the coAing sequence for the mature polypeptide; the coAing sequence for the mature polypeptide and additional coding sequence such as a transmembrane (TM) or intra-cellular domain; the co~ing sequence for the mature polypeptide (and optionally additional coAin~ sequence) and non-coAing sequence, such as introns or non-coAing sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
Thus, the term "polynucleotide ~ncoAi ng a polypeptide n encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the hereinabove described polynucleotides which ~ncoAe for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure l or the W096~9437 PCT~S95/07173 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, 37:76, (1984)).
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the co~in~ region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA
library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least ~0 bases and may contain, for example, 50 or more bases. The probe may also be used to identify a cDN~
clone corresron~ing to a full length transcript and a genomic, clone or clones that contain 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 c~mplementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The - present invention further relates to polynucleotides which hybridize to the hereinAhove-described seqll~nc~s if there is at least 70%, preferably at least 90%, and more preferably at least 95~ identity between the se~l~nce~. The present invention particularly relates to polynucleotides which hybridize under stringent condition~ to the her~in~hove-described polynucleotides. As herein used, the term n~tringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides _g_ W096~9437 PCT/U~S/~7173 polypeptide Pnro~e~ by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturalIy occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides PnroA-ng the same mature polypeptide as shown in Figure 1 ~SEQ ID N0:2) or the same mature polypeptide Pnro~e~ by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SBQ ID
N0:2) or the polypeptide PnCo~d by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As herPtnAhove indicated, the polynucleotide may have a roAi n~ gequence which is a naturally occurring allelic-variant of the co~i ng sequence shown in ~igure 1 (S~Q ID~
N0:1) or of the coding sequence of the deposited clone. 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 of the encoded polypeptide.
The polynucleotides may also PnroAe for a soluble form of the G-protein chemokine receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved from the TM and intracellular domain of the full-length polypeptide of the present invention.
The polynucleotides of the present invention may also have the coding seguence fused in frame to a marker sequence which allows for purification of the polypeptide 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 polypeptide 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~m,m~l ian -a-w096~9437 PCT/~S35~173 The present invention further relates to a G-protein chemokine receptor polypeptide which has the deduced amino acid sequence of Figure 1 (S~Q ID N0:2) or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
The tenms "fragment, n nderivativell and "analog" when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which either retains sub~tantially the same biological function or activity as such polypeptide, i.e. functions as a G-protein rhemokine receptor, or retains the ability to bind the ligand or the receptor even though the polypeptide does not function as a G-protein chemokine receptor, for example, a ~oluble form of the receptor. An analog includes a ~lv~Lotein which can be activated by cleavage of the ~v~otein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a ~ynthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polypeptide of Figure 1 (SBQ ID N0:2) or that encoAe~ by the deposited cDNA may be ~i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one 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 half-life of 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 membrane bound, yet still binds ligands to the membrane bound receptor. Such fragments, derivatives W096/39437 PCT~S95/07173 which hybridize to the herein~hove described polynucléotides in a preferred embodiment PncoAe polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO:1) or the deposited cDNA(s).
Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as herPin~hove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SBQ ID NO:1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR
primer.
Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which PnCOAP~ the polypeptide of SBQ ID NO:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides PncoA~PA by such polynucleotides.
The deposit(s~ referred to herein will be ma;nt~tned under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. 112.
The sequence of the polynucleotides cont~tnp~ in the deposited materials, as well as the amino acid sequence 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 required to make, or sell the deposited materials, and no such license is hereby granted.
W096/39437 PCT/U~9SI~7173 hybridize to the polynucleotide sequences of the present invention.
In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and for detecting an altered level of the soluble form of the receptor polypeptides.
In accordance with yet a further aspect of the present invention, there are provided processes for utilizing such receptor polypeptides, or polynucleotides encoding 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.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure l shows the cDNA sequence and the corresponding deduced amino acid sequence of the G-protein coupled receptor of the present invention. The st~n~rd one-letter abbreviation for amino acids is used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).
Figure 2 illustrates an amino acid alignment of the G-protein rh~mokine receptor of the present invention and the human MCP-l receptor.
In accor~ance with an a~pect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure l (SBQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. on June l, l995.
The polynucleotide of this invention was discovered in a cDNA library derived from human monocytes. It is W096~9437 PCT~Sss/07173 and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to hu~oye~leity~
The polypeptides of the present invention include the polypeptide of SBQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70%
similarity (preferably a 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably a 90% s~ rity (more preferably a 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably a 95% s~ rity (still more preferably a 90% identity) to the polypeptide of SEQ ID NO:2 and to portions of such polypeptide with such portion of the polypeptide generally cont~intng at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "Si~il~rity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The term "gene n means the segment of DNA involved in pro~llcing a polypeptide chain; it includes regions preceding and following the ro~ing region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons).
The term n isolated" means that the material is removed from its original environment (e.g., the natural envi.ol~-,ellt W096~9437 PCT/U~S~7173 if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a li~ing ~n~-l iS not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
The polypeptides of the present invention include the polypeptide of SBQ ID NO:2 (in particular the mature polypeptide) a~ well as polypeptides which have at lea~t 70%
similarity (preferably at least 70~ identity) to the polypeptide of S8Q ID NO:2 and more preferably at least 90%
similarity (more preferably at least 90% identity) to the polypeptide of SBQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include portion~
of such polypeptides with such portion of the polypeptide generally ~ont~in~ng at least 30 amino acids and more preferably at least 50 amino acids.
AS known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. ~ragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells W096~9437 PCT~S95/07173 which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant 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 fonm of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by reComh;n~nt techniques. 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, nQnchromosomal and synthetic DNA seguences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be uQed as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA
~equence is inserted into an appropriate restriction Pn~onnrlease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) W096/39437 PCT~Sss/07173 (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage 1~mhAA PL
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also contains a ribosome h; n~i ng site for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably cont~in-one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such a~ tetracycline or ampicillin resistance in B. coli.
The vector cont~i ni ng the a~ o~riate DNA sequence as herPin~hove described, as well as an a~ro~riate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
As representative examples of a~lu~riate hosts, there may be mentioned: bacterial cells, such as E. coli, StreptomYces, S~l~onella t~phimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9;
~nir-l cells such as CH0, COS or ~owes mel~n~~-; adenovirus;
plant cells, 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 rec~in~nt 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 w096~9437 PCT~S95/07173 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. Bacte~_al: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKR223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSV~3, 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 a~u~riate vectors are P~K232-8 and PCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PRI 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 a~ riate 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 cont~in~n~ the above-described constructs. The host cell can be a higher 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 a~ a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEA~-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 ~nn~r to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of WOs6~s437 PCT~S95/07173 the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in ~mm~lian 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 deri~ed from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory ~n~ , Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA PncoAing the polypeptides of the present invention by higher eukaryotes is increased by inserting an Pnh~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 including the SV40 Pnh~ncer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early pl~..,o~er enhancer, the polyoma enhAncer on the late side of the replication origin, and adenovirus Pnh~ncers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance yene 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 o~,~o~ers can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ~-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. ~ptionally, the heterologous sequence W096~9437 PCT~S95/07173 can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
Useful expression ~ectors 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 replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, S~ nella ty~h~ rium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC
37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and G~M1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone"
sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by 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 purification.
W O 96/39437 PCT/U~7S~7173 Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
Various m~mm~l ian cell culture systems can also be employed to express recombinant protein. Examples of --mm~lian 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 ~3HK cell lines. ~ lian expression vectors will comprise an origin of replication, a suitable promoter and Pnh~ncer, and also any necessary ribosome hi n~i ng site~, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5~ fl~nking nontran~cribed seguences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The G-protein chemokine receptor polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation PYch~nge 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 protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification ~teps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinan~ techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and m~m~lian cells in w096~9437 pcT~s9s/o7l73 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 for discovery of treatments and diagnostics to human disease.
The G-protein chemokine receptors of the present invent.ion may be employed in a process for screening for compounds which activate (agonists) or inhibit activation (antagonists) of the receptor polypeptide of the present invention .
In general, such screening procedures involve providing o~liate cells which express the receptor polypeptide of the present invention on the surface thereof. Such cells include cells from mammals, yeast, drosophila or E. Coli. In particular, a polynucleotide Pnco~ing the receptor of the present invention is employed to transfect cells to thereby express the G-protein chemokine receptor. The expressed receptor is then contacted with a test compound to observe hin~ing, stimulation or inhibition of a functional response.
One such screening procedure involves the use of melAnophore~ which are transfected to express the G-protein chemokine receptor of the present invention. Such a screening.technique is described in PCT WO 92/01810 published ~ebruary 6, 1992.
Thus, for example, such assay may be employed for screening for a compound which inhihits activation of the receptor polypeptide of the present invention by contacting the melAnsrhore cells which encode the receptor with both the receptor ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor.
W096~9437 PCT~S95/07173 The screen may be employed for 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 techniques include the use of cells which express the G-protein ch~mokine receptor (for example, transfected 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 19~9). For example, c~--~ounds may be contacted with a cell which expresses the receptor polypeptide of the present invention and a second messenger response, e.g.
signal transduction or pH changes, may be measured to determine whether the potential ~..~ound activates or inhibits the receptor.
Another such screening technique involves intro~-lctng, RNA encoding the G-protein chemokine receptor into xenopus oocytes to transiently express the receptor. The receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for co~pounds which are thought to i nht hi t activation of the receptor.
Another screening technique involves expressing the G-protein chemokine receptor in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embryonic kidney cells, etc. The screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.
Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention antagonists by determining inhibition hinrltng of labeled ligand to cells which have the receptor on the O 96~9437 PCT/U',~0~173 surface thereof. Such a method involves transfecting a eukaryotic cell with DNA encoding the G-protein chPmokine receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. Tf the compound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inh; h; ted.
An antibody may antagonize a G-protein ch~m~kine receptor of the present invention, or in some cases an oligopeptide, which bind to the G-protein chemokine receptor but does not elicit a second messenger response such that the activity of the G-protein ~hP~okine receptors is prevented.
Antibodies include anti-idiotypic antibodies which recognize unique determinants generally associated with the antigen-hin~ing gite of an antibody. Potential antagonist compounds also include proteins which are closely related to the ligand of the G-protein chPmokine receptors, i.e. a fragment of the ligand, which have lost biological function and when binding to the G-protein chemokine receptor elicit no response.
An antisense construct prepared through the use of antisense technology, may be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on hin~ing of a polynucleotide to DNA or RNA. For example, the 5~ coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisen~e RNA oligonucleotide of from about 10 to 40 base pairs in length. 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);
W096~9437 PCT~S95/07173 and Dervan et al., Science, 251: 1360 (1991)), tnereby preventing transcription and the production of G-protein chemokine receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vi~o and blocks translation of mRN~
molecules into G-protein coupled receptor (antisense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Bxpression, 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 inhibit production of G-protein rhPm~kine receptor.
A small molecule which binds to the G-protein chemokine receptor, making it inaccessible to ligands such that normal biological activity is ~Leve~lted, for example small peptides or peptide-like molecules, may also be used to inh; h~ t activation of the receptor polypeptide of the present invention.
A soluble form of the G-protein chemokine receptor, e.g.
a fragment of the receptors, may be used to inh;hit activation of the receptor by h~ nrling to the ligand to a polypeptide of the present invention and preventing the ligand from interacting with membrane bound G-protein chemokine receptors.
The compounds which bind to and activate the G-protein rh~m~kine receptors of the present invention may be employed to stimul~te haematopoiesis, wound healing, coagulation, angiogenesis, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-immNne diseases, parasitic infections, psoriasis, and to stimulate growth factor activity.
The compounds which bind to and inhibit the G-protein chemokine receptors of the present invention may be employed to treat allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, hist~ine and IgE-mediated allergic reactions, prostaglandin-independent fever, bone w096~9437 PCT~S95/07173 marrow failure, silicosis, sarcoidosis, rheumatoid arth_itis, shock and hyper-eosinophilic syndrome.
The compounds may be employed in co~hin~tion with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of 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 formulation should suit the mode of j ni stration-The invention also provides a pharmaceutical pack or kitcomprising one or more cont~iners filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such csnt~iner~s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the compounds of the present invention may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositions may be ~*mj ni stered in a co.~v~ient ~-nner such as by the topical, inL a~ous, intraperiton~al, intramuscular, subcutaneous, intranasal or intradermal (applicable?) routes. The pharmaceutical compositions are ~ministered in an amount which is effective for treating and/or prophylaxis of the specific indication.
In general, the pharmaceutical compositions will be ~i ni stered in an amount of at least about lO ~g/kg body weight and in most cases they will be ~ini stered in an amount not in excess of about 8 mg/~g body weight per day.
In most cases, the dosage is from about lO ~g/kg to about l mg/kg body weight daily, taking into account the routes of stration, symptoms, etc. (CONFIR~ nOS~S) The G-protein rh~mokine receptor polypeptides and antagonists or agonists which are polypeptides, may also be W096~9437 PCT~Sss/07173 employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy.~
Thus, for example, cells from a patient may be engineered with a polynucleotide ~DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle cont~inin~ RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle cont~inin~ RNA
PnC~Aing the polypeptide of the present invention may be inistered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings 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 u~ed to engineer cells in vivo after combination with a suitable delivery vehicle.
RetrQviruses from which the retroviral plasmid vectors hereinabove 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 Virus, avian leukosis virus, gibbon ape leukemia virus, human im~ moAPficiency virus, adenoviru~, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
wos6~9437 PcT~ss5/07173 The vector includes one Qr more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ~-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 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 sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promotcrs 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; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoter~; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpe~ Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove 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 of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H2, ~CRE, ~CRIP, GP+E-~6, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Thera~Y, Vol. 1, pgs. S-14 (1990), which is incorporated herein by reference W096~9437 PCT~S95/07173 in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP04 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a lipo~ome, or coupled to a lipid, and then ~ministered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in ~ivo. The transduced eukaryotic cells will express the nucleic acid sequence(s~ encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothPliAl cells, and bronch;Al epithelial cells.
The present invention also provide~ a method for determining whether a ligand not known to be cArAhle of hi nrling to a G-protein chem~kine receptor can bind to such receptor which comprises contacting a mammalian cell which expre~se~ a G-protein chemokine receptor with the ligand under conditions permitting hin~li ng of ligands to the G-protein chemokine receptor, detecting the presence of a ligand which binds to the receptor and thereby determining whether the ligand binds to the G-protein chemokine receptor.
The systems herPin~hove described for determining agonist~
and/or antagonists may also be employed for determining ligands which bind to the receptor.
This invention also provides a method of detecting expression of a G-protein che~okine receptor polypeptide of the present invention on the surface of a cell by detecting the presence of mRNA coding for the receptor which comprises obtAining total mRNA from the cell and contacting the mRNA so w096~9437 PCT~S95/07173 obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 10 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA
hybridized to the probe, and thereby detecting the expression of the receptor by the cell.
The present invention also provides a method for identifying receptors related to the receptor polypeptides of the present invention. These related receptors may be identified by homology to a G-protien che~okine receptor polypeptide of the present invention, by low stringency cross hybridization, or by identifying receptors that interact with related natural or synthetic ligands and or elicit similar behaviors after genetic or pharmacological blockade of the chemokine receptor polypeptides of the present invention.
~ ragments of the genes may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the genes of the present invention, or which have similar biological activity. Probes of this type are at least 20 bases, preferably at least 30 ba~es and most preferably at least 50 bases or more. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that cont~ i n the complete gene of the present invention including regulatory and promoter regions, exons and introns.
An example of a screen of this type cumprises 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~ of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The present invention also contemplates the use of the genes of the present invention as a diagnostic, for example, W O 96~9437 some diseases result from inherited defective genes. These genes can be detected by comparing the sequences of the defective gene with that of a normal one. Subsequently, one can verify that a "mutant" gene is associated with abnormal receptor activity. In addition, one can insert mutant receptor genes into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, expression on MacConkey plates, complementation experiments, in a receptor deficient strain of HEK293 cells) as yet another means to verify or identify mutations. Once "mutant" genes have been identified, one can then screen population for carriers of the "mutant" receptor gene.
Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be ob~ine~ from a patient's cells, including but not limited to such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR
~Saiki, et al., Nature, 324:163-166 1986) prior to analysis.
RNA or cDNA may al~o be used for the same purpose. As an example, PCR primers complimentary to the nucleic acid of the instant invention can be used to identify and analyze mutations in the gene of the present invention. 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 radio labeled RNA of the invention or alternatively, radio labeled antisense DNA sequences of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Such a diagnostic would be particularly useful for prenatal or even neonatal testing.
Sequence differences between the reference gene and "mutants~ may be revealed by the direct DNA sequencing W O 96~9437 PCT~S95/07173 method. In addition, cloned DNA se~ments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequence primer is used with double stranded PCR
product or a single stranded template molecule generated by a modified PCR. The sequence detenmination is performed by conventional procedures with radio labeled nucleotide or b an automatic se~1encing procedure with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection of alterations in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Sequences changes at specific 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 of, or are characterized by changes in gene expression which can be detected by changes in the mRNA. Alternatively, the genes of the present invention can be used as a reference to identify individuals expressing a decrease of functions associated with receptors of this type.
The present invention also relates to a diagnostic assay for detecting altered levels of soluble forms of the G-proein chemokine receptor polypeptides of the present invention in various tissues. Assays used to detect levels of the soluble receptor polypeptides in a sample derived from a host are well known to those of skill in the art and include radioi~nmo~says, competitive-hi n~i ng assays, Western blot analysis and preferably as ELISA assay.
An ELISA assay initially comprises preparing an antibody specific to antigens of the G-protein chemokine receptor polypeptides, 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 W096~9437 PCT~S95/07173 example a horseradish peroxidase enzyme. A sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any G-protein ch~mokine receptor proteins attached to the polystyrene dish. All unbound monoclonal antibody-is washed ou' with bu~fer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the Le~o~Ler antibody to any monoclonal antibody bound to G-protein chemokine 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 measu~...e-~t of the amount of G-protein chemokine receptor proteins present in a given volume of patient sample when compared against a st~n~rd curve.
The sequences of 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 chromosome marking reagents based on actual sequence 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, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 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 DN~, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell W096~9437 PCT~S95/07173 hybrids containing individual human chromosomes. Only those hybrids cont~ining 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 ~Anner. 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
clone to a metAphA~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 50 or 60 bases.
~or a review of this technique, see Verma et al., Human rhromosomes: a ~mlAl of Basic Techniques, Pe~yd~ Press, New York (1988).
Once a seguence 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. McXusick, M~n~Plidn 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 unaffected 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 to be the causative agent of the disease.
wo 96/39437 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 cenes. (This assumes 1 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 immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chimeric, single chain, and hl-m-nized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by ~inisterinc the polypeptides to an Ani~-l, preferably a nonhuman. The antibody so obt~ine~l will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies hi n~li 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 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 human B-cell hybridoma technique (Rozbor et al., 1983, Immunology Today 4:72), and the ~BV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
W096~9437 PCT~S95/07173 Techniques described fo- the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immllnogenic polypeptide products of this invention. Also, trans-enic mice may be used to express hl~m~nized antibodies tO iT"mlmogenic 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.
In order to facilitate underst~n~ing of the following examples certain frequently occur.ing methods and/or tenms will be described.
"Plasmids~l are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion~ of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically l ~g of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 ~l of buffer solution. For the ~urpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~g of DNA are digested with 20 to 2sO units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the w096~9437 PCT~Sgs/07173 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 electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D.
et al ., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming 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 buffers and conditions with lO units to T4 DN~ liga~e ( n ligase") per 0.5 ~g of approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology,.52:4S6-457 ~1973).
ExamDle 1 Bacterial ExPression and Purification of HDGNRlO
The DN~ sequence encoding for HDGNRlO, ATCC # _ is initially amplified using PCR oligonucleotide primers correspon~ing to the 5~ and sequences of the procesged HDOENR10 protein (minus the signal peptide sequence) and the vector sequences 3' to the HDGNRlO gene. Additional nucleotides corresponding to HDGNRlO were added to the 5' and 3' sequences respectively. The 5l oligonucleotide primer has PCT~S95/07173 the sequence 5' CGGAATTCCTCCATGGATT~TCAA~l~l~A 3' contains an BcoRI restriction enzyme site followed by 18 nucleotides of HDGNR10 coding sequence starting from the presumed terminal amino acid of the processed protein codon. The 3' sequence 5' CGGAAGCTTCGTCACAAGCCCACAGATAT 3' contains complementary sequences to c HindIII site and is followed by 18 nucleotides of HDGNR10 coding sequence. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chat~worth, CA, 91311). pQE-9 encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome bin~ing site (~3S), a 6-His tag and restriction enzyme sites. pQE-9 was then digested with EcoRI and HindIII. The amplified sequences were ligated into pQE-9 and were inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture was then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sa,.~Look, J. et al., Molecular Cloning: A Laboratory ~nt~
Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan').
Transformants are identified by their ability to grow on LB
plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired constructs were grown Gvernight (0/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 of 1:100 to 1:250. The cells were grown to an optical density 600 (O.D.~) of between 0.4 and 0.6. IPTG
(nI~o~lo~yl-B-D-thiogalacto pyranoside") was then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the ?/O leading tO increased gene expression. Cells 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. After clarification, solubilized HDGNR10 was purified from this solution by chromatography on a Nickel-Chelate - column under conditions that allow for tight binding by proteins cont~ining the 6-His tag. Hochuli, E. et al., J.
Chromatography 411:177-184 (1984). HDGNR10 was eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.
~xample 2 ExDression of Recombinant HDGNR10 in COS cells The expression of plasmid, HDGNR10 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a S~40 intron and polyadenylation site. A DN~
fragment encoding the entire HDGNR10 precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore, the recom~inant 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 reCom~nAnt protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
- The DNA sequence encoding fo- HDGNR10, ATCC # _ , was constructed by PCR using two primers: the S' primer 5~ GTCC
.
W096~9437 PCT~S95/07173 AAGCTTGCCACCATG&ATTATCAAGTGTCA 3' and contains a HindIII site followed by 18 nucleotides of HDGNR10 coding sequence starting from the initiation codon; the 3' seguence 5' CTAGCTCGAGTCAAGCGTA~~ A~ ATG~GTAGCACAAGCCCACAGATATTTC
It is well established that many medically significant biological processes are mediated by proteins participating in signaL transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 (1991)). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG
proteins. Some examples of these ?roteins include the GPC
receptors, such as tho~e for adrenergic agents and ~o~ine (Kobilka, B.K., et al., PN~S, 84:46-50 (1987); Robilka, 9.K., et al., Science 238:650-656 (1987); 9unzow, J.R., et al., Nature, 336:783-787 (1g88)), G-proteins themselve~, ef~ector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, ar.d actuator proteins, e.g., protein W096~9437 PCT~S95/07173 kinase A and protein kinase C ~Simon, M.I., et al., Science, 252:802-8 (l99l)).
For example, in one form of signal transduction, the effect of hormone hln~;ng iS activation of an enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP, and GTP also influences hormone hin~l;ng. A G-protein connects the hormone receptors to adenylate cyclase. G-protein was shown to ~Ych~nge GTP for bound GDP when activated by hon~one receptors. The GTP-carrying form then binds to an activated adenylate cyclase. Hydrolysis of GTP
to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
The ...~,~Lane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane a-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
G-protein coupled receptors have been characterized as including these seven conserved hydroph~bic stretches of about 20 -to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein family of coupled receptors includes doramine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, hist~mine, thrombin, kinin, follicle stimulating honmone, opsins, endothelial differentiation gene-l receptor and rhodop~ins, odorant, cytomegalovirus receptors, etc.
Wos6/3s437 PCT~S95/07173 G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion ch~nnels and transporters (see, Johnson et al., Endoc., Rev., 10:317-331 (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 G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host.
Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are 8-10 kd in size. In general, chemokines ~Yh;h; t 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine~
residues that form two disulfide bondg. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta.
In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the "C-X-C"
subfamily. In the beta subfamily, the two cysteines are in an adjacent po~ition and are, therefore, referred to as the "C-C~ subfamily. Thus far, at least nine different members of this family have been identified in humans.
The intercrine cytokines P}-h;h; t a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophil8, T lymphocytes, ha~oph;ls and fibrobla~ts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. TheQe activities include stimulation of histamlne release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cells, enhanced binding of complement proteins, induced expression W O 96~9437 PCT/U~5/'~7173 of granulocyte adhesion molecules and complement recéptors, and respiratory bur6t. In addition to their involvement in inflammation, certain chemokines 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-8 ~IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cell~.
In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and di6eage condition6, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.
In accordance with one a6pect of the present invention, there are provided novel mature receptor polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The receptor polypeptides of the present invention are of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules PncoAing the receptor polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA a6 well a6 antisense- analog6 thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with a further aspect of the present invention, there are provided processes for producing such receptor polypeptides by recombinant technique6 compri6ing culturing recombinant prokaryotic and/or eukaryotic ho6t cells, cont~ining nucleic acid sequences encoAing the receptor polypeptides of the present invention, under conditions promoting expre6sion of said polypeptides and subsequent recovery of said polypeptide6.
w096~9437 PCT/U535l~7173 In accordance with yet a further aspect of the present invention, there are provided antibcdies against such receptor polypeptides.
In accordance with another aspect of the present invention there are provided methods of screening for compounds which bind to and activate or inhibit activation of - the receptor polypeptides of the present invention.
In accordance with still another embodiment of the present invention there are provided processes of ~ministering compounds to a host which bind to and activate the receptor polypeptide of the present invention which are useful in stimulating haematopoiesis, wound healing, coagulation, angiogenesi~, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-immune diseases, parasitic infections, psoriasis, and to stimulate growth factor activity.
In accordance with another aspect of the present invention there is provided a method of administering the receptor polypeptides of the present invention via gene therapy to treat conditions related to undeLex~ession of the polypeptides or underexpression of a ligand for the receptor polypeptide.
In accordance with still another embodiment of the present invention there are provided processes of ~mi ni stering co~ro~nA~ to a host which bind to and inhibit activation of the receptor polypeptides of the present invention which are useful in the prevention and/or treatment of allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and Ig8-mediated allergic reactions, prostagl ~nAi n - independent fever, bone marrow failure, silicosis, sarcoidosis, rheumatoid arthritis, shock and hyper-eosin~rhilic syndrome.
In accordance with yet another aspect of,the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically W096~9437 PCT~S95/07173 structurallv related to the G protein-coupled receptor family. It cont~inc an open re~ing frame ~nco~in~ a protein of 352 amino acid residues. The protein exhibits the highest degree of homology to a human MCP-l receptor with 70.1 %
identity and 82.9 % similarity over a 347 amino acid stretch.
The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coAin~ strand or non-co~ing (anti-sense) strand. The coAing sequence which ~n~oAes the mature polypeptide may be identical to the ro~i ng sequence shown in Figure l (SBQ ID
NO:l) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the re~ln~ncy or degeneracy of the genetic code, encodes the-same mature polypeptide as the DNA of Figure l (SBQ ID NO~
or the deposited cDNA.
The polynucleotide which PncoAP~ for the mature polypeptide of Figure l or for the mature polypeptide PncoAeA
by the deposited cDNA may include: only the coAing sequence for the mature polypeptide; the coAing sequence for the mature polypeptide and additional coding sequence such as a transmembrane (TM) or intra-cellular domain; the co~ing sequence for the mature polypeptide (and optionally additional coAin~ sequence) and non-coAing sequence, such as introns or non-coAing sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
Thus, the term "polynucleotide ~ncoAi ng a polypeptide n encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the hereinabove described polynucleotides which ~ncoAe for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure l or the W096~9437 PCT~S95/07173 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, 37:76, (1984)).
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the co~in~ region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA
library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least ~0 bases and may contain, for example, 50 or more bases. The probe may also be used to identify a cDN~
clone corresron~ing to a full length transcript and a genomic, clone or clones that contain 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 c~mplementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The - present invention further relates to polynucleotides which hybridize to the hereinAhove-described seqll~nc~s if there is at least 70%, preferably at least 90%, and more preferably at least 95~ identity between the se~l~nce~. The present invention particularly relates to polynucleotides which hybridize under stringent condition~ to the her~in~hove-described polynucleotides. As herein used, the term n~tringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides _g_ W096~9437 PCT/U~S/~7173 polypeptide Pnro~e~ by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturalIy occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides PnroA-ng the same mature polypeptide as shown in Figure 1 ~SEQ ID N0:2) or the same mature polypeptide Pnro~e~ by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SBQ ID
N0:2) or the polypeptide PnCo~d by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As herPtnAhove indicated, the polynucleotide may have a roAi n~ gequence which is a naturally occurring allelic-variant of the co~i ng sequence shown in ~igure 1 (S~Q ID~
N0:1) or of the coding sequence of the deposited clone. 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 of the encoded polypeptide.
The polynucleotides may also PnroAe for a soluble form of the G-protein chemokine receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved from the TM and intracellular domain of the full-length polypeptide of the present invention.
The polynucleotides of the present invention may also have the coding seguence fused in frame to a marker sequence which allows for purification of the polypeptide 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 polypeptide 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~m,m~l ian -a-w096~9437 PCT/~S35~173 The present invention further relates to a G-protein chemokine receptor polypeptide which has the deduced amino acid sequence of Figure 1 (S~Q ID N0:2) or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
The tenms "fragment, n nderivativell and "analog" when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which either retains sub~tantially the same biological function or activity as such polypeptide, i.e. functions as a G-protein rhemokine receptor, or retains the ability to bind the ligand or the receptor even though the polypeptide does not function as a G-protein chemokine receptor, for example, a ~oluble form of the receptor. An analog includes a ~lv~Lotein which can be activated by cleavage of the ~v~otein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a ~ynthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polypeptide of Figure 1 (SBQ ID N0:2) or that encoAe~ by the deposited cDNA may be ~i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one 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 half-life of 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 membrane bound, yet still binds ligands to the membrane bound receptor. Such fragments, derivatives W096/39437 PCT~S95/07173 which hybridize to the herein~hove described polynucléotides in a preferred embodiment PncoAe polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO:1) or the deposited cDNA(s).
Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as herPin~hove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SBQ ID NO:1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR
primer.
Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which PnCOAP~ the polypeptide of SBQ ID NO:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides PncoA~PA by such polynucleotides.
The deposit(s~ referred to herein will be ma;nt~tned under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. 112.
The sequence of the polynucleotides cont~tnp~ in the deposited materials, as well as the amino acid sequence 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 required to make, or sell the deposited materials, and no such license is hereby granted.
W096/39437 PCT/U~9SI~7173 hybridize to the polynucleotide sequences of the present invention.
In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and for detecting an altered level of the soluble form of the receptor polypeptides.
In accordance with yet a further aspect of the present invention, there are provided processes for utilizing such receptor polypeptides, or polynucleotides encoding 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.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure l shows the cDNA sequence and the corresponding deduced amino acid sequence of the G-protein coupled receptor of the present invention. The st~n~rd one-letter abbreviation for amino acids is used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).
Figure 2 illustrates an amino acid alignment of the G-protein rh~mokine receptor of the present invention and the human MCP-l receptor.
In accor~ance with an a~pect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure l (SBQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. on June l, l995.
The polynucleotide of this invention was discovered in a cDNA library derived from human monocytes. It is W096~9437 PCT~Sss/07173 and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to hu~oye~leity~
The polypeptides of the present invention include the polypeptide of SBQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70%
similarity (preferably a 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably a 90% s~ rity (more preferably a 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably a 95% s~ rity (still more preferably a 90% identity) to the polypeptide of SEQ ID NO:2 and to portions of such polypeptide with such portion of the polypeptide generally cont~intng at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "Si~il~rity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The term "gene n means the segment of DNA involved in pro~llcing a polypeptide chain; it includes regions preceding and following the ro~ing region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons).
The term n isolated" means that the material is removed from its original environment (e.g., the natural envi.ol~-,ellt W096~9437 PCT/U~S~7173 if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a li~ing ~n~-l iS not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
The polypeptides of the present invention include the polypeptide of SBQ ID NO:2 (in particular the mature polypeptide) a~ well as polypeptides which have at lea~t 70%
similarity (preferably at least 70~ identity) to the polypeptide of S8Q ID NO:2 and more preferably at least 90%
similarity (more preferably at least 90% identity) to the polypeptide of SBQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include portion~
of such polypeptides with such portion of the polypeptide generally ~ont~in~ng at least 30 amino acids and more preferably at least 50 amino acids.
AS known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. ~ragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells W096~9437 PCT~S95/07173 which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant 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 fonm of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by reComh;n~nt techniques. 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, nQnchromosomal and synthetic DNA seguences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be uQed as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA
~equence is inserted into an appropriate restriction Pn~onnrlease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) W096/39437 PCT~Sss/07173 (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage 1~mhAA PL
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also contains a ribosome h; n~i ng site for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably cont~in-one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such a~ tetracycline or ampicillin resistance in B. coli.
The vector cont~i ni ng the a~ o~riate DNA sequence as herPin~hove described, as well as an a~ro~riate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
As representative examples of a~lu~riate hosts, there may be mentioned: bacterial cells, such as E. coli, StreptomYces, S~l~onella t~phimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9;
~nir-l cells such as CH0, COS or ~owes mel~n~~-; adenovirus;
plant cells, 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 rec~in~nt 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 w096~9437 PCT~S95/07173 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. Bacte~_al: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKR223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSV~3, 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 a~u~riate vectors are P~K232-8 and PCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PRI 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 a~ riate 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 cont~in~n~ the above-described constructs. The host cell can be a higher 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 a~ a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEA~-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 ~nn~r to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of WOs6~s437 PCT~S95/07173 the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in ~mm~lian 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 deri~ed from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory ~n~ , Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA PncoAing the polypeptides of the present invention by higher eukaryotes is increased by inserting an Pnh~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 including the SV40 Pnh~ncer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early pl~..,o~er enhancer, the polyoma enhAncer on the late side of the replication origin, and adenovirus Pnh~ncers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance yene 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 o~,~o~ers can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ~-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. ~ptionally, the heterologous sequence W096~9437 PCT~S95/07173 can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
Useful expression ~ectors 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 replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, S~ nella ty~h~ rium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC
37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and G~M1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone"
sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by 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 purification.
W O 96/39437 PCT/U~7S~7173 Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
Various m~mm~l ian cell culture systems can also be employed to express recombinant protein. Examples of --mm~lian 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 ~3HK cell lines. ~ lian expression vectors will comprise an origin of replication, a suitable promoter and Pnh~ncer, and also any necessary ribosome hi n~i ng site~, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5~ fl~nking nontran~cribed seguences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The G-protein chemokine receptor polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation PYch~nge 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 protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification ~teps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinan~ techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and m~m~lian cells in w096~9437 pcT~s9s/o7l73 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 for discovery of treatments and diagnostics to human disease.
The G-protein chemokine receptors of the present invent.ion may be employed in a process for screening for compounds which activate (agonists) or inhibit activation (antagonists) of the receptor polypeptide of the present invention .
In general, such screening procedures involve providing o~liate cells which express the receptor polypeptide of the present invention on the surface thereof. Such cells include cells from mammals, yeast, drosophila or E. Coli. In particular, a polynucleotide Pnco~ing the receptor of the present invention is employed to transfect cells to thereby express the G-protein chemokine receptor. The expressed receptor is then contacted with a test compound to observe hin~ing, stimulation or inhibition of a functional response.
One such screening procedure involves the use of melAnophore~ which are transfected to express the G-protein chemokine receptor of the present invention. Such a screening.technique is described in PCT WO 92/01810 published ~ebruary 6, 1992.
Thus, for example, such assay may be employed for screening for a compound which inhihits activation of the receptor polypeptide of the present invention by contacting the melAnsrhore cells which encode the receptor with both the receptor ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor.
W096~9437 PCT~S95/07173 The screen may be employed for 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 techniques include the use of cells which express the G-protein ch~mokine receptor (for example, transfected 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 19~9). For example, c~--~ounds may be contacted with a cell which expresses the receptor polypeptide of the present invention and a second messenger response, e.g.
signal transduction or pH changes, may be measured to determine whether the potential ~..~ound activates or inhibits the receptor.
Another such screening technique involves intro~-lctng, RNA encoding the G-protein chemokine receptor into xenopus oocytes to transiently express the receptor. The receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for co~pounds which are thought to i nht hi t activation of the receptor.
Another screening technique involves expressing the G-protein chemokine receptor in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embryonic kidney cells, etc. The screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.
Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention antagonists by determining inhibition hinrltng of labeled ligand to cells which have the receptor on the O 96~9437 PCT/U',~0~173 surface thereof. Such a method involves transfecting a eukaryotic cell with DNA encoding the G-protein chPmokine receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. Tf the compound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inh; h; ted.
An antibody may antagonize a G-protein ch~m~kine receptor of the present invention, or in some cases an oligopeptide, which bind to the G-protein chemokine receptor but does not elicit a second messenger response such that the activity of the G-protein ~hP~okine receptors is prevented.
Antibodies include anti-idiotypic antibodies which recognize unique determinants generally associated with the antigen-hin~ing gite of an antibody. Potential antagonist compounds also include proteins which are closely related to the ligand of the G-protein chPmokine receptors, i.e. a fragment of the ligand, which have lost biological function and when binding to the G-protein chemokine receptor elicit no response.
An antisense construct prepared through the use of antisense technology, may be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on hin~ing of a polynucleotide to DNA or RNA. For example, the 5~ coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisen~e RNA oligonucleotide of from about 10 to 40 base pairs in length. 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);
W096~9437 PCT~S95/07173 and Dervan et al., Science, 251: 1360 (1991)), tnereby preventing transcription and the production of G-protein chemokine receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vi~o and blocks translation of mRN~
molecules into G-protein coupled receptor (antisense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Bxpression, 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 inhibit production of G-protein rhPm~kine receptor.
A small molecule which binds to the G-protein chemokine receptor, making it inaccessible to ligands such that normal biological activity is ~Leve~lted, for example small peptides or peptide-like molecules, may also be used to inh; h~ t activation of the receptor polypeptide of the present invention.
A soluble form of the G-protein chemokine receptor, e.g.
a fragment of the receptors, may be used to inh;hit activation of the receptor by h~ nrling to the ligand to a polypeptide of the present invention and preventing the ligand from interacting with membrane bound G-protein chemokine receptors.
The compounds which bind to and activate the G-protein rh~m~kine receptors of the present invention may be employed to stimul~te haematopoiesis, wound healing, coagulation, angiogenesis, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-immNne diseases, parasitic infections, psoriasis, and to stimulate growth factor activity.
The compounds which bind to and inhibit the G-protein chemokine receptors of the present invention may be employed to treat allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, hist~ine and IgE-mediated allergic reactions, prostaglandin-independent fever, bone w096~9437 PCT~S95/07173 marrow failure, silicosis, sarcoidosis, rheumatoid arth_itis, shock and hyper-eosinophilic syndrome.
The compounds may be employed in co~hin~tion with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of 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 formulation should suit the mode of j ni stration-The invention also provides a pharmaceutical pack or kitcomprising one or more cont~iners filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such csnt~iner~s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the compounds of the present invention may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositions may be ~*mj ni stered in a co.~v~ient ~-nner such as by the topical, inL a~ous, intraperiton~al, intramuscular, subcutaneous, intranasal or intradermal (applicable?) routes. The pharmaceutical compositions are ~ministered in an amount which is effective for treating and/or prophylaxis of the specific indication.
In general, the pharmaceutical compositions will be ~i ni stered in an amount of at least about lO ~g/kg body weight and in most cases they will be ~ini stered in an amount not in excess of about 8 mg/~g body weight per day.
In most cases, the dosage is from about lO ~g/kg to about l mg/kg body weight daily, taking into account the routes of stration, symptoms, etc. (CONFIR~ nOS~S) The G-protein rh~mokine receptor polypeptides and antagonists or agonists which are polypeptides, may also be W096~9437 PCT~Sss/07173 employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy.~
Thus, for example, cells from a patient may be engineered with a polynucleotide ~DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle cont~inin~ RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle cont~inin~ RNA
PnC~Aing the polypeptide of the present invention may be inistered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings 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 u~ed to engineer cells in vivo after combination with a suitable delivery vehicle.
RetrQviruses from which the retroviral plasmid vectors hereinabove 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 Virus, avian leukosis virus, gibbon ape leukemia virus, human im~ moAPficiency virus, adenoviru~, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
wos6~9437 PcT~ss5/07173 The vector includes one Qr more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ~-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 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 sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promotcrs 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; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoter~; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpe~ Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove 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 of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H2, ~CRE, ~CRIP, GP+E-~6, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Thera~Y, Vol. 1, pgs. S-14 (1990), which is incorporated herein by reference W096~9437 PCT~S95/07173 in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP04 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a lipo~ome, or coupled to a lipid, and then ~ministered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in ~ivo. The transduced eukaryotic cells will express the nucleic acid sequence(s~ encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothPliAl cells, and bronch;Al epithelial cells.
The present invention also provide~ a method for determining whether a ligand not known to be cArAhle of hi nrling to a G-protein chem~kine receptor can bind to such receptor which comprises contacting a mammalian cell which expre~se~ a G-protein chemokine receptor with the ligand under conditions permitting hin~li ng of ligands to the G-protein chemokine receptor, detecting the presence of a ligand which binds to the receptor and thereby determining whether the ligand binds to the G-protein chemokine receptor.
The systems herPin~hove described for determining agonist~
and/or antagonists may also be employed for determining ligands which bind to the receptor.
This invention also provides a method of detecting expression of a G-protein che~okine receptor polypeptide of the present invention on the surface of a cell by detecting the presence of mRNA coding for the receptor which comprises obtAining total mRNA from the cell and contacting the mRNA so w096~9437 PCT~S95/07173 obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 10 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA
hybridized to the probe, and thereby detecting the expression of the receptor by the cell.
The present invention also provides a method for identifying receptors related to the receptor polypeptides of the present invention. These related receptors may be identified by homology to a G-protien che~okine receptor polypeptide of the present invention, by low stringency cross hybridization, or by identifying receptors that interact with related natural or synthetic ligands and or elicit similar behaviors after genetic or pharmacological blockade of the chemokine receptor polypeptides of the present invention.
~ ragments of the genes may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the genes of the present invention, or which have similar biological activity. Probes of this type are at least 20 bases, preferably at least 30 ba~es and most preferably at least 50 bases or more. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that cont~ i n the complete gene of the present invention including regulatory and promoter regions, exons and introns.
An example of a screen of this type cumprises 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~ of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The present invention also contemplates the use of the genes of the present invention as a diagnostic, for example, W O 96~9437 some diseases result from inherited defective genes. These genes can be detected by comparing the sequences of the defective gene with that of a normal one. Subsequently, one can verify that a "mutant" gene is associated with abnormal receptor activity. In addition, one can insert mutant receptor genes into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, expression on MacConkey plates, complementation experiments, in a receptor deficient strain of HEK293 cells) as yet another means to verify or identify mutations. Once "mutant" genes have been identified, one can then screen population for carriers of the "mutant" receptor gene.
Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be ob~ine~ from a patient's cells, including but not limited to such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR
~Saiki, et al., Nature, 324:163-166 1986) prior to analysis.
RNA or cDNA may al~o be used for the same purpose. As an example, PCR primers complimentary to the nucleic acid of the instant invention can be used to identify and analyze mutations in the gene of the present invention. 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 radio labeled RNA of the invention or alternatively, radio labeled antisense DNA sequences of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Such a diagnostic would be particularly useful for prenatal or even neonatal testing.
Sequence differences between the reference gene and "mutants~ may be revealed by the direct DNA sequencing W O 96~9437 PCT~S95/07173 method. In addition, cloned DNA se~ments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequence primer is used with double stranded PCR
product or a single stranded template molecule generated by a modified PCR. The sequence detenmination is performed by conventional procedures with radio labeled nucleotide or b an automatic se~1encing procedure with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection of alterations in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Sequences changes at specific 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 of, or are characterized by changes in gene expression which can be detected by changes in the mRNA. Alternatively, the genes of the present invention can be used as a reference to identify individuals expressing a decrease of functions associated with receptors of this type.
The present invention also relates to a diagnostic assay for detecting altered levels of soluble forms of the G-proein chemokine receptor polypeptides of the present invention in various tissues. Assays used to detect levels of the soluble receptor polypeptides in a sample derived from a host are well known to those of skill in the art and include radioi~nmo~says, competitive-hi n~i ng assays, Western blot analysis and preferably as ELISA assay.
An ELISA assay initially comprises preparing an antibody specific to antigens of the G-protein chemokine receptor polypeptides, 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 W096~9437 PCT~S95/07173 example a horseradish peroxidase enzyme. A sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any G-protein ch~mokine receptor proteins attached to the polystyrene dish. All unbound monoclonal antibody-is washed ou' with bu~fer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the Le~o~Ler antibody to any monoclonal antibody bound to G-protein chemokine 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 measu~...e-~t of the amount of G-protein chemokine receptor proteins present in a given volume of patient sample when compared against a st~n~rd curve.
The sequences of 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 chromosome marking reagents based on actual sequence 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, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 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 DN~, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell W096~9437 PCT~S95/07173 hybrids containing individual human chromosomes. Only those hybrids cont~ining 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 ~Anner. 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
clone to a metAphA~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 50 or 60 bases.
~or a review of this technique, see Verma et al., Human rhromosomes: a ~mlAl of Basic Techniques, Pe~yd~ Press, New York (1988).
Once a seguence 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. McXusick, M~n~Plidn 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 unaffected 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 to be the causative agent of the disease.
wo 96/39437 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 cenes. (This assumes 1 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 immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chimeric, single chain, and hl-m-nized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by ~inisterinc the polypeptides to an Ani~-l, preferably a nonhuman. The antibody so obt~ine~l will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies hi n~li 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 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 human B-cell hybridoma technique (Rozbor et al., 1983, Immunology Today 4:72), and the ~BV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
W096~9437 PCT~S95/07173 Techniques described fo- the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immllnogenic polypeptide products of this invention. Also, trans-enic mice may be used to express hl~m~nized antibodies tO iT"mlmogenic 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.
In order to facilitate underst~n~ing of the following examples certain frequently occur.ing methods and/or tenms will be described.
"Plasmids~l are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion~ of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically l ~g of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 ~l of buffer solution. For the ~urpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~g of DNA are digested with 20 to 2sO units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the w096~9437 PCT~Sgs/07173 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 electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D.
et al ., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming 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 buffers and conditions with lO units to T4 DN~ liga~e ( n ligase") per 0.5 ~g of approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology,.52:4S6-457 ~1973).
ExamDle 1 Bacterial ExPression and Purification of HDGNRlO
The DN~ sequence encoding for HDGNRlO, ATCC # _ is initially amplified using PCR oligonucleotide primers correspon~ing to the 5~ and sequences of the procesged HDOENR10 protein (minus the signal peptide sequence) and the vector sequences 3' to the HDGNRlO gene. Additional nucleotides corresponding to HDGNRlO were added to the 5' and 3' sequences respectively. The 5l oligonucleotide primer has PCT~S95/07173 the sequence 5' CGGAATTCCTCCATGGATT~TCAA~l~l~A 3' contains an BcoRI restriction enzyme site followed by 18 nucleotides of HDGNR10 coding sequence starting from the presumed terminal amino acid of the processed protein codon. The 3' sequence 5' CGGAAGCTTCGTCACAAGCCCACAGATAT 3' contains complementary sequences to c HindIII site and is followed by 18 nucleotides of HDGNR10 coding sequence. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chat~worth, CA, 91311). pQE-9 encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome bin~ing site (~3S), a 6-His tag and restriction enzyme sites. pQE-9 was then digested with EcoRI and HindIII. The amplified sequences were ligated into pQE-9 and were inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture was then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sa,.~Look, J. et al., Molecular Cloning: A Laboratory ~nt~
Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan').
Transformants are identified by their ability to grow on LB
plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired constructs were grown Gvernight (0/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 of 1:100 to 1:250. The cells were grown to an optical density 600 (O.D.~) of between 0.4 and 0.6. IPTG
(nI~o~lo~yl-B-D-thiogalacto pyranoside") was then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the ?/O leading tO increased gene expression. Cells 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. After clarification, solubilized HDGNR10 was purified from this solution by chromatography on a Nickel-Chelate - column under conditions that allow for tight binding by proteins cont~ining the 6-His tag. Hochuli, E. et al., J.
Chromatography 411:177-184 (1984). HDGNR10 was eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.
~xample 2 ExDression of Recombinant HDGNR10 in COS cells The expression of plasmid, HDGNR10 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a S~40 intron and polyadenylation site. A DN~
fragment encoding the entire HDGNR10 precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore, the recom~inant 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 reCom~nAnt protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
- The DNA sequence encoding fo- HDGNR10, ATCC # _ , was constructed by PCR using two primers: the S' primer 5~ GTCC
.
W096~9437 PCT~S95/07173 AAGCTTGCCACCATG&ATTATCAAGTGTCA 3' and contains a HindIII site followed by 18 nucleotides of HDGNR10 coding sequence starting from the initiation codon; the 3' seguence 5' CTAGCTCGAGTCAAGCGTA~~ A~ ATG~GTAGCACAAGCCCACAGATATTTC
3~ contains complementary sequences to an XhoI site, translation stop codon, HA tag and the last 18 nucleotides of the HDGNR10 coding sequence ~not including the stop codon).
Therefore, the PCR product contains a HindIII site HDGNR10 coding seguence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an XhoI site. The PCR amplified DNA fragment and the vector, pcDNAItAmp, were digested with HindIII and XhoI restriction enzyme and ligated. The ligation mixture was transfonmed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and ~mi ned by restriction analysis for the presence of the correct fragment. ~or expression of the recombinant HDGNR10, COS cells were transfected with the expression vector by DBAE-DEXTRAN
method. (J. Sa,.L~ook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory ~nl~l, Cold Spring Laboratory Pre~s, (1989)). The expression of the HDGNR10 HA protein was detected by radiolabelling and ~ noprecipitation method.
(E. Harlo~, D. Lane, Antibodies: A Laboratory ~nll~l, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with 35S-cysteine two days post transfection.
Culture media were then collected and cells were lysed with deteryent (RIPA buffer (150 ~M 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 specific monoclonal antibody.
Proteins precipitated were analyzed on 15% SDS-PAGE gels.
W096/39437 PCT~S95/07173 Exam~le 3 Cloninq and ex~ression of HDGNR10 usinq the baculovirus ex~ression s~stem The DNA sequence encoding the full length HDGNR10 protein, ATCC # , was amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5' CGGGATCCCTCCATGGATTAT
CAA~l~l~A 3' and contains a BamHI restriction enzyme site followed by 4 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (J. Mol.
Biol. 1987, 196, 947-950, Kozak, M.), and just behind the first 18 nucleotides of the HDGNR10 gene (the initiation codon for translation is "ATG").
The 3' primer has the sequence 5' CGGGATCCCGCT
CACAAGCCCACAGATAT 3' and contains the cleavage site for the restriction endonuclease BamHI and 18 nucleotides complementary to the 3' non-translated sequence of the HDGNR10 gene. The amplified sequences were isolated from a 1% agarose gel using a commercially available kit (nGeneclean," BIO 101 Inc., La Jolla, Ca.). The fragment was then digested with the ~n~Qnt~clease 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 HDGNR10 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimer.~al Station Bulletin No. 1555).
This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction en~m1clease BamHI. The polyadenylation site of - the simian virus (S~)40 is used for efficient polyadenylation. For an easy select~on of recombinant w096~9437 PCT~Sss/07173 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 both sides by viral sequences for the cell-mediated homologous recombination of co-transfected 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 was digested with the restriction enzyme BamHI and then ~erhosphorylated 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 HB101 cel~s were then transfonmed and bacteria identified that contained the plasmid (pBacHDGNR10) with the HDGNR10 gene using the enzyme BamHI. The sequence of the cloned fragment was confirmed by DNA sequencing.
S ~g of the plasmid pBacHDGNR10 were co-transfected with 1.0 ~g of a commercially available linearized baculovirus ("BaculoGold~ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)).
l~g of BaculoGold~ virus DNA and S ~g of the plasmid pBacHDGNR10 were mixed in a sterile well of a microtiter plate contAining 50 ~l of serum free Grace~s medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~l Lipofectin plus 90 ~l Grace's mediu~ were added, mixed and incubated for 15 minutes at room _emperature. 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 W096/39437 PCT~S95/07173 was then incubated for 5 hours at 27~C. After 5 hours the transfection solution was removed from the plate and l ml of Grace's insect medium supplemented with 10% fetal calf serum was added. The plate was put back into an incu~ator and cultivation continued at 27~C for four days.
After four days the supernatant was collected and a plaque assay performed similar as described by Summers and Smith (supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gai'hersburg) was used which allows an easy isolation of blue stained plaques. (A
detailed description of a "plaque assay" can also be found in the user~s guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10) .
Four days after the serial dilution, the viruses were added to the cells, blue stained plaques were picked with the tip of an Eppendorf pipette. The agar cont~ining the recombinant viruses was then resuspended in an Eppendorf tube cont~ining 200 ~l of Grace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculoviruses was used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of the~e culture dishes were harvested and then stored at 4~C.
Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated F8S. The cells were infected with the recombinant baculovirus V-HDGNRl0 at a multiplicity of infection (MOI) of 2. Six hours later the medium was removed and replaced with SF900 II medium minus methionine and cy~teine (Life Technologies Inc., Gaithersburg). 42 hours later 5 ~Ci of 35S-methionine and 5 ~Ci 35S cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.
Example 4 --~1--W O 96~9437 PCT/U~,S/~7173 Ex~ression via Gene TherapY
Fibroblasts are obtained from a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added.
This is then incubated at 37~C for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively. The 5' primer ccntains an EcoRI site. and the 3' primer contains a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditior.s appropriate for ligation of the two fragments. The ligation mixture is used to transform bacteria B 101, which are then plated onto agar-containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
w096~9437 PCT~S95/07173 The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector cont~ining the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles cont~ining the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblas~ cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
The engineered fibroblasts are then injec~ed into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.
Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.
W096~9437 PCT~S95/07173 SEQUENCE LISTING
(l) GENERAL INFORMATION:
(i) APPLICANT: Li, ET AL.
(ii) TITLE OF INv~NllON: Human G-Protein Chemokine Receptor (iii) NUMBER OF SEQUENCES:
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) C'OUN1KY: USA
(F) ZIP: 07Go8 (v) CO~U1~K READABLE rOR~i:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COM~ul~;K: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: ~-ORD PE~F~CT 5.l (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: concurrently (C) CLASSIFICATION:
(vii) ATTORNEY/AGh~T INFORMATION:
(A) NAME: FERRA~G, GREGORY D.
~B) REGISTRATION NUMBER: 36,134 (C) REFE~REN OE /DûCXET ~JMBER: 325800-(viii) TELECOMMUNICATION INFORMATION:
(A) TELhPHONE: 20~-994-1700 (B) TELEFAX: 201-394-1744 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCr. CHARACTEP~ISI'I.S
(A) LENC:TH: 1414 BA~;E ~AIRS
(B) TY~E: NUCLEIC ACIB
(C) STRANDEDNESS: SINGLE
(~) TO~OLOGY: LINEAR
(ii) MOLECULr. TYPE: ~DNA
(xi) SEQUEN-E DESCRIPTION SrQ ,D NO:l:
GTGAGATGG. GCTTTCATGA A~TCCC:CI~. ~A~GAGCC~A GCTCTCCATC TAG-GGACAG 60 GGAAGCTAGC AGCAAACCTT CC~ ~ACTA CGAAACTTCA TTG-TTGGCC CAAAAGAGAG 120 TTAATTCAAT GTAGACATCT ATGTAGGCAA TTAAAAACCT ATTGATGTAT AAAPr~5 m 130 GCATTCATGG AGGGCAACTA AATACATTCT AGGAC'ATTAT AAAAGATCAC TTTTTATTTA 240~GCACAGGGT GGAACAAG ATG GAT TAT CAA GTG TCA AGT CCA ATC TAT GAC 291 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr Ser Glu Pro Cys Pro Lys Ile Asn Val Lys Gln ATC GCA GCC CGC CTC CTG CCT CCG CTC TAC TCA CTG GTG -TC ATC TT, 387 Ile Ala Ala Arg Leu Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val I1E Leu Ile Leu Iie Asn Cys Gln Arg Leu Glu Ser Met Thr Asp I~e Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu Leu Thr Vai Pro Phe Trp Ala His ~yr Ala Ala GCC CAG TGG GAC TTT GGA AAT ACA ATG rrGT CAA CTC TTG ACA GGG CTC 579Ala Gln Trp Asp Phe Gly Asn Thr Met Cys Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe Phe Ser Gly Ile Phe Phe Ile Ile Gln Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val H_s Ala Val Phe Ala Leu Lys Ala AGG ACG GTC ACC r~-l GGG GTG GTG ACA AGT GTG ATC ACT ''GG GTG GTG 723 Arg Thr VaL Thr Phe Gly Val Val Thr Ser Val Ile Thr Trp Val Val GCT GTG TTT GCG ' CT CTC CCA GGA ATC ATC TTT ACC AGA TCT CAA AAA 771Ala Val Phe Ala Ser Leu Pro Gly Ile Ile Phe Tnr Arg Ser Gln Lys GAA GGT CTT CAT TAC ACC TGC AGC TCT CAT T~T CCA TAC AGT CAG TAT 819 Glu Gly Leu His Tyr Thr cys Ser Ser His Phe Pro I~r Ser Gln Tyr CAA TTC TGG AAG AAT TTC CAG ACA TT~ AAG A-.A G.C ATC '..G GGG CTG 867 Gln Phe Trp Lys Asn Phe Gln Thr ~eu Lys i_e Val ,ie Leu Gly Leu GTC CTG CCG,CTG CTT GTC ATG G.C A._ TGC TA- TCG GGA ATC CTA AAA 915 Val Leu Pro Leu Leu Val Met Val ,ie Cys Tyr Ser Giy Ile Leu Lys ACT CTG CTT CGG TGT CGA AAT ~A~ AAC- AAG AGG CAC AGG GC'. GT~ AGG 963 Thr Leu Leu Arg Cys Arg Asn G;u Lys Lys Arg H1s Arg Ala Val Arg CTT ATC TTC ACC ATC ATG A-T G'~T T~'.' 1-1-. C'~C TTC 'GG GCT CCC TAC 1011 Leu Ile Phe Thr Ile Met Iie Val '.~r Phe ~eu ~he .:-p Ala Pro Tyr AAC ATT GTC CTT CTC CTG AAC ACC TTC ~AG ~A TTC L~ GGC CTG AAT lOS9 Asn Ile Val Leu Leu Leu Asn Tnr 2ne Gln G-u Pne Phe Gly Leu Asn AAT TGC AGT AGC ''CT AAC AGG TTG G~C CAA G~ ATG Gi~G GTG ACA GAG 1107 Asn Cys Ser Ser Ser Asn Arg Leu ~sF G~n A~a Met Gin Val Thr Glu ACT CTT GGG ATG ACG CAC TGC TGC A" C AAC C;'C ATC ATC TA'; GCC TTT 1155 Thr Leu Gly Met Thr His Cys Cy5 I_e Asn ~ro Iie lle I~r Ala Phe CA 022l69l2 l997-ll-27 GTC GGG GAG AAG ~C AGA AAC TAC CTC TTA GTC TTC TTC CAA AAG CAC . 1203 Val Gly Glu Lys Phe Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys Hls Ile Ala Lys Arg Phe Cys Lys Cys Cys Ser Ile Phe Gin Gln Glu ~la CCC GAG CGA GCA AGC TCA G~T TAC ACC CGA TCC ACT GGG GAG CAG GAA 1299 Pro Glu Arg Ala Ser Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Glu Ile Ser Val Gly Leu TGCACATGGC TTAGl~ l~A TACACAGCC~ GGG~-lGG5GG l~GGGl~GAA GAG~l--l-l-~-l 1414 (2) INFORMATION FOR SEQ ID NO:2:
(i) S~Qu~CE CHARACTERISTICS
(A) LENGTH: AMINO ACIDS
~L) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: EROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
~et Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr 15~hr Ser Glu Pro Cys Pro Lys Ile Asn Val Lys Gln Ile Ala Ala 30~rg Leu Leu Ero Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe 45~al Gly Asn Met Leu Val Ile Leu Ile Leu Ile Asn Cys Gln Arg 60~eu Glu Ser Met Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser 75~fip Leu Phe Phe Leu Leu .hr Val Pro Phe Trp Ala His Tyr Ala 90~la Ala Gln Trp Asp Pne Gly Asn Thr Met Cys Leu Leu Thr Gly 100 105~eu Tyr Phe Ile Gly Pne Phe Ser Gly Ile Phe Phe Ile Ile Gln 110 11~ 120~eu Leu Thr Ile Asp Arg ~yr ~eu Ala Ile Val ~is Ala Val Phe 125 13C 135~la Leu Lys Ala Arg Thr Val '.h-; Phe Gly Val Val Thr Ser Val 140 14, 150~le Thr Trp Val Val Ala Val Phe ~la Ser Le~ Pro Gly Ile Ile 155 160 165~he Thr Arg Ser Gln Lys G.u Gly Leu Xis Tyr Thr cys Ser Ser 170 1/5 180~is Phe Pro Tyr Ser Gln Tyr Gln Phe Tr~ Lys ~sn Phe Gln Thr 185 l9C 195~eu Lys lle Val Ile LeL Gly Le-~ Val Le-~ Pro Leu Le~ Val Met 2~G 2G5 210 -4~-al Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu Arg Cys Arg ~sn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile Phe Thr Ile ~et Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn Ile Val Leu ~eu Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu Asn Asn Cys Ser ~er Ser Asn Arg Leu Asp Gln Ala Met Gln Val Thr Glu Thr Leu ~ly Met Thr His Cys Cys I;e Asn Pro Ile Ile Tyr Ala Phe Val ~ly Glu Lys Phe Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His ~le Ala Lys Arg Phe Cys Lys C~ys Cys Ser Ile Phe Gln Gln Glu 32~ 3,-5 330 ~la Pro Glu Arg Ala Ser Ser Jal Tyr rh~~ Arg Ser Thr Gly Glu ~ln Glu Ile Ser Val Gly Leu
Therefore, the PCR product contains a HindIII site HDGNR10 coding seguence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an XhoI site. The PCR amplified DNA fragment and the vector, pcDNAItAmp, were digested with HindIII and XhoI restriction enzyme and ligated. The ligation mixture was transfonmed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and ~mi ned by restriction analysis for the presence of the correct fragment. ~or expression of the recombinant HDGNR10, COS cells were transfected with the expression vector by DBAE-DEXTRAN
method. (J. Sa,.L~ook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory ~nl~l, Cold Spring Laboratory Pre~s, (1989)). The expression of the HDGNR10 HA protein was detected by radiolabelling and ~ noprecipitation method.
(E. Harlo~, D. Lane, Antibodies: A Laboratory ~nll~l, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with 35S-cysteine two days post transfection.
Culture media were then collected and cells were lysed with deteryent (RIPA buffer (150 ~M 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 specific monoclonal antibody.
Proteins precipitated were analyzed on 15% SDS-PAGE gels.
W096/39437 PCT~S95/07173 Exam~le 3 Cloninq and ex~ression of HDGNR10 usinq the baculovirus ex~ression s~stem The DNA sequence encoding the full length HDGNR10 protein, ATCC # , was amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5' CGGGATCCCTCCATGGATTAT
CAA~l~l~A 3' and contains a BamHI restriction enzyme site followed by 4 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (J. Mol.
Biol. 1987, 196, 947-950, Kozak, M.), and just behind the first 18 nucleotides of the HDGNR10 gene (the initiation codon for translation is "ATG").
The 3' primer has the sequence 5' CGGGATCCCGCT
CACAAGCCCACAGATAT 3' and contains the cleavage site for the restriction endonuclease BamHI and 18 nucleotides complementary to the 3' non-translated sequence of the HDGNR10 gene. The amplified sequences were isolated from a 1% agarose gel using a commercially available kit (nGeneclean," BIO 101 Inc., La Jolla, Ca.). The fragment was then digested with the ~n~Qnt~clease 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 HDGNR10 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimer.~al Station Bulletin No. 1555).
This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction en~m1clease BamHI. The polyadenylation site of - the simian virus (S~)40 is used for efficient polyadenylation. For an easy select~on of recombinant w096~9437 PCT~Sss/07173 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 both sides by viral sequences for the cell-mediated homologous recombination of co-transfected 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 was digested with the restriction enzyme BamHI and then ~erhosphorylated 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 HB101 cel~s were then transfonmed and bacteria identified that contained the plasmid (pBacHDGNR10) with the HDGNR10 gene using the enzyme BamHI. The sequence of the cloned fragment was confirmed by DNA sequencing.
S ~g of the plasmid pBacHDGNR10 were co-transfected with 1.0 ~g of a commercially available linearized baculovirus ("BaculoGold~ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)).
l~g of BaculoGold~ virus DNA and S ~g of the plasmid pBacHDGNR10 were mixed in a sterile well of a microtiter plate contAining 50 ~l of serum free Grace~s medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~l Lipofectin plus 90 ~l Grace's mediu~ were added, mixed and incubated for 15 minutes at room _emperature. 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 W096/39437 PCT~S95/07173 was then incubated for 5 hours at 27~C. After 5 hours the transfection solution was removed from the plate and l ml of Grace's insect medium supplemented with 10% fetal calf serum was added. The plate was put back into an incu~ator and cultivation continued at 27~C for four days.
After four days the supernatant was collected and a plaque assay performed similar as described by Summers and Smith (supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gai'hersburg) was used which allows an easy isolation of blue stained plaques. (A
detailed description of a "plaque assay" can also be found in the user~s guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10) .
Four days after the serial dilution, the viruses were added to the cells, blue stained plaques were picked with the tip of an Eppendorf pipette. The agar cont~ining the recombinant viruses was then resuspended in an Eppendorf tube cont~ining 200 ~l of Grace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculoviruses was used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of the~e culture dishes were harvested and then stored at 4~C.
Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated F8S. The cells were infected with the recombinant baculovirus V-HDGNRl0 at a multiplicity of infection (MOI) of 2. Six hours later the medium was removed and replaced with SF900 II medium minus methionine and cy~teine (Life Technologies Inc., Gaithersburg). 42 hours later 5 ~Ci of 35S-methionine and 5 ~Ci 35S cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.
Example 4 --~1--W O 96~9437 PCT/U~,S/~7173 Ex~ression via Gene TherapY
Fibroblasts are obtained from a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added.
This is then incubated at 37~C for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively. The 5' primer ccntains an EcoRI site. and the 3' primer contains a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditior.s appropriate for ligation of the two fragments. The ligation mixture is used to transform bacteria B 101, which are then plated onto agar-containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
w096~9437 PCT~S95/07173 The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector cont~ining the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles cont~ining the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblas~ cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
The engineered fibroblasts are then injec~ed into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.
Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.
W096~9437 PCT~S95/07173 SEQUENCE LISTING
(l) GENERAL INFORMATION:
(i) APPLICANT: Li, ET AL.
(ii) TITLE OF INv~NllON: Human G-Protein Chemokine Receptor (iii) NUMBER OF SEQUENCES:
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) C'OUN1KY: USA
(F) ZIP: 07Go8 (v) CO~U1~K READABLE rOR~i:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COM~ul~;K: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: ~-ORD PE~F~CT 5.l (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: concurrently (C) CLASSIFICATION:
(vii) ATTORNEY/AGh~T INFORMATION:
(A) NAME: FERRA~G, GREGORY D.
~B) REGISTRATION NUMBER: 36,134 (C) REFE~REN OE /DûCXET ~JMBER: 325800-(viii) TELECOMMUNICATION INFORMATION:
(A) TELhPHONE: 20~-994-1700 (B) TELEFAX: 201-394-1744 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCr. CHARACTEP~ISI'I.S
(A) LENC:TH: 1414 BA~;E ~AIRS
(B) TY~E: NUCLEIC ACIB
(C) STRANDEDNESS: SINGLE
(~) TO~OLOGY: LINEAR
(ii) MOLECULr. TYPE: ~DNA
(xi) SEQUEN-E DESCRIPTION SrQ ,D NO:l:
GTGAGATGG. GCTTTCATGA A~TCCC:CI~. ~A~GAGCC~A GCTCTCCATC TAG-GGACAG 60 GGAAGCTAGC AGCAAACCTT CC~ ~ACTA CGAAACTTCA TTG-TTGGCC CAAAAGAGAG 120 TTAATTCAAT GTAGACATCT ATGTAGGCAA TTAAAAACCT ATTGATGTAT AAAPr~5 m 130 GCATTCATGG AGGGCAACTA AATACATTCT AGGAC'ATTAT AAAAGATCAC TTTTTATTTA 240~GCACAGGGT GGAACAAG ATG GAT TAT CAA GTG TCA AGT CCA ATC TAT GAC 291 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr Ser Glu Pro Cys Pro Lys Ile Asn Val Lys Gln ATC GCA GCC CGC CTC CTG CCT CCG CTC TAC TCA CTG GTG -TC ATC TT, 387 Ile Ala Ala Arg Leu Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val I1E Leu Ile Leu Iie Asn Cys Gln Arg Leu Glu Ser Met Thr Asp I~e Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu Leu Thr Vai Pro Phe Trp Ala His ~yr Ala Ala GCC CAG TGG GAC TTT GGA AAT ACA ATG rrGT CAA CTC TTG ACA GGG CTC 579Ala Gln Trp Asp Phe Gly Asn Thr Met Cys Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe Phe Ser Gly Ile Phe Phe Ile Ile Gln Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val H_s Ala Val Phe Ala Leu Lys Ala AGG ACG GTC ACC r~-l GGG GTG GTG ACA AGT GTG ATC ACT ''GG GTG GTG 723 Arg Thr VaL Thr Phe Gly Val Val Thr Ser Val Ile Thr Trp Val Val GCT GTG TTT GCG ' CT CTC CCA GGA ATC ATC TTT ACC AGA TCT CAA AAA 771Ala Val Phe Ala Ser Leu Pro Gly Ile Ile Phe Tnr Arg Ser Gln Lys GAA GGT CTT CAT TAC ACC TGC AGC TCT CAT T~T CCA TAC AGT CAG TAT 819 Glu Gly Leu His Tyr Thr cys Ser Ser His Phe Pro I~r Ser Gln Tyr CAA TTC TGG AAG AAT TTC CAG ACA TT~ AAG A-.A G.C ATC '..G GGG CTG 867 Gln Phe Trp Lys Asn Phe Gln Thr ~eu Lys i_e Val ,ie Leu Gly Leu GTC CTG CCG,CTG CTT GTC ATG G.C A._ TGC TA- TCG GGA ATC CTA AAA 915 Val Leu Pro Leu Leu Val Met Val ,ie Cys Tyr Ser Giy Ile Leu Lys ACT CTG CTT CGG TGT CGA AAT ~A~ AAC- AAG AGG CAC AGG GC'. GT~ AGG 963 Thr Leu Leu Arg Cys Arg Asn G;u Lys Lys Arg H1s Arg Ala Val Arg CTT ATC TTC ACC ATC ATG A-T G'~T T~'.' 1-1-. C'~C TTC 'GG GCT CCC TAC 1011 Leu Ile Phe Thr Ile Met Iie Val '.~r Phe ~eu ~he .:-p Ala Pro Tyr AAC ATT GTC CTT CTC CTG AAC ACC TTC ~AG ~A TTC L~ GGC CTG AAT lOS9 Asn Ile Val Leu Leu Leu Asn Tnr 2ne Gln G-u Pne Phe Gly Leu Asn AAT TGC AGT AGC ''CT AAC AGG TTG G~C CAA G~ ATG Gi~G GTG ACA GAG 1107 Asn Cys Ser Ser Ser Asn Arg Leu ~sF G~n A~a Met Gin Val Thr Glu ACT CTT GGG ATG ACG CAC TGC TGC A" C AAC C;'C ATC ATC TA'; GCC TTT 1155 Thr Leu Gly Met Thr His Cys Cy5 I_e Asn ~ro Iie lle I~r Ala Phe CA 022l69l2 l997-ll-27 GTC GGG GAG AAG ~C AGA AAC TAC CTC TTA GTC TTC TTC CAA AAG CAC . 1203 Val Gly Glu Lys Phe Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys Hls Ile Ala Lys Arg Phe Cys Lys Cys Cys Ser Ile Phe Gin Gln Glu ~la CCC GAG CGA GCA AGC TCA G~T TAC ACC CGA TCC ACT GGG GAG CAG GAA 1299 Pro Glu Arg Ala Ser Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Glu Ile Ser Val Gly Leu TGCACATGGC TTAGl~ l~A TACACAGCC~ GGG~-lGG5GG l~GGGl~GAA GAG~l--l-l-~-l 1414 (2) INFORMATION FOR SEQ ID NO:2:
(i) S~Qu~CE CHARACTERISTICS
(A) LENGTH: AMINO ACIDS
~L) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: EROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
~et Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr 15~hr Ser Glu Pro Cys Pro Lys Ile Asn Val Lys Gln Ile Ala Ala 30~rg Leu Leu Ero Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe 45~al Gly Asn Met Leu Val Ile Leu Ile Leu Ile Asn Cys Gln Arg 60~eu Glu Ser Met Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser 75~fip Leu Phe Phe Leu Leu .hr Val Pro Phe Trp Ala His Tyr Ala 90~la Ala Gln Trp Asp Pne Gly Asn Thr Met Cys Leu Leu Thr Gly 100 105~eu Tyr Phe Ile Gly Pne Phe Ser Gly Ile Phe Phe Ile Ile Gln 110 11~ 120~eu Leu Thr Ile Asp Arg ~yr ~eu Ala Ile Val ~is Ala Val Phe 125 13C 135~la Leu Lys Ala Arg Thr Val '.h-; Phe Gly Val Val Thr Ser Val 140 14, 150~le Thr Trp Val Val Ala Val Phe ~la Ser Le~ Pro Gly Ile Ile 155 160 165~he Thr Arg Ser Gln Lys G.u Gly Leu Xis Tyr Thr cys Ser Ser 170 1/5 180~is Phe Pro Tyr Ser Gln Tyr Gln Phe Tr~ Lys ~sn Phe Gln Thr 185 l9C 195~eu Lys lle Val Ile LeL Gly Le-~ Val Le-~ Pro Leu Le~ Val Met 2~G 2G5 210 -4~-al Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu Arg Cys Arg ~sn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile Phe Thr Ile ~et Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn Ile Val Leu ~eu Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu Asn Asn Cys Ser ~er Ser Asn Arg Leu Asp Gln Ala Met Gln Val Thr Glu Thr Leu ~ly Met Thr His Cys Cys I;e Asn Pro Ile Ile Tyr Ala Phe Val ~ly Glu Lys Phe Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His ~le Ala Lys Arg Phe Cys Lys C~ys Cys Ser Ile Phe Gln Gln Glu 32~ 3,-5 330 ~la Pro Glu Arg Ala Ser Ser Jal Tyr rh~~ Arg Ser Thr Gly Glu ~ln Glu Ile Ser Val Gly Leu
Claims (20)
1. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide as set forth in SEQ ID NO:2;
(b) a polynucleotide encoding a mature polypeptide encoded by the DNA contained in ATCC Deposit No. ;
(c) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a) or (b); and (d) a polynucleotide fragment of the polynucleotide of (a), (b) or (c).
(a) a polynucleotide encoding the polypeptide as set forth in SEQ ID NO:2;
(b) a polynucleotide encoding a mature polypeptide encoded by the DNA contained in ATCC Deposit No. ;
(c) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a) or (b); and (d) a polynucleotide fragment of the polynucleotide of (a), (b) or (c).
2. The polynucleotide of claim 1 wherein the polynucleotide is DNA.
3. A vector containing the DNA of Claim 2.
4. A host cell transformed or transfected with the vector of Claim 3.
5. A process for producing a polypeptide comprising:
expressing from the host cell of Claim 4 the polypeptide encoded by said DNA.
expressing from the host cell of Claim 4 the polypeptide encoded by said DNA.
6. A process for producing cells capable of expressing a polypeptide comprisins transforming or transfecting the cells with the vector of Claim 3.
7. A receptor polypeptide comprising a member selected from the group consisting of:
(i) a polypeptide having the deduced amino acid sequence of SEQ ID NO:2 and fragments, analogs and derivatives thereof; and (ii) a polypeptide encoded by the cDNA of ATCC
Deposit No. and fragments, analogs and derivatives of said polypeptide.
(i) a polypeptide having the deduced amino acid sequence of SEQ ID NO:2 and fragments, analogs and derivatives thereof; and (ii) a polypeptide encoded by the cDNA of ATCC
Deposit No. and fragments, analogs and derivatives of said polypeptide.
8. The polypeptide of Claim 7 wherein the polypeptide has the deduced amino acid sequence of SEQ ID
NO:2.
NO:2.
9. An antibody against the polypeptide of claim 7 selected from the group consisting of an antibody which agonizes the activity of the polypeptide and an antibody which antagonizes the activity of the polypeptide.
10. A compound which activates the polypeptide of claim 7.
11. A compound which inhibits activation the polypeptide of claim 7.
12. 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 10.
administering to the patient a therapeutically effective amount of the compound of claim 10.
13. 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 11.
administering to the patient a therapeutically effective amount of the compound of claim 11.
14. The method of claim :2 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to tne patient DNA
encoding said agonist and expressing said agonist in vivo.
encoding said agonist and expressing said agonist in vivo.
15. The method of claim 13 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 .
encoding said antagonist and expressing said antagonist in vivo .
16. A method for identifying compounds which bind to and activate the receptor polypeptide of claim 7 comprising:
contacting a cell expressing on the surface thereof the receptor polypeptide, said receptor 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 receptor polypeptide; and identifying if the compound is an effective agonist by detecting the signal produced by said second component.
contacting a cell expressing on the surface thereof the receptor polypeptide, said receptor 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 receptor polypeptide; and identifying if the compound is an effective agonist by detecting the signal produced by said second component.
17. A method for identifying compounds which bind to and inhibit activation the polypeptide of claim 7 comprising:
contacting a cell expressing on the surface thereof the receptor polypeptide, said receptor 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 to be screened under conditions to permit binding to the receptor polypeptide; and determining whether the compound inhibits activation of the polypeptide by detecting the absence of a signal generated from the interaction of the ligand with the polypeptide.
contacting a cell expressing on the surface thereof the receptor polypeptide, said receptor 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 to be screened under conditions to permit binding to the receptor polypeptide; and determining whether the compound inhibits activation of the polypeptide by detecting the absence of a signal generated from the interaction of the ligand with the polypeptide.
18. A process for diagnosing a disease or a susceptibility to a disease related to an under-expression of the polypeptide of claim 7 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide.
determining a mutation in the nucleic acid sequence encoding said polypeptide.
19. The polypeptide of Claim 7 wherein the polypeptide is a soluble fragment of the polypeptide and is capable of binding a ligand for the receptor.
20. A diagnostic process comprising:
analyzing for the presence of the polypeptide of claim 19 in a sample derived from a host.
analyzing for the presence of the polypeptide of claim 19 in a sample derived from a host.
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002562211A CA2562211A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
CA002562162A CA2562162A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
CA002216912A CA2216912A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002216912A CA2216912A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002562162A Division CA2562162A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
CA002562211A Division CA2562211A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2216912A1 true CA2216912A1 (en) | 1996-12-12 |
Family
ID=4161544
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002216912A Abandoned CA2216912A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
CA002562162A Abandoned CA2562162A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
CA002562211A Abandoned CA2562211A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002562162A Abandoned CA2562162A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
CA002562211A Abandoned CA2562211A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
Country Status (1)
Country | Link |
---|---|
CA (3) | CA2216912A1 (en) |
-
1995
- 1995-06-06 CA CA002216912A patent/CA2216912A1/en not_active Abandoned
- 1995-06-06 CA CA002562162A patent/CA2562162A1/en not_active Abandoned
- 1995-06-06 CA CA002562211A patent/CA2562211A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
CA2562162A1 (en) | 1996-12-12 |
CA2562211A1 (en) | 1996-12-12 |
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