WO2003069348A2 - USE OF MIP-3α AND ITS RECEPTOR TO TREAT ARTHRITIS - Google Patents

Abstract

The present invention relates to novel uses for the chemokine MIP-3α and its receptor CCR6 to treat and diagnose osteoarthritis. In particular, MIP-3α is shown to be expressed in cartilage tissue and chondrocytes derived from individuals having osteoarthritis, whereas normally the chemokine is not expressed in such cells or tissues. These findings indicate that both the MIP-3α chemokine and the CCR6 receptor are useful targets, e.g., for the development of therapeutics to treat, ameliorate and/or modulate conditions such as osteoarthritis. Accordingly, the invention provides screening methods for identifying candidate compounds that modulate MIP-3α binding to its receptor and may therefore be used to treat osteoarthritis. Therapeutic methods and pharmaceutical compositions that inhibit MIP-3α expression or activity or, alternatively, inhibit CCR6 expression or activity are also provided. Finally, the invention also provides diagnostic and prognostic methods for identifying individuals having osteoarthritis by detecting MIP-3α expression.

Description

USE OF MIP-3α AND ITS RECEPTOR TO TREAT ARTHRITIS

FIELD OF THE INVENTION

The present invention relates to novel uses for the chemokine MIP-3α and its receptor CCR6. In particular, the invention relates to compositions and methods that use MIP-3α and/or CCR6 to diagnose osteoarthritis (OA), as well as such other (e.g. similar or related) disorders as may be discussed herein. Similarly, the invention relates to the use of MIP-3α and/or CCR6 to screen for therapeutic agents to treat, prevent, or ameliorate OA.

BACKGROUND OF THE INVENTION

Osteoarthritis (OA), also called degenerative joint disease, is the most common type of arthritis. It is associated with degradation and breakdown of cartilage in joints and commonly occurs in the hips, knees and spine. Also, it often affects the finger joints, the joint at the base of the thumb, and the joint at the base of the big toe.

Osteoarthritis affects almost 16 million men and women in the United States. The chance of developing osteoarthritis increases with age. Most people over age 60 have osteoarthritis to some degree, but its severity varies, and some people develop more severe symptoms than others. Osteoarthritis causes the cartilage in a joint to become stiff and lose its elasticity, making it more susceptible to damage. Over time, the cartilage may wear away in some areas, greatly decreasing its ability to act as a shock-absorber. As the cartilage wears away, tendons and ligaments stretch, causing pain. If the condition worsens, the bones could rub against each other. Symptoms of osteoarthritis include joint aching and soreness, especially with movement, pain after overuse or after long periods of inactivity, and bony enlargements in the middle and end joints of the fingers (which may or may not be painful).

Current diagnosis of osteoarthritis is based on a combination of the patient's description of symptoms, the location and pattern of pain, and certain findings upon physical examination. The use of X-rays to confirm the diagnosis and to reveal the extent of joint damage, is also prevalent. If fluid has accumulated in the joints, fluid from the joint may be removed (called joint aspiration) and examined under a microscope to rule out other diseases. Osteoarthritis is usually treated by medications (to alleviate pain) exercise, application of heat or cold to the painful joint, use of supportive devices such as crutches or canes, and weight control. Surgery may be helpful to relieve pain when other treatment options have not been effective. The type of treatment prescribed will depend on several factors including the patient's age, activities and occupation, overall health, medical history and severity of the condition.

Chemokines are a group of cytokines that are primarily known to induce adhesion and directional migration of specific cell types to extravascular sites of inflammation (Rossi et al., Ann. Rev. Immunol. 2000; 18:217-42). An important role for them in the induction of inflammatory pain has also been suggested (See, Lotz et al., Anesthesia: biologic foundations 1997; Cunha et al., Br. J. Pharmacol. 1991; 104: 765-67). There is a recent report which suggests that chemokines also contribute to the degradation of cartilage in OA (Borzi et al., Arthritis and Rheumatism 2000;43: 1734-41). Gene expression analysis demonstrated increased expression of a large number of chemokines in OA cartilage indicating that chemokines may play a role in joint disease (Pulsatelli et ai, J. Rheumatol. 1999; 26:1992-2001, Borzi et al, FEBS Lett. 1999; 455:238-42). In fact, chemokines were shown to induce expression of MMP-3 in isolated human chondrocytes (Borzi et al., Arthritis and Rheumatism 2000;43: 1734-41).

It has been determined that there is a large family (>20 members) of structurally related chemoattractant chemokines. Chemokines consists of 8, 10, and 12kD proteins and are separated into four families: the CXC, CC, C and the CX₃C family, which are categorized based upon the position of the first two amino-terminal proximal cysteines (C) and the intervening amino acids (Rossi & Ziotnik, Ann. Rev. Immunol. 2000; 18:217-242). Each chemokine contains four cysteine residues and two internal disulfide bonds. Chemokines can be grouped into two subfamilies, based on whether the two amino terminal cysteine residues are adjacent to each other or are separated by any amino acid. Of the identified chemokine receptors, all of these belong to the seven transmembrane G protein- coupled receptor family. All of these receptors (to date, there have been 18 identified) mediate the binding and signaling of more than one chemokine (Bagglioni, J. Intern. Med. 2001; 250:91-104). Macrophage inhibitory protein-3 alpha (MIP-3α) belongs to the CC or β family and is also known in the art as LARC, ckβ4 and Exodus- 1 (Heishima et al., J. Biol. Chem. 1997, 272:5846-53; U.S. Patent No. 5,504, 003). Macrophage inflammatory proteins are proteins that are produced by macrophages and lymphocytes in response to stimuli such as bacteria, and exhibit pro-inflammatory properties. Therefore, these molecules may have diagnostic and therapeutic utility for detecting and treating infections, auto-immune diseases, or any other inflammatory condition.

MIP-3α is expressed in lung, liver, lymphoid tissue, activated monocytes, endothelial cells, fibroblasts, dendritic and T cells. MTP-3α binds specifically only to the CCR6 receptor, a unique feature that makes this chemokine attractive for drug recovery. CCR6 is expressed by resting memory T cells, B cells and dendritic cells (Heishima et al, J. Biol. Chem. 1997, 272:5846-5843; Power et al, J. Exp. Med. 1997, 186:825-35). Recently, increased amounts of MIP-3α were detected in synovial fluids and tissue samples of patients with rheumatoid arthritis, as well as expression of the MIP-3α gene in cells from synovial fluid (Matsui et al. Cain. Exp. Immunol. 2001, 125(1):155). However, MIP-3α has not been observed in tissues from patients with OA.

Since chemokines are involved in at least three components of arthritis pathogenesis (joint inflammation, pain induction and matrix degradation), inhibitors of chemokine action may have value in the management of rheumatoid arthritis (RA) and OA. Therefore, there is a need to identify biological agents, including particular chemokines, that are involved in cartilage degradation, and disorders such as OA, that are associated with cartilage degradation. Further, there is a need to develop therapeutic compounds to inhibit cartilage degradation and treat disorders (such as OA) that are associated with cartilage degradation.

SUMMARY OF THE INVENTION

The present invention relates to novel uses for the chemokine MIP-3α and its receptor, CCR6. In particular, Applicants have discovered that MIP-3α is expressed in cells and tissue derived from the cartilage of individuals having osteoarthritis, whereas the chemokine is normally expressed at only very low or undetectable levels in those tissues. Because MIP-3α specifically binds to a receptor, CCR6, that is also expressed in these tissues, this surprising discovery demonstrates that MIP-3α and its receptor may be used in novel methods to diagnose and/or treat osteoarthritis. Similarly, these findings support novel screening assays that use MIP-3α chemokine and/or its receptor (i.e., CCR6) to identify compounds that may be used to treat or modulate osteoarthritis.

Provided herein, therefore, as part of the present invention, are screening methods for identifying compounds that may be used to treat osteoarthritis. These methods comprise, in preferred embodiments, contacting a test compound to a reaction mixture that contains an MIP-3α chemokine (i.e., MIP-3α protein or polypeptide) and its receptor, the CCR6 receptor. In preferred embodiments, MIP-3α is a polypeptide having an amino acid sequence which may comprise the sequence set forth here in SEQ ID NO: 12, in SEQ ID NO:7 or in SEQ ID NO: 10. However, any MIP-3α homolog, ortholog, variant, etc. may be used in these methods, as can fusion constructs of those polypeptides (for example a fusion construct as described in the Examples, infra). For instance, the MIP-3α polypeptide may have an amino acid sequence that is substantially homologous ie.g., at least 75%, 80%, 85%, 90%, 95% or 99% identical) to SEQ ID NO: 12, or it may be encoded by a nucleic acid molecule that hybridizes ie.g., under stringent or other hybridization conditions defined herein) to the complement of a nucleic acid molecule that encodes the amino acid sequence set forth in SEQ ID NO: 12 ie.g., the nucleotide sequence set forth in SEQ ID NO:l 1). Preferably, however, the MIP-3α polypeptide used in these methods will be capable of binding to a CCR6 receptor under normal ie.g., physiological conditions), thereby forming a binding complex.

The CCR6 receptor used in such screening methods is preferably a polypeptide having the amino acid sequence set forth in SEQ ID NO: 14. However, any CCR6 homolog, ortholog, variant, etc. may be used in these methods, as can fusion constructs of those polypeptides. For instance, the CCR6 polypeptide may have an amino acid sequence that is substantially homologous ie.g., at least 75%, 80%, 85%, 90%, 95% or 99% identical) to SEQ ID NO: 14, or it may be encoded by a nucleic acid molecule that hybridizes ie.g., under stringent or other hybridization conditions defined herein) to the complement of a nucleic acid molecule that encodes the amino acid sequence set forth in SEQ ID NO: 14 ie.g., the nucleotide sequence set forth in SEQ ID NO: 13). Fragments of a CCR6 receptor may also be used, such as fragments corresponding to one or more distinct domains ie.g., an extracellular domain) of the CCR6 receptor. Preferably, however, the CCR6 receptor or fragment thereof that is used in these methods will be capable of binding to a MIP-3α chemokine under normal (e.g., physiological conditions), thereby forming a binding complex.

As part of the screening methods of this invention, the test compound is contacted to the reaction mixture under conditions that permit formation of a binding complex, and formation of such a binding complex is detected by a user. The level of binding complex formed in the presence of the test compound is then compared to the level of binding complex formed in the absence of the test compound ie.g., from a previous control experiment in which binding complexes were detected in the reaction mixture under identical conditions, but in the absence of a test compound). According to these methods, when it is determined that a decreased level or amount of the binding complex is formed in the presence of the test compound, the test compound is identified as a compound that modulates binding of MIP-3α to its receptor and, as such, is identified as a compound that may be used to treat osteoarthritis.

In another embodiment, the invention provides diagnostic and prognostic methods for identifying individuals who have osteoarthritis or who are at risk of developing osteoarthritis. Such methods involve detecting expression of either an MIP-3α gene or, alternatively, an MD?-3α gene product (such as an MIP-3α polypeptide or an MIP-3α mRNA that encodes the MIP-3α polypeptide). Preferably, the MIP-3α gene or gene product is detected in a sample of cartilage tissue or cells ie.g., in chondrocyte cells) that are derived from an individual who is either suspected of having osteoarthritis or who is suspected of being at risk for developing osteoarthritis. Detection of the MIP-3α gene or its gene product in such tissue indicates that the individual is one who either has or is at increased risk of having osteoarthritis. It is noted that the MIP-3α gene or gene product detected in these methods may comprise any of the MIP-3α nucleic acid or amino acid sequences discussed supra. Also, the MIP-3α gene or gene product may be detected using any MIP-3α specific probe (including, e.g., an antibody or nucleic acid probe) or primer. Such probes and primers include, but are not limited to, ones having any of the nucleic acid sequences provided here at SEQ ID NOS:l-6 and 8-9.

In still other embodiments, the invention provides therapeutic methods that may be used to treat osteoarthritis in an individual. These methods comprise administering to the individual an effective amount of one or more compounds that modulate binding of MIP- 3α polypeptide to its receptor ie.g., to a CCR6 receptor). In other embodiments, the therapeutic methods of the invention may also comprise administering a compound to an individual that inhibits or reduces expression of either an MIP-3α gene or its gene product in the individual or, alternatively, a compound that inhibits or reduces expression of either a CCR6 or its gene product in the individual. Preferably, the MIP-3α polypeptide and its receptor are ones expressed in cartilage tissue of the individual being treated. Such polypeptides may therefore comprise, e.g., any of the MIP-3α and CCR6 amino acid sequences described supra.

The invention also provides pharmaceutical compositions that may be used in therapeutic methods of the invention (for example, in a method to treat osteoarthritis in an individual). These pharmaceutical compositions comprise an effective amount of one or more compounds that modulate binding of an MIP-3α polypeptide to its receptor ie.g., to a CCR6 receptor). However, the therapeutic compounds of the invention may comprise an effective amount of one or more compounds that inhibit expression of an MIP-3α gene or its gene product in the individual, or they may comprise an effective amount of one or more compounds that inhibit expression of a CCR6 gene or its gene product in the individual. Preferably, the MIP-3α polypeptide and its receptor are ones expressed in cartilage tissue of the individual being treated. Such polypeptides may therefore comprise, e.g., any of the MIP- 3α and CCR6 amino acid sequences described supra. In preferred embodiments, a pharmaceutical composition of the invention will also contain one or more physiologically acceptable carriers or excipients.

DETAILED DESCRIPTION OF THE INVENTION

Definitions The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.

General Definitions. As used herein, the term "isolated" means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism. An isolated material may be, but need not be, purified.

The term "purified" as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.

Methods for purification are well-known in the art. For example, nucleic acids can be purified by precipitation, chromatography (including preparative solid phase chromatography, oligonucleotide hybridization, and triple helix chromatography), ultracentrifugation, and other means. Polypeptides and proteins can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation and salting-out chromatography, extraction, and countercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence, or a sequence that specifically binds to an antibody, such as FLAG and GST. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents. Cells can be purified by various techniques, including centrifugation, matrix separation (e.g., nylon wool separation), panning and other immunoselection techniques, depletion (e.g., complement depletion of contaminating cells), and cell sorting (e.g., fluorescence activated cell sorting [FACS]). Other purification methods are possible. A purified material may contain less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated. The "substantially pure" indicates the highest degree of purity which can be achieved using conventional purification techniques known in the art.

A "sample" as used herein refers to a biological material which can be tested, e.g., for the presence of MIP-3α polypeptides, MIP-3α nucleic acids, CCR6 polypeptides, or CCR6 nucleic acids, e.g., to identify cells that specifically express the MIP-3α gene and its gene product. Such samples can be obtained from any source, including tissue. In preferred embodiments samples are obtained, e.g., in a biopsy, from patients having osteoarthritis or from tissue that is suspected of being arthritic. In one particularly preferred embodiment samples are obtained from cartilage of patients having or suspected of having OA.

For the purposes herein a "section" of a tissue sample is meant a single part or piece of a tissue sample, e.g., a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis according to the present invention, provided that it is understood that the present invention comprises a method whereby the same section of tissue sample may be analyzed at both morphological and molecular levels, or may be analyzed with respect to both protein and nucleic acid.

The term "molecule" means any distinct or distinguishable structural unit of matter comprising one or more atoms, and includes, for example, polypeptides and polynucleotides.

The term "therapeutically effective dose" refers to that amount of a compound or compositions that is sufficient to result in a desired activity. Thus, as used to describe a vaccine, a therapeutically effective dose refers to the amount of a compound or compositions (e.g., an antigen) that is sufficient to produce an effective immune response.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction (for example, gastric upset, dizziness and the like) when administered to an individual. Preferably, and particularly where a vaccine is used in humans, the term "pharmaceutically acceptable" may mean approved by a regulatory agency (for example, the U.S.

Food and Drug Agency) or listed in a generally recognized pharmacopeia for use in animals (for example, the U.S. Pharmacopeia).

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Exemplary suitable pharmaceutical carriers are described in "Reminington's Pharmaceutical Sciences" by E.W. Martin. As used herein "chemokine" refers to proteins which are proinflammatory cytokines that are chemoattractants and activators of specific types of leukocytes. Further details with respect to chemokine activity can be found, for example, in U.S. Patent No. 5,688,92 and Baggiolini et al, Advances in Immunology 1994, 55:97-179.

The term "cartilage," as used herein and as commonly understood in the art, refers to connective tissue in animals (including mammals, such as mice, rats, humans, etc.). Cartilage is characterized by an extracellular matrix that contains collagen type II and large amounts of proteoglycans, including aggrecan. The proteoglycans found in cartilage may be characterized as having attachment sites for glycosamino glycans (GAGs) including, e.g., chondroitin sulfate. Cartilage is produced by chondrocyte cells (also referred to herein as "chondrocytes"). These cells normally secrete the extracellular matrix of collagen and can be found within that matrix, e.g., in samples of collagen tissue.

Arthritis is a general term used to describe diseases, disorders and other conditions that are associated with the destruction of tissue, particularly articular cartilage (i.e., cartilage tissue surrounding the joints). Osteoarthritis (often indicated here by the abbreviation "OA") is a non-inflammatory degenerative joint disease that is characterized by the degredation of articular cartilage. By contrast, rheumatoid arthritis (often indicated herein by the abbreviation "RA") is an inflammatory conditions associated with the destruction of articular cartilage and other tissue surrounding the joints.

Molecular Biology Definitions. In accordance with the present invention, there may be employed conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook, Fitsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (referred to herein as "Sambrook et al, 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins, eds. 1984); Animal Cell Culture (R.I. Freshney, ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B.E. Perbal, A Practical Guide to Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

The term "polynucleotide" or "nucleic acid molecule" as used herein refers to a polymeric molecule having a backbone that supports bases capable of hydrogen bonding to typical polynucleotides, wherein the polymer backbone presents the bases in a manner to permit such hydrogen bonding in a specific fashion between the polymeric molecule and a typical polynucleotide (e.g., single-stranded DNA). Such bases are typically inosine, adenosine, guanosine, cytosine, uracil and thymidine. Polymeric molecules include "double stranded" and "single stranded" DNA and RNA, as well as backbone modifications thereof (for example, ethylphosphonate linkages).

Thus, a "polynucleotide" or "nucleic acid" sequence is a series of nucleotide bases (also called "nucleotides"), generally in DNA and RNA, and means any chain of two or more nucleotides. A nucleotide sequence frequently carries genetic information, including the information used by cellular machinery to make proteins and enzymes. The terms include genomic DNA, cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This includes single- and double-stranded molecules; i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example, thio-uracil, thio-guanine and fluoro- uracil.

The polynucleotides herein may be flanked by natural regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'-non-coding regions and the like. The nucleic acids may also be modified by many means known in the art. Non- limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.) and alkylators to name a few. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidite linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin and the like. Other non-limiting examples of modification which may be made are provided, below, in the description of the present invention.

A "polypeptide" is a chain of chemical building blocks called amino acids that are linked together by chemical bonds called "peptide bonds". The term "protein" refers to polypeptides that contain the amino acid residues encoded by a gene or by a nucleic acid molecule (e.g., an mRNA or a cDNA) transcribed from that gene either directly or indirectly. Optionally, a protein may lack certain amino acid residues that are encoded by a gene or by an mRNA. For example, a gene or mRNA molecule may encode a sequence of amino acid residues on the N-terminus of a protein (i.e., a signal sequence) that is cleaved from, and therefore may not be part of, the final protein. A protein or polypeptide, including an enzyme, may be a "native" or "wild-type", meaning that it occurs in nature; or it may be a "mutant", "variant" or "modified", meaning that it has been made, altered, derived, or is in some way different or changed from a native protein or from another mutant.

A "ligand" is, broadly speaking, any molecule that binds to another molecule. In preferred embodiments, the ligand is either a soluble molecule or the smaller of the two molecules or both. The other molecule is referred to as a "receptor". In preferred embodiments, both a ligand and its receptor are molecules (preferably proteins or polypeptides) produced by cells. Preferably, a ligand is a soluble molecule and the receptor is an integral membrane protein (i.e., a protein expressed on the surface of a cell).

The binding of a ligand to its receptor is frequently a step of signal transduction within a cell. Other exemplary ligand-receptor interactions include, but are not limited to, binding of a hormone to a hormone receptor (for example, the binding of estrogen to the estrogen receptor) and the binding of a neurotransmitter to a receptor on the surface of a neuron.

"Amplification" of a polynucleotide, as used herein, denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of PCR see Saiki et al, Science 1988, 239:487.

"Chemical sequencing" of DNA denotes methods such as that of Maxam and Gilbert (Maxam-Gilbert sequencing; see Maxam & Gilbert, Proc. Natl. Acad. Sci. U.S.A. 1977, 74:560), in which DNA is cleaved using individual base-specific reactions.

"Enzymatic sequencing" of DNA denotes methods such as that of Sanger (Sanger et al, Proc. Natl Acad. Sci. U.S.A. 1977, 74:5463) and variations thereof well known in the art, in a single-stranded DNA is copied and randomly terminated using DNA polymerase.

A "gene" is a sequence of nucleotides which code for a functional "gene product". Generally, a gene product is a functional protein. However, a gene product can also be another type of molecule in a cell, such as an RNA (e.g., a tRNA or a rRNA). For the purposes of the present invention, a gene product also refers to an mRNA sequence which may be found in a cell. For example, measuring gene expression levels according to the invention may correspond to measuring mRNA levels. A gene may also comprise regulatory ( .e., non-coding) sequences as well as coding sequences. Exemplary regulatory sequences include promoter sequences, which determine, for example, the conditions under which the gene is expressed. The transcribed region of the gene may also include untranslated regions including introns, a 5'-untranslated region (5'-UTR) and a 3'-untranslated region (3'-UTR).

A "coding sequence" or a sequence "encoding" an expression product, such as a RNA, polypeptide, protein or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein or enzyme; i.e., the nucleotide sequence "encodes" that RNA or it encodes the amino acid sequence for that polypeptide, protein or enzyme. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently found, for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

A coding sequence is "under the control of or is "operatively associated with" transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into RNA, which is then trans-RNA spliced (if it contains introns) and, if the sequence encodes a protein, is translated into that protein.

The term "express" and "expression" means allowing or causing the information in a gene or DNA sequence to become manifest, for example producing RNA (such as rRNA or mRNA) or a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed by a cell to form an "expression product" such as an RNA (e.g., a mRNA or a rRNA) or a protein. The expression product itself, e.g., the resulting RNA or protein, may also said to be "expressed" by the cell.

The term "transfection" means the introduction of a foreign nucleic acid into a cell. The term "transformation" means the introduction of a "foreign" (i.e., extrinsic or extracellular) gene, DNA or RNA sequence into a host cell so that the host cell will express the introduced gene or sequence to produce a desired substance, in this invention typically an RNA coded by the introduced gene or sequence, but also a protein or an enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a "cloned" or "foreign" gene or sequence, may include regulatory or control sequences (e.g., start, stop, promoter, signal, secretion or other sequences used by a cell's genetic machinery). The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been "transformed" and is a "transformant" or a "clone". The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell or cells of a different genus or species.

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors may include plasmids, phages, viruses, etc. and are discussed in greater detail below.

A "cassette" refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct." A common type of vector is a "plasmid," which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. The term "host cell" means any cell of any organism that is selected, modified, transformed, grown or used or manipulated in any way for the production of a substance by the cell. For example, a host cell may be one that is manipulated to express a particular gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays that are described infra. Host cells may be cultured in vitro or one or more cells in a non-human animal (e.g., a transgenic animal or a transiently transfected animal).

The term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells such as Sf9, Hi5 or S2 cells and Baculovirus vectors, Drosophila cells (Schneider cells) and expression systems and mammalian host cells and vectors.

The term "heterologous" refers to a combination of elements not naturally occurring. For example, the present invention includes chimeric RNA molecules that comprise an rRNA sequence and a heterologous RNA sequence which is not part of the rRNA sequence. In this context, the heterologous RNA sequence refers to an RNA sequence that is not naturally located within the ribosomal RNA sequence. Alternatively, the heterologous RNA sequence may be naturally located within the ribosomal RNA sequence, but is found at a location in the rRNA sequence where it does not naturally occur. As another example, heterologous DNA refers to DNA that is not naturally located in the cell, or in a chromosomal site of the cell. Preferably, heterologous DNA includes a gene foreign to the cell. A heterologous expression regulatory element is a regulatory element operatively associated with a different gene that the one it is operatively associated with in nature.

The terms "mutant" and "mutation" mean any detectable change in genetic material, e.g., DNA, or any process, mechanism or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., RNA, protein or enzyme) expressed by a modified gene or DNA sequence. The term "variant" may also be used to indicate a modified or altered gene, DNA sequence, RNA, enzyme, cell, etc.; i.e., any kind of mutant. For example, the present invention relates to altered or "chimeric" RNA molecules that comprise an rRNA sequence that is altered by inserting a heterologous RNA sequence that is not naturally part of that sequence or is not naturally located at the position of that rRNA sequence. Such chimeric RNA sequences, as well as DNA and genes that encode them, are also referred to herein as "mutant" sequences.

"Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. "Function-conservative variants" of a polypeptide or polynucleotide are those in which a given amino acid residue in the polypeptide, or the amino acid residue encoded by a codon of the polynucleotide, has been changed or altered without altering the overall conformation and function of the polypeptide. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the polypeptide. "Function- conservative variants" of a given polypeptide also include polypeptides that have at least 60% amino acid sequence identity to the given polypeptide as determined, e.g., by the BLAST or FASTA algorithms. Preferably, function-conservative variants of a given polypeptide have at least 75%, more preferably at least 85% and still more preferably at least 90% amino acid sequence identity to the given polypeptide and, preferably, also have the same or substantially similar properties (e.g., of molecular weight and/or isoelectric point) or functions (e.g., biological functions or activities) as the native or parent polypeptide to which it is compared. A functional conservative variant of a polypeptide may also share one or more conserved domains or sequence motifs with that polypeptide.

The term "homologous", in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a "common evolutionary origin", including proteins from superfamilies (e.g., the immunoglobulin superfamily) in the same species of organism, as well as homologous proteins from different species of organism (for example, myosin light chain polypeptide, etc.; see, Reeck et al, Cell 1987, 50:667). Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.

In specific embodiments, two nucleic acid sequences are "substantially homologous" or "substantially similar" when at least about 80%, and more preferably at least about 90% or at least about 95% of the nucleotides match over a defined length of the nucleic acid sequences, as determined by a sequence comparison algorithm known such as BLAST, FASTA, DNA Strider, CLUSTAL, etc.

Similarly, in particular embodiments of the invention, two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 80% of the amino acid residues are identical, or when greater than about 90% of the amino acid residues are similar (i.e., are functionally identical).

As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, preferably no more than 100 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with P-nucleotides or nucleotides to which a label, such as biotin or a fluorescent dye (for example, Cy3 or Cy5) has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of MIP-3α, or to detect the presence of nucleic acids encoding MIP-3α. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.

The present invention provides antisense nucleic acids which may be used to inhibit expression of a MDP-3α gene or its gene product. An "antisense nucleic acid" is a single stranded nucleic acid molecule which, on hybridizing with complementary bases in an RNA or DNA molecule, inhibits the latter's role. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a countertranscript or mRNA-interfering complementary nucleic acid. As presently used, "antisense" broadly includes RNA-RNA interactions, RNA-DNA interactions, triple helix interactions, ribozymes and RNase-H mediated arrest. Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. Patent No. 5,814,500; U.S. Patent No. 5,811,234), or alternatively they can be prepared synthetically (e.g., U.S. Patent No. 5,780,607). Other specific examples of antisense nucleic acid molecules of the invention are provided infra.

A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength isee Sambrook et al, supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T_m (melting temperature) of 55EC, can be used, e.g., 5x SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher T_m, e.g., 40% formamide, with 5x or 6x SSC. High stringency hybridization conditions correspond to the highest T_m, e.g., 50% formamide, 5x or 6x SSC. Ix SSC is solution containing 0.15M NaCl, 0.015M Na-citrate. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T_m) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T_m have been derived isee Sambrook et al, supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al, supra, 11.7-11.8). A minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.

In a specific embodiment, the term "standard hybridization conditions" refers to a T_m of 55°C, and utilizes conditions as set forth above. In a preferred embodiment, the T_m is 60°C; in a more preferred embodiment, the T_m is 65°C. In a specific embodiment, "high stringency" refers to hybridization and/or washing conditions at 68°C in 0.2x SSC, at 42°C in 50% formamide, 4x SSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.

As an alternative to antisense, sequence-specific degradation MIP-3α gene or its gene product with oligonucleotides can also be triggered by chemically synthesized short RNA duplexes by an RNA interference (RNAi) mechanism. RNA interference is a process of sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNA that is homologous in sequence to the silenced gene. In an analgous fashion to the antisense mechanism, the effect of introducing permutated base pairs in the short RNA duplexes, such that the oligonucleotides are no longer sequence identical to the target mRNA reveals that mismatches are ineffective inhibitors of the target mRNA. Representative examples are described in Elbashir et al., 2001, Nature 411, 494-498. Particularly advantageous are short double stranded RNAs having a length of 19 to 25 nucleotides. Most preferred are double stranded RNAs having a length of 21 to 23 nucleotides. The dsRNA may be blunt ended or ligated at or on at least one end with either loops composed of ribonucleotides or deoxyribonucleotides or a chemical synthetic linker (WO00/44895). In a preferred embodiment, the ribonucleic acid contains 3 '-end nucleotide overhangs on the antisense strand and / or the sense strands of the dsRNA of at least one ribonucleotide or deoxyribonucleotide, or modified nucleotide. Preferred are overhangs with 1 , 2, 3 or 4 nucleotides. The overhangs may contain both ribonucleotide(s) and deoxyribonucleotide(s) which in addition may contain modified sugar moieties. The overhang may be of any sequence, but in a preferred embodiment, the overhang is complementary to the target mRNA strand. In another preferred embodiment the overhang contains at least one UU group or dTdT group. In another preferred embodiment, the overhang on the antisense strand has the penultimate overhanging nucleotide complementary to the mRNA target strand. Preferably, such an overhang is a 2-nucleotides overhang. In a further preferred embodiment, the overhang is composed of 4 Us. In another preferred embodiment, the extreme 3 '-position of the siRNA is a hydroxyl group. Additionally, the 5'-end may be a hydroxyl or phosphate group.

MIP-3α Polypeptides

The present invention relates to a polypeptide, referred to herein as MIP-3α, that has been previously described, (see, for example, U.S. Patent No. 5,504,003). In one specific embodiment, a MIP-3α polypeptide of the invention is derived from a human cell or has an amino acid sequence of a polypeptide derived from a human cell. For example, a human MIP-3α polypeptide of the invention may comprise the amino acid sequence set forth in SEQ. ID. NO. 12 (Genbank Accession Number AAB61534).

MIP-3α polypeptides of the invention also include polypeptides comprising an amino acid sequence of one or more epitopes or domains of a full length MIP-3α polypeptide, such as epitopes or domains of the full length MIP-3α polypeptide set forth in SEQ ID NO. 12. An epitope of a MIP-3α polypeptide represents a site on the polypeptide against which an antibody may be produced and to which the antibody binds. Therefore, polypeptides comprising the amino acid sequence of a MJP-3α epitope are useful for making antibodies to a MIP-3α polypeptide. Preferably, an epitope comprises a sequence of at least 5, more preferably at least 10, 15, 20, 25, or 50 amino acid residues in length. Thus, polypeptides of the invention that comprise epitopes of a MJP-3α polypeptide preferably contain an amino acid sequence corresponding to at least 5, at least 10, at least 15, at least 20, at least 25 or at least 50 amino acid residues of a full length MJP-3α polypeptide sequence.

The MIP-3α polypeptides of the invention also include analogs and derivatives of the full length MIP-3α polypeptides (e.g., of SEQ ID NO. 12). Analogs and derivatives of the MIP-3α polypeptides of the invention have the same or homologous characteristics of MIP-3α polypeptides set forth above.

A MTP-3α chimeric or fusion polypeptide may also be prepared in which the MIP-3α portion of the fusion polypeptide has one or more characteristics of the MIP-3α polypeptide. Such fusion polypeptides therefore represent embodiments of the MTP-3α polypeptides of this invention. Such fusion polypeptides may also comprise the amino acid sequence of a marker polypeptide; for example FLAG, a histidine tag, glutathione S- transferase (GST), or Fc portion of an IgG. Additionally, MIP-3α fusion polypeptides may comprise amino acid sequences that increase solubility of the polypeptide, such as a thioreductase amino acid sequence or the sequence of one or more immunoglobulin proteins (e.g., IgGl or IgG2).

MIP-3α analogs or variants can also be made by altering encoding nucleic acid molecules, for example by substitutions, additions or deletions. Preferably such altered nucleic acid molecules encode functionally similar molecules (i.e., molecules that perform one or more MTP-3α functions or have one or more MIP-3α bioactivities).

Amino acid residues, other than ones that are specifically identified herein as being conserved, may differ among variants of a protein or polypeptide. Accordingly, the percentage of protein or amino acid sequence similarity between any two MJP-3α polypeptides of similar function may vary. Typically, the percentage of protein or amino acid sequence similarity between different MJP-3α polypeptide variants may be from 70% to 99%, as determined according to an alignment scheme such as the Cluster Method and/or the MEGALIGN or GCG alignment algorithm. Function-conservative variants of the present invention, as defined above, include, not only variants of the full length MIP-3α polypeptides of the invention (e.g., variants of a polypeptide comprising the sequence set forth in SEQ ID NO. 12, but also include function-conservative variants of modified MIP-3α polypeptides (e.g., truncations and deletions) and of fragments (e.g., corresponding to domains or epitopes) of full length MB°-3α polypeptides.

In yet other embodiments, an analog of a MIP-3α polypeptide is an allelic variant or mutant of a MDP-3α polypeptide. The terms allelic variant and mutant, when used herein to describe a polypeptide, refer to a polypeptide encoded by an allelic variant or mutant gene. Thus, the allelic variant and mutant MIP-3α polypeptides of the invention are polypeptides encoded by allelic variants or mutants of the MTP-3α nucleic acid molecules of the present invention.

In yet other embodiments, an analog of a MTP-3α polypeptide is a substantially homologous polypeptide from the same species (e.g., allelic variants) or from another species (e.g., an orthologous polypeptide). For example, MIP-3α homologs and orthologs have been identified in mammals such as humans, mice, rats and hamsters. MIP-3α homologs of the invention may, however, be from any species of animal, including other species of mammals (e.g., rabbit, guinea pig, dog, cat, sheep, goat, pig, horse, and cow to name a few).

While the above exemplary variants of a MJP-3α polypeptide are described in terms of the MIP-3α polypeptide set forth in SEQ ID NO. 12, it is understood that variant MIP-3α polypeptides of the invention include other MTP-3oc polypeptides having equivalent amino acid substitutions, deletions or insertions. For example, the variant MTP-3α polypeptides of the invention also include fragments of the full length MTP-3α polypeptide set forth in SEQ ID NO. 12 that have one or more of the amino acid substitutions, deletions or insertions described above for the full length MIP-3α polypeptide.

Other homologous or variant MTP-3α polypeptide sequences include allelic or species variants of the specific MIP-3α polypeptide sequence set forth in SEQ ID NO. 12. Sequences that are substantially homologous can be readily identified by comparing the sequences using standard software packages available in sequence data banks, including the BLAST algorithms (e.g., BLASTP, BLASTN, BLASTX), FASTA, DNA Strider, the GCG pileup program, CLUSTAL, and other such programs which are known in the art or are described herein.

In other embodiments, variants of a MIP-3α polypeptide (including analogs and homologs) are polypeptides encoded by nucleic acid molecules that hybridize to the complement of a nucleic acid molecule encoding a MIP-3α polypeptide (e.g., in a Southern hybridization experiment under defined conditions). For example, in a particular embodiment analogs and/or homologs of a MIP-3α polypeptide comprise amino acid sequences encoded by nucleic acid molecules that hybridize to a complement of a MTP-3α nucleic acid sequence, such as the coding sequence set forth in SEQ ID NO. 11 according to the definition of hybridization described supra.

In still other embodiments, variants (including analogs, homologs and orthologs) of a MTP-3α polypeptide can also be identified by isolating variant MIP-3α genes, e.g., by PCR using degenerate oligonucleotide primers designed on the basis of amino acid sequences of the MTP-3α polypeptide and as described below.

Derivatives of the MJP-3α polypeptides of the invention further include, but are by no means limited to, phosphorylated MTP-3α, myristylated MTP-3α, methylated MIP-3oc and other MIP-3α polypeptides that are chemically modified. MTP-3α polypeptides of the invention also include labeled variants; for example, radio-labeled with iodine or phosphorous (see, e.g., EP 372707B) or other detectable molecule such as, but by no means limited to, biotin, a fluorescent dye (e.g., Cy5 or Cy3), a chelating group complexed with a metal ion, a chromophore or fluorophore, a gold colloid, a particle such as a latex bead, or attached to a water soluble polymer.

Chemical modification of a biologically active component or components of MIP-3α nucleic acids or polypeptides may provide additional advantages under certain circumstances. See, for example, U.S. Patent No. 4,179,337 issued December 18, 1970 to Davis et al. Also, for a review see Abuchowski et al, in Enzymes as Drugs (J.S. Holcerberg and J. Roberts, eds. 1981), pp. 367-383. A review article describing protein modification and fusion proteins is also found in Fracis, Focus on Growth Factors 1992, 3:4-10, Mediscript: Mountview Court, Friern Barnet Lane, London N20, OLD, UK.

MIP-3α Nucleic Acids

In general, a MTP-3α nucleic acid molecule of the present invention comprises a nucleic acid sequence that encodes a MJP-3α polypeptide as defined, supra, the complement of a nucleic acid sequence that encodes a MIP-3α polypeptide, and fragments thereof. Thus, in one preferred embodiment the MTP-3α nucleic acid molecules of the invention comprise a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO. 12. In a particularly preferred aspect of this embodiment, the MIP-3α nucleic acid has a nucleotide sequence that comprises the coding portion (i.e., an open reading frame or "ORF") of the nucleotide sequence set forth in GenBank Accession No. U64197.1 (GI: 1778716) and provided here at SEQ ID NO: 11.

In still other embodiments, the MIP-3α nucleic acid molecules of the invention comprise nucleic acid sequences that encode one or more domains of a MTP-3α polypeptide.

The MIP-3α nucleic acid molecules of the invention also include nucleic acids which comprise a sequence encoding one or more fragments of a MIP-3α polypeptide sequence (e.g., of the polypeptide sequence set forth in SEQ ID NO. 12). The MJP-3α nucleic acid molecules of the invention also include nucleic acid molecules that comprise coding sequences for modified MTP-3α polypeptides (e.g., having amino acid substitutions, deletions or truncations) and for variants (including allelic variants, analogs and homologs from the same or different species) of MTP-3α polypeptides. In preferred embodiments, such nucleic acid molecules have at least 50%, preferably at least 75% and more preferably at least 90% sequence identity to a MIP-3α coding sequence (e.g., to the MTP-3α coding sequence set forth in SEQ ID NO. 11).

In addition, the MIP-3α nucleic acid molecules of the invention may also be ones that hybridize to a MTP-3α nucleic acid molecule, e.g., in a Southern blot assay under defined conditions. For example, in specific embodiments a MIP-3α nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to a complement of a MEP-3α nucleic acid sequence, such as the coding sequence set forth in SEQ ID NO. 11 under any of the hybridization conditions described above. Alternatively, a nucleic acid molecule of the invention may hybridize, under the same defined hybridization conditions, to the complement of a fragment of a nucleotide sequence encoding a full length MTP-3α polypeptide.

In other embodiments, the nucleic acid molecules of the invention comprise fragments of a full length MIP-3α nucleic acid sequence. Such MIP-3α nucleic acid fragments comprise a nucleotide sequence that corresponds to a sequence of at least 10 nucleotides, preferably at least 15 nucleotides and more preferably at least 20 nucleotides of a full length coding MIP-3α nucleotide sequence. In specific embodiments, the fragments correspond to a portion (e.g., of at least 10, 15 or 20 nucleotides) of the MIP-3α coding sequence set forth in SEQ ED NO. 11. In other preferred embodiments, the MIP-3α nucleic acid fragments comprise sequences of at least 10, preferably at least 15, and more preferably at least 20 nucleotides that are complementary and/or hybridize to a full length coding MJP-3α nucleic acid sequence (e.g., in the sequence set forth in SEQ ID NO. 1 1) or to a fragment thereof. Suitable hybridization conditions for such oligonucleotides are described supra. Nucleic acid molecules comprising such fragments are useful, for example, as oligonucleotide probes and primers (e.g., PCR primers) to detect and amplify other nucleic acid molecules encoding a MJP-3α polypeptide, including genes the encode variant MD°-3α polypeptides such as MJP-3α analogs and homologs. Oligonucleotide fragments of the invention may also be used, e.g., as antisense nucleic acids to modulate levels of MTP-3α gene expression or transcription in cells.

The nucleic acid molecules of the invention also include "chimeric" MJP-3a nucleic acid molecules. Such chimeric nucleic acid molecules are polynucleotides which comprise at least one MP-3α nucleic acid sequence (which may be any of the full length or partial MJP-3α nucleic acid sequences described above), and also at least one non-MJP-3α nucleic acid sequence. For example, the non-MJP-3α nucleic acid sequence may be a heterologous regulatory sequence (for example a promoter sequence) that is derived from another, non-MIP-3α gene and is not normally associated with a naturally occurring MTP-3α gene. The non-MIP-3α nucleic acid sequence may also be a coding sequence of another, non- MTP-3α polypeptide such as FLAG, a histidine tag, glutathione S-transferase (GST), hemaglutinin, β-galactosidase, thioreductase or an immunoglobulin domain or domains (for examples, an Fc region). In preferred embodiments, a chimeric nucleic acid molecule of the invention encodes a MIP-3α fusion polypeptide of the invention.

MTP-3α nucleic acid molecules of the invention, whether genomic DNA, cDNA or otherwise, can be isolated from any source including, for example, cDNA or genomic libraries derived from a cell or cell line from an organism that has a MTP-3α gene. In the case of cDNA libraries, such libraries are preferably derived from a cell or cell line that expresses a MJP-3α gene. Methods for obtaining MIP-3α genes are well known in the art, as described above (see, e.g., Sambrook et al, 1989, supra).

The DNA may be obtained by standard procedures known in the art from cloned DNA (for example, from a DNA "library"), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein. In one preferred embodiment, the DNA is obtained from a "subtraction" library to enrich the library for cDNAs of genes specifically expressed by a particular cell type or under certain conditions. Use of such a subtraction library may increase the likelihood of isolating cDNA for a particular gene, such as MTP-3α. In still other embodiments, a library may be prepared by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA or fragments thereof purified from the desired cell (See, for example, Sambrook et al, 1989, supra; Glover, D.M. ed., 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd. Oxford, U.K. Vols. I and II).

In one embodiment, a cDΝA library may be screened for a MJP-3α nucleic acid by identifying cDΝA inserts that encode a polypeptide which is homologous or substantially similar to a MJP-3α polypeptide, such as the polypeptide set forth in SEQ ID NO. 12 or a fragment thereof. Similarly, a cDNA library may be screened for a MJP-3α nucleic acid by identifying cDNA inserts having a nucleic acid sequence that is homologous or substantially similar to a MIP-3α nucleic acid sequence, such as the nucleic acid sequence set forth in SEQ ID NO. 1 1 or a fragment thereof.

Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions. Clones derived from cDNA generally will not contain intron sequences. Whatever the source, the gene is preferably molecularly cloned into a suitable vector for propagation of the gene. Identification of the specific DNA fragment containing the desired MJP-3α gene may be accomplished in a number of ways. For example, a portion of a MTP-3α gene can be purified and labeled to prepare a labeled probe (Benton & Davis, Science 1977, 196: 180; Grunstein & Hogness, Proc. Natl. Acad. Sci. U.S.A. 1975, 72:3961). Those DNA fragments with substantial homology to the probe, such as an allelic variant from another individual, will hybridize. In a specific embodiment, highest stringency hybridization conditions are used to identify a homologous MJP-3α gene.

The genes encoding MTP-3α derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned MTP-3α gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al, 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of MIP-3α, care should be taken to ensure that the modified gene remains within the same translational reading frame as the MIP-3α gene, uninterrupted by translational stop signals, in the gene region where the desired activity is encoded.

Additionally, the MJP-3α-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Modifications can also be made to introduce restriction sites and facilitate cloning the MIP-3α gene into an expression vector. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C, et al, J. Biol. Chem. 253:6551, 1978; Zoller and Smith, DNA 3:479-488, 1984; Oliphant et al, Gene 44: 177, 1986; Hutchinson et al, Proc. Natl. Acad. Sci. U.S.A. 83:710, 1986), use of TAB^" linkers (Pharmacia Corp., Peapack, NJ), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, pKK plasmids (Clonetech, Palo Alto, CA), pET plasmids (Novagen, Inc., Madison, WI), pRSET or pREP plasmids, pcDNA (Invitrogen, Carlsbad, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), etc. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini. These ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated. Preferably, the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, a shuttle vector, which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences from the yeast 2m plasmid.

CCR6 Polypeptides

The present invention also relates to a polypeptide, referred to herein as CCR6, that has been previously described (Varona et al, FEBS Lett. 1998; 440(1-2): 188-94). CCR6 is the receptor for Exodus- 1 or MIP-3α. In one specific embodiment, a CCR6 polypeptide of the invention is derived from a human cell or has an amino acid sequence of a polypeptide derived from a human cell. For example, a human CCR6 polypeptide of the invention may comprise the amino acid sequence set forth in GenBank Accession No. XP_033840.1 (GI No. 14755138) and provided here at SEQ. ID. NO. 14.

CCR6 polypeptides of the invention also include, in other embodiments, variants, including (homologs, orthologs, derivatives, mutants, and chimeric or fusion polypeptides) of the CCR6 amino acid sequence set forth in SEQ ID NO. 14, as well as fragments and truncated forms of those amino acid sequences. Such CCR6 variants, fragments, truncated forms etc. are defined as provided above for MJP-3cc.

CCR6 Nucleic Acids

A CCR6 nucleic acid molecule of the present invention comprises a nucleic acid sequence that encodes a CCR6 polypeptide as defined, supra, the complement of a nucleic acid sequence that encodes a CCR6 polypeptide, and fragments thereof. Thus, in one preferred embodiment the CCR6 nucleic acid molecules of the invention comprise a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO. 14. Such sequences are known in the art and include, e.g., the nucleotide sequence set forth in GenBank Accession No. XM_033840.1 (GI No. 14755137), which is provided here at SEQ ID NO: 13.

CCR6 nucleic acids of the invention also include, in other embodiments, nucleic acids that encode a variant, fragmented or truncated CCR6 polypeptide as described, supra. For example, the CCR6 nucleic acid molecule of this invention include variant (e.g. orthologs or homologs) nucleic acid sequences that hybridize to SEQ ID NO. 13 (or its equivalent) under conditions such as the ones defined above for MIP-3α. Alternatively, variant CCR6 nucleic acids of the invention may share a specified level of nucleotide sequence identity (e.g. as specified supra for MIP-3α variants) with the CCR6 nucleotide sequence set forth in SEQ ID NO. 13). The invention provides still other CCR6 variants, fragments, truncated forms, etc. that are defined as provided above for MIP-3α.

Expression of MJP-3α and CCR6 Polypeptides

A nucleotide sequence coding for MJP-3α or CCR6 , for antigenic fragments, derivatives or analogs of MTP-3cc or CCR6, or for functionally active derivatives of MIP-3 or CCR6 (including a chimeric protein) may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Thus, a nucleic acid encoding an MIP-3α or CCR6 polypeptide of the invention can be operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences. Such vectors can be used to express functional or functionally inactivated MJP-3α or CCR6 polypeptides.

The necessary transcriptional and translational signals can be provided on a recombinant expression vector.

Potential host-vector systems include but are not limited to mammalian or other vertebrate cell systems transfected with expression plasmids or infected with virus (e.g., vaccinia virus, adenovirus, adeno-associated virus, he es virus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used.

Expression of a MJP-3α protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control MTP-3α gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist and Chambon, Nature 1981, 290:304- 310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, Cell 1980, 22:787-797), the herpes thymidine kinase promoter (Wagner et al, Proc. Natl. Acad. Sci. U.S.A. 1981, 78: 1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al, Nature 1982, 296:39-42); prokaryotic expression vectors such as the b- lactamase promoter (Villa-Komaroff, et al, Proc. Natl Acad. Sci. U.S.A. 1978, 75:3727- 3731), or the tac promoter (DeBoer, et al, Proc. Natl. Acad. Sci. U.S.A. 1983, 80:21-25, 1983); see also "Useful proteins from recombinant bacteria" in Scientific American 1980, 242:74-94. Still other useful promoter elements which may be used include promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and transcriptional control regions that exhibit hematopoietic tissue specificity, in particular: beta-globin gene control region which is active in myeloid cells (Mogra et al. , Nature 1985, 315:338-340; Kollias et al, Cell 1986, 46:89-94), hematopoietic stem cell differentiation factor promoters, erythropoietin receptor promoter (Maouche et al , Blood 1991, 15:2557), etc.

In another embodiment, the invention provides methods for expressing MIP-3α or CCR6 polypeptides by using a non-endogenous promoter to control expression of an endogenous MIP-3α or CCR6 genes within a cell. Endogenous MTP-3α or CCR6 genes within a cell are MTP-3α or CCR6 gene of the present invention which are ordinarily (i.e., naturally) found in the genome of that cell. A non-endogenous promoter, however, is a promoter or other nucleotide sequence that may be used to control expression of a gene but is not ordinarily or naturally associated with the endogenous MTP-3α or CCR6 gene. As an example, methods of homologous recombination may be employed (preferably using non- protein encoding MJP-3oc or CCR6 nucleic acid sequences of the invention) to insert an amplifiable gene or other regulatory sequence in the proximity of an endogenous MJP-3α or CCR6 gene. The inserted sequence may then be used, e.g., to provide for higher levels of MD°-3α or CCR6 gene expression than normally occurs in that cell, or to overcome one or more mutations in the endogenous MIP-3α or CCR6 regulatory sequences which prevent normal levels of MJP-3α or CCR6 gene expression. Such methods of homologous recombination are well known in the art. See, for example, International Patent Publication No. WO 91/06666, published May 16, 1991 by Skoultchi; International Patent Publication No. WO 91/099555, published July 11, 1991 by Chappel; and International Patent Publication No. WO 90/14092, published November 29, 1990 by Kucheriapati and Campbell.

Soluble forms of the protein can be obtained by collecting culture fluid, or solubilizing inclusion bodies, e.g., by treatment with detergent, and if desired sonication or other mechanical processes, as described above. The solubilized or soluble protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-dimensional gel electrophoresis, chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.

Preferred vectors are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, and other recombinant viruses with desirable cellular tropism. Thus, a gene encoding a functional or mutant MTP-3α or CCR6 protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both.

Antibodies to MJP-3α and CCR6

Antibodies to MTP-3α and/or CCR6 are useful, inter alia, for diagnostic and therapeutic methods, as set forth below. According to the invention, MIP-3α or CCR6 polypeptides produced, e.g., recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as an immunogen to generate antibodies that recognize the MIP-3α or CCR6 polypeptides. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. Such an antibody is preferably specific for (i.e., specifically binds to) a human MJP-3α or CCR6 polypeptide of the present invention. However, the antibody may, alternatively, be specific for a MIP-3α or CCR6 ortholog from some other species of organism, preferably another species of mammal such as mouse, rat or hamster, to name a few. The antibody may recognize wild-type or mutant forms of MTP-3α or CCR6, or both.

Various procedures known in the art may be used for the production of polyclonal antibodies to MB°-3α or CCR6 polypeptides or derivatives or analogs thereof. For the production of antibody, various host animals can be immunized by injection with the MIP-3α or CCR6 polypeptides, or derivatives (e.g., fragments or fusion proteins) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, the MIP-3α or CCR6 polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG ibacille Calmette-Guerin) and Corynebacterium parvum. For preparation of monoclonal antibodies directed toward the MIP-3α or CCR6 polypeptides, or fragments, analogs, or derivatives thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature 1975, 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today 1983, 4:72; Cote et al, Proc. Natl. Acad. Sci. U.S.A. 1983, 80:2026-2030), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (International Patent Publication No. WO 89/12690). In fact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al, J. Bacteriol 1984, 159:870; Neuberger et al, Nature 1984, 312:604-608; Takeda et al, Nature 1985, 314:452-454) may also be used. Briefly, such techniques comprise splicing the genes from an antibody molecule from a first species of organism (e.g., a mouse) that is specific for a MIP-3α polypeptide together with genes from an antibody molecule of appropriate biological activity derived from a second species of organism (e.g., from a human). Such chimeric antibodies are within the scope of this invention.

Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

According to the invention, techniques described for the production of single chain antibodies (U.S. Patent Nos. 5,476,786, 5,132,405, and 4,946,778) can be adapted to produce MTP-3α polypeptide-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al, Science 1989, 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for either an MJP-3α or CCR6 polypeptide, or its derivatives, or analogs.

In the production and use of antibodies, screening for or testing with the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

Commercial antibodies to MTP-3α and CCR6 that can be used to practice the invention are available from several sources, including R&D Systems, Minneapolis, MN (see the Examples, infra).

The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the MJP-3α or CCR6 polypeptides, e.g., for Western blotting, imaging MTP-3α or CCR6 polypeptides in situ, measuring levels thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art. Such antibodies can also be used in assays for ligand binding, e.g., as described in US Patent No. 5,679,582. Antibody binding generally occurs most readily under physiological conditions, e.g., pH of between about 7 and 8, and physiological ionic strength. The presence of a carrier protein in the buffer solutions stabilizes the assays. While there is some tolerance of perturbation of optimal conditions, e.g., increasing or decreasing ionic strength, temperature, or pH, or adding detergents or chaotropic salts, such perturbations generally decrease binding stability. In still other embodiments, anti-MJJP-3α or CCR6 antibodies may also be used to isolate cells which express a MIP-3α or CCR6 polypeptides by panning or related immunoadsorption techniques.

In a specific embodiment, antibodies that agonize or antagonize the activity of a MIP-3α or CCR6 polypeptides can be generated. In particular, intracellular single chain Fv antibodies can be used to regulate (inhibit) MJP-3α activity (Marasco et al, Proc. Natl. Acad. Sci. U.S.A. 1993, 90:7884-7893; Chen., Mol Med. Today 1997, 3:160-167; Spitz et al, Anticancer Res. 1996, 16:3415-22; Indolfi et al, Nat. Med. 1996, 2:634-635; Kijma et α/., Pharmacol Ther. 1995, 68:247-267). Such antibodies can be tested using the assays described infra for identifying ligands.

Applications and Uses

Described herein are various novel applications and uses for the MTP-3α and CCR6 gene and gene products, including novel applications and uses for MIP-3α and CCR6 nucleic acids (described above), for MJP-3α polypeptides (described, supra) and for antibodies directed against such MJJP-3α and CCR6 polypeptides and nucleic acids (described supra). Applicants have discovered that the MIP-3α gene and gene product is expressed at elevated levels in cells derived from patients with OA compared with healthy subjects. Levels of MJP-3α gene or gene product expression may be similarly elevated in other types of diseased tissue, as discussed in detail infra.

For example, the data presented in the Examples, infra, shows that MIP-3α polypeptide is associated with cartilage tissue of patients with OA. Indeed, MIP-3α protein is found in normal cartilage tissue only at very low or undetectable levels. Previous work by other researchers has found elevated levels of MJP-3α in synovium and tissue of patients with rheumatoid arthritis, but failed to observe MTP-3α in tissues from patients with OA (Matsui et al Cain. Exp. Immunol. 2001, 125(1): 155). As a result of this unexpected discovery, Applicants have determined that the MIP-3α gene and its gene product may be used as a tissue specific marker to detect and/or identify OA cartilage or tissue. Accordingly, the MJP- 3α nucleic acids and polypeptides described supra may actually be used in methods for detecting OA, e.g., in diagnostic and prognostic applications by using the MTP-3α gene or its gene product to detect MTP-3α expression in a sample such as a tissue sample (e.g., from a biopsy) from an individual. Methods are provided herein that use MIP-3α nucleic acids and polypeptides to detect cartilage degradation, such as degradation that is associated with OA and other arthritic conditions.

The MTP-3α gene and its gene product may also be used in therapeutic regimes, e.g., to treat individuals suffering from OA.

In addition, the MJP-3α gene and its gene product as well as the CCR6 gene and its gene product may serve as drug targets for the development of therapeutics for use in the treatment of individuals suffering from OA. Methods are provided that use MIP-3α nucleic acids and/or polypeptides to screen for compounds, including candidate therapeutic compounds such as drugs, that may be used to treat or prevent cartilage degradation and/or conditions such as OA. For instance, methods are described, infra, which use compounds that interfere with or modulate binding of MIP-3α to a CCR6 receptor. In other embodiments, such methods may use compounds that modulate downstream signaling events from the binding of MIP-3α to a CCR6 receptor. Such compounds may be readily identified by persons skilled in the art, for example by using the screening methods of this invention.

Screening Assays. Using screening assays such as those described below, it is possible to identify compounds that bind to or otherwise interact with MJP-3α and/or CCR6 gene products, including intracellular compounds (for example, proteins or portions of proteins) that interact with MTP-3α and/or CCR6 gene products, natural and synthetic ligands or receptors for an MIP-3α or CCR6 gene product, compounds that interfere with the interaction of a MIP-3α gene product (for example, compounds that interfere with specific binding of a MTP-3α gene product to CCR6 or another receptor), and compounds that modulate the activity of a MJP-3(X gene (for example, by modulating the level of MJP-3oc gene expression) or the activity (for example, the bioactivity) of a MTP-3α polypeptide or other MIP-3α gene products and their receptor (e.g., CCR6). The screening assays of this invention may therefore be used to identify compounds that specifically bind to a MJP-3α gene or gene product to modulate MIP-3α expression. For example, the screening assays described here may therefore be used to identify compounds that bind to a promoter or other regulatory sequence of a MJP-3α gene, and so may modulate the level of MTP-3α gene expression (see, for example, Platt, J. Biol. Chem. 1994, 269:28558-28562). The screening assays may also be used to identify compounds that bind to and thereby stabilize a MTP-3α nucleic acid or polypeptide. In addition, these screening assays may be used to identify compounds that inhibit or modulate such binding interactions and which are therefore useful, e.g., as agonists or antagonists for MIP-3α binding to a specific transcription factor or enhancer, or for MTP-3α binding to a stabilizer. Compounds identified in these or similar screening assays may therefore be used to treat diseases and disorders that are associated with abnormal MTP-3α expression and/or abnormal levels of MIP-3α polypeptides or nucleic acids, including, but not limited to, OA.

Classes of compounds that may be identified by such screening assays include, but are not limited to, small molecules (e.g., organic or inorganic molecules which are less than about 2 kDa in molecular weight, are more preferably less than about 1 kDa in molecular weight, and/or are able to cross the blood-brain barrier or gain entry into an appropriate cell and affect expression of either a MIP-3α gene or of some gene involved in a MIP-3α regulatory pathway) as well as macromolecules (e.g., molecules greater than about 2 kDa in molecular weight). Compounds identified by these screening assays may also include nucleic acids, peptides and polypeptides. Examples of such compounds (including peptides) include but are not limited to: soluble peptides; fusion peptide members of combinatorial libraries (such as ones described by Lam et al, Nature 1991, 354:82-84; and by Houghten et al, Nature 1991, 354:84-86); members of libraries derived by combinatorial chemistry, such as molecular libraries of D- and/or L-configuration amino acids; phosphopeptides, such as members of random or partially degenerate, directed phosphopeptide libraries (see, e.g., Songyang et al, Cell 1993, 72:767-778); antibodies, including but not limited to polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies; antibody fragments, including but not limited to Fab, F(ab')₂, Fab expression library fragments, and epitope-binding fragments thereof. Nucleic acids used in these screening assays may be DNA or RNA, or synthetic nucleic acids (described in the Definitions, supra). Particular examples include, but are by no means limited to, antisense nucleic acids and ribozymes, as well as double-stranded and triple helix nucleic acid molecules.

Assays for binding compounds. In vitro systems can be readily designed to identify compounds capable of binding the MJP-3α gene products of the present invention. Such compounds can be useful, for example, in modulating the expression, stability or activity of a wild-type MEP-3α gene product or, alternatively, to modulate the expression, stability or activity of a mutant or other variant MIP-3α gene product.

Generally, such screening assays involve preparation of a reactive mixture comprising a MTP-3α gene product and a test compound under conditions and for a time sufficient to allow the two compounds to interact (e.g., bind), thereby forming a complex that may be detected. The assays may be conducted in any of a variety of different ways. For example, one embodiment comprises anchoring a MTP-3α polypeptide or a test compound onto a solid phase and detecting complexes of the MIP-3α polypeptide and the test compound that are on the solid phase at the end of the reaction and after removing (e.g., by washing) unbound compounds. For example, in one preferred embodiment of such a method, a MTP-3α gene product may be anchored onto a solid surface and a labeled compound (e.g., labeled according to any of the methods described supra) is contacted to the surface. After incubating the test compound for a sufficient time and under sufficient conditions that a complex may form between the MJP-3α gene product and the test compound, unbound molecules of the test compound are removed from the surface (e.g., by washing) and labeled molecules which remain are detected.

In another, alternative embodiment, molecules of one or more different test compounds are attached to the solid phase and molecules of a labeled MTP-3α polypeptide may be contacted thereto. In such embodiments, the molecules of different test compounds are preferably attached to the solid phase at a particular location on the solid phase so that test compounds that bind to a MTP-3α polypeptide may be identified by determining the location of bound MIP-3α polypeptides on the solid phase or surface. Assaysfor compounds that interact with MJP -3a Any of a variety of known methods for detecting protein-protein interactions may also be used to detect and/or identify proteins that interact with a MIP-3α and CCR6 gene products. For example, co- immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns as well as other techniques known in the art may be employed. Proteins which may be identified using such assays include, but are not limited to, extracellular proteins, such as MJP-3α specific receptors and CCR6 ligands, as well as intracellular proteins such as signal transducing proteins.

Compounds, including other cellular proteins and nucleic acids, that interact with MIP-3α and/or CCR6 may themselves be used in the methods of this invention, e.g., to modulate MIP-3α or CCR6 activity and to treat or prevent cartilage degradation. Alternatively, such interacting compounds may, themselves, be used in the screening assays of this invention to identify other compounds that modulate MJP-3α activity (e.g., binding to a CCR6 receptor and downstream signal events resulting therefrom) and could, in turn, be used to treat or prevent cartilage degradation.

As an example, and not by way of limitation, an expression cloning assay may be used to identify MTP-3α specific receptors and other proteins that specifically interact with a MIP-3α gene product. In such assays, a cDNA expression library may be generated from any cell line that expresses a MB°-3α specific receptor, e.g., CCR6, (for example, leukocyte cells, such as monocytes, B lymphocytes and T lymphocytes, and dendritic cells). Clones from such an expression library may then be transfected or infected into cells that do not normally express a MJP-3α specific receptor. Cells that are transfected with a clone that encodes a MJP-3α specific receptor may then express this gene product, and can be identified and isolated using standard techniques such as FACS or using magnetic beads that have a MTP-3α polypeptide (for example, a MJP-3α-Fc fusion polypeptide) attached thereto.

Alternatively, a MJP-3α specific receptors or ligands may be isolated from a cell line, including any of the MIP-3α receptor expressing cell lines as described above, using immunoprecipitation techniques that are well known in the art. MIP-3α specific receptors and/or ligands may also be isolated using any of the screening assays discussed, supra for identifying MTP-3α binding compounds. For example, a MTP-3α-Fc fusion polypeptide may be bound or otherwise attached to a solid surface, and a labeled compound (e.g., a candidate MIP-3α receptor or ligand) may be contacted to the surface for a sufficient time and under conditions that permit formation of a complex between the MTP-3α-Fc fusion polypeptide and the test compound. Unbound molecules of the test compound can then be removed from the surface (e.g., by washing), and labeled compounds that remain bound can be detected.

Once so isolated, standard techniques may be used to identify any protein detected in such assays. For example, at least a portion of the amino acid sequence of a protein that interacts with the MJP-3α gene product can be ascertained using techniques well known in the art, such as the Edman degradation technique (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman&Co., New York, pages 34-49).

Once such proteins have been identified, their amino acid sequence may be used as a guide for the generation of oligonucleotide mixtures to screen for gene sequences encoding such proteins; e.g., using standard hybridization or PCR techniques described supra. See, for example, Ausubel supra; and PCR Protocols: A Guide to Methods and Applications, Innis et al, eds., Academic Press, Inc., New York (1990) for descriptions of techniques for the generation of such oligonucleotide mixtures and their use in screening assays.

Other methods are known in the art which result in the simultaneous identification of genes that encode a protein that interacts with a MJP-3α polypeptide. For example, expression libraries may be probed with a labeled MJP-3α polypeptide.

As another example and not by way of limitation, a two-hybrid system may be used to detect protein interactions with a MIP-3α gene product in vivo. Briefly, utilizing such a system, plasmids may be constructed which encode two hybrid proteins, one of which preferably comprises of the DNA-binding domain of a transcription activator protein fused to a MIP-3α gene product. The other hybrid protein preferably comprises an activation domain of the transcription activator protein used in the first hybrid, fused to an unknown protein that is encoded by a cDNA recombined into the plasmid library as part of a cDNA library. Both the DNA-binding domain fusion plasmid and the cDNA library may be co-transformed into a strain of Saccharomyces cerevisiae or other suitable organism which contains a reporter gene (for example, HBS, lacZ, HIS3 or GFP). Preferably, the regulatory region of this reporter gene comprises a binding site for the transcription activator moiety of the two hybrid proteins. In such a two-hybrid system, the presence of either of the two hybrid proteins alone cannot activate transcription of the reporter gene. Specifically, the DNA-binding domain hybrid protein cannot activate transcription because it cannot localize to the necessary activation function. Likewise, the activation domain hybrid protein cannot activate transcription because it cannot localize to the DNA binding site on the reporter gene. However, interaction between the two hybrid proteins, reconstitutes that functional transcription activator protein and results in expression of the reporter gene. Thus, in a two-hybrid system such as the one described here in detail, an interaction between a MIP-3α polypeptide (i.e., the MTP-3α polypeptide fused to the transcription activator's DNA binding domain) and a test polypeptide ( .e., a protein fused to the transcription activator's DNA binding domain) may be detected by simply detecting expression of a gene product of the reporter gene. cDNA libraries for screening in such two-hybrid and other assays may be made according to any suitable technique known in the art. As a particular and non-limiting example, cDNA fragments may be inserted into a vector so that they are translationally fused to the transcriptional activation domain of GAL4, and co-transformed along with a "bait" MJP-3α-GAL4 fusion plasmid into a strain of Saccharomyces cerevisiae or other suitable organism that contains a HIS 3 gene driven by a promoter that contains a GAL4 activation sequence. A protein from this cDNA library, fused to the GAL4 transcriptional activation domain, which interacts with the MJP-3α polypeptide moiety of the MTP-3α-GAL4 fusion will reconstitute and active GAL4 protein, and can thereby drive expression of the HIS3 gene. Colonies that express the HIS3 gene may be detected by their growth on petri dishes containing semi-solid agar based media lacking histidine. The cDNA may then be purified from these strains, sequenced and used to identify the encoded protein which interacts with the MIP-3α polypeptide. Once compounds have been identified which bind to a MIP-3α or CCR6 gene product of the invention, the screening methods described in these methods may also be used to identify other compounds (e.g., small molecules, peptides and proteins) which bind to these binding compounds. Such compounds may also be useful for modulating MJP-3ct and or CCR6 -related bioactivities, for example by binding to a natural MIP-3α receptor, ligand or other binding partner, and preventing its interaction with a MIP-3α gene product. For instance, these compounds could be tested for their ability to inhibit the binding of MTP-3α- Fc to cell lines which express the MIP-3α specific receptor (CCR6).

Assays for compounds that interfere with a MIP —3a ligand interaction. As noted supra, a MJP-3α gene product of the invention may interact with one or more molecules (e.g., with an MIP-3α specific receptor) in vivo or in vitro. For example, in preferred embodiments, the MIP-3α gene product binds specifically with a CCR6 gene product of the invention. Compounds that disrupt or otherwise interfere with this binding interaction are therefore useful in modulating biological activity or activities that are associated with MIP-3α, including for example, cartilage degradation. Such compounds may therefore be useful, e.g., to treat disorders such as OA that are associated with abnormal levels of MTP-3α expression and/or activity.

Such compounds include, but are not limit to, compounds identified according to the screening assays described supra, for identifying compounds that bind to a MTP-3α gene product, including any of the numerous exemplary classes of compounds described therein.

In general, assays for identifying compounds that interfere with the interaction between a MTP-3α gene product and a binding partner (e.g., a receptor or ligand) involve preparing a test reaction mixture that contains the MJP-3α gene product and its binding partner under conditions and for a time sufficient for the MJP-3α gene product and its binding partner to bind and form a complex. i order to test a compound for inhibitory activity (i.e., for the ability to inhibit formation of the binding complex or to disrupt the binding complex once formed), the test compound preferably is also present in the test reaction mixture. In one exemplary embodiment, the test compound may be initially included in the test reaction mixture with the MJP-3α gene product and its binding partner. Alternatively, however, the test compound may be added to the test reaction mixture at a later time, subsequent to the addition of the MIP-3α gene product and its binding partner. In preferred embodiments, one or more control reaction mixtures, which do not contain the test compound, may also be prepared. Typically, a control reaction mixture will contain the same MTP-3α gene product and binding partner that are in the test reaction mixture, but will not contain a test compound. A control reaction mixture may also contain a placebo, not present in the test reaction mixture, in place of the test compound. The formation of a complex between the MIP-3α gene product and the binding partner may then be detected in the reaction mixture. The formation of such a complex in the absence of the test compound (e.g., in a control reaction mixture) but not in the presence of the test compound, indicates that the test compound is one which interferes with or modulates the interaction of a MTP-3α polypeptide and a binding partner.

Such assays for compounds that modulate the interaction of a MTP-3α gene product and a binding partner may be conducted in a heterogenous format or, alternatively, in a homogeneous format. Heterogeneous assays typically involve anchoring either a MJP-3α gene product or a binding partner onto a solid phase and detecting compounds anchored to the solid phase at the end of the reaction. Thus, such assays are similar to the solid phase assays described supra for detecting and/or identifying MTP-3α nucleic acids and gene products and for detecting or identifying MJP-3α binding partners. Indeed, those skilled in the art will recognize that many of the principles and techniques described above for those assays may be modified and applied without undue experimentation in the solid phase assays described here, for identifying compounds that modulate interaction(s) between and MTP-3α gene product and a binding partner.

Regardless of the particular assay used, the order to which reactants are added to a reaction mixture may be varied; for example, to identify compounds that interfere with the interaction of a MIP-3α gene product with a binding partner by competition, or to identify compounds that disrupt a preformed binding complex. Compounds that interfere with the interaction of a MTP-3α gene product with a binding partner by competition may be identified by conducting the reaction in the presence of a test compound. Specifically, in such assays a test compound may be added to the reaction mixture prior to or simultaneously with the MIP-3α gene product and the binding partner. Test compounds that disrupt preformed complexes of a MJP-3α gene product and a binding partner may be tested by adding the test compound to a reaction mixture after complexes have been formed.

The screening assays described herein may also be practiced using peptides or polypeptides that correspond to portions of a full length MJP-3α polypeptide or protein, or with fusion proteins comprising such peptide or polypeptide sequences. For example, screening assays for identifying compounds the modulate interactions of a MIP-3α polypeptide with a binding partner may be practiced using peptides or polypeptides corresponding to particular regions or domains of a full length MIP-3α polypeptide that bind to a binding partner (e.g., receptor "binding sites").

A variety of methods are known in the art that may be used to identify specific binding sites of a MJP-3α polypeptide. For example, binding sites may be identified by mutating a MJP-3α gene and screening for disruptions of binding as described above. A gene encoding the binding partner may also be mutated in such assays to identify mutations that compensate for disruptions from the mutation to the MIP-3α gene. Sequence analysis of these mutations can then reveal mutations that correspond to the binding region of the two proteins.

In an alternative embodiment, a protein (e.g., a MIP-3α protein or a protein binding partner to a MJP-3α protein) may be anchored to a solid surface or support using the methods described hereinabove. Another labeled protein which binds to the protein anchored to the solid surface may be treated with a proteolytic enzyme, and its fragments may be allowed to interact with the protein attached to the solid surface, according to the methods of the binding assays described supra. After washing, short, labeled peptide fragments of the treated protein may remain associated with the anchored protein. These peptides can be isolated and the region of the full length protein from which they are derived may be identified by the amino acid sequence. JJI still other embodiments, compounds that interfere with a MTP-3α-receptor interaction may also be identified by screening for compounds that modulate binding of a MJP-3α polypeptide (for example, a MIP-3α-Fc fusion polypeptide) to cells that express a MJP-3α specific receptor, such as leukocyte cells (including monocytes, B lymphocytes and T lymphocytes, including CD8 and CD4 cells).

Diagnostic and Prognostic Applications

A variety of methods can be employed for diagnostic and prognostic methods using reagents such as the MIP-3α and CCR6 nucleic acids and polypeptides described supra as well as antibodies directed against such MD?-3α and CCR6 nucleic acids and polypeptides. For example, using the methods described here it is possible to detect expression of a MTP-3α nucleic acid or protein in cells or tissues from an individual, such as in cells or tissues in a sample (e.g., from a biopsy) obtained or derived from an individual subject or patient. As explained above, MIP-3α nucleic acids and polypeptides are expressed at elevated levels in tissues from patients having OA.

Thus, using the methods described here (as well as other methods known in the art) a skilled artisan may detect elevated levels of a MIP-3α nucleic acid or polypeptide in a sample of cells or tissue from an individual, and may thereby detect and/or identify cells or tissue in that sample as being symptomatic of OA. In certain preferred embodiments the particular type of tissue identified in such methods is cartilage tissue. By using such methods to detect such cells or tissue in an individual, a skilled user may thereby diagnose the presence of OA in that individual.

In preferred embodiments the methods described herein are performed using pre-packaged diagnostic kits. Such kits may comprise at least one specific MEP-3α nucleic acid or a MIP-3α specific antibody reagent. In preferred embodiments, a kit will also contain instructions for its use, e.g., to detect diseased cells or tissues, or to diagnose a disorder (such as OA) associated with abnormal MTP-3α expression. In preferred embodiments, such instructions may be packaged directly with the kit. Li other embodiments, however, instructions may be provided separately. For example, the invention provides embodiments of kits where instructions for using the kit may be downloaded, e.g., from the internet. A kit of the invention may also comprise, preferably in separate contains, suitable buffers and other solutions to use the reagents (e.g., MJP-3α specific nucleic acid or antibody) to detect MTP-3CC. The kit and any reagent(s) contained therein may be used, for example, in a clinical setting, to diagnose patients exhibiting or suspected of having OA.

A sample comprising a cell of any cell type or tissue of any tissue type in which a MJP-3α gene is expressed may also be used in such diagnostic methods, e.g., for detection of MIP-3α gene expression or of MJP-3α gene products (such as MJ_P-3α proteins), as well as for identifying cells, e.g. chondrocytes, that express a MIP-3α gene or a MTP-3α gene product. Thus, in one embodiment, the methods described herein may be performed in situ, e.g., using cells or tissues obtained from an individual such as in a biopsy. Such methods may be useful, for example, in surgical procedures where it is desirable to identify arthritic tissue without removing benign, healthy tissue.

The methods described herein are not limited to diagnostic applications, but may also be used in prognostic applications, e.g., to monitor the progression of a disease (such as OA) that is associated with abnormal MIP-3α expression, or to monitor a therapy thereto. Accordingly, prognostic methods of the invention may comprise, in one exemplary embodiment, monitoring MIP-3α nucleic acid or polypeptide levels in an individual during the course of a treatment or therapy (for example, a drug treatment or exercise regimen) for OA. Similarly, the methods of the invention may also be used to detect and identify diseased cells and tissue (e.g. cells overexpressing MIP-3 compared to non OA cells or tissue) during the course of a therapy. In such embodiments, decreasing numbers of diseased cells is generally indicative of an effective treatment. The methods of the invention may further be used, e.g., to screen candidate drugs or compounds and identify ones that may be effective, e.g., as anti-OA drugs. Such methods may be performed in vivo ie.g., using an animal model) or in vitro (for example, in a cell culture assay). In one embodiment such methods may comprise contacting a candidate compound to a cell and identifying whether MIP-3α expression by the cell has been inhibited. In another embodiment, a compound may be contacted to a cell or administered to an organism, and extracellular levels of MIP-3α nucleic acid or polypeptide may be measured (for example, in cell culture media for cell culture assays, or in blood or other body fluid in an animal model assay).

Detection of AΩP-3a nucleic acids. The diagnostic and prognostic methods of the invention include methods for assaying the level of MTP-3α gene expression. A variety of methods known in the art may be used to detect assay levels of MTP-3α nucleic acid sequences in a sample. For example, RNA from a cell type or tissue that is known or suspected to express the MJP-3α gene may be isolated and tested utilizing hybridization or PCR techniques known in the art. The isolated cells may be, for example, cells derived from a cell culture or from an individual. The analysis of cells taken from a cell culture may be useful, e.g., to test the effect of compounds on the expression of a MTP-3α gene, or alternatively, to verify that the cells are ones of a particular cell type that expresses a MIP-3α gene.

As an example, and not by way of limitation, diagnostic methods for the detection of MTP-3α nucleic acids can involve contacting and incubating nucleic acids (including recombinant DNA molecules, cloned genes or degenerate variants thereof) obtained from a sample with one or more labeled nucleic acid reagents, such as recombinant MTP-3α DNA molecules, cloned genes or degenerate variants thereof, under conditions favorable for specifically annealing or hybridizing these reagents to their complementary sequences in the sample nucleic acids. After incubation, all non-annealed or non-hybridized nucleic acids are removed. The presence of nucleic acids that have hybridized, if any such molecules exist, is then detected and the level of MIP-3α nucleic acid sequences to which the nucleic acid reagents have annealed may be compared to the annealing pattern or level expected from a control sample (e.g., from a sample of normal, non-OA cells or tissues) to determine whether MTP-3α nucleic acid is expressed at an elevated level.

In a preferred embodiment of such a detection scheme, the nucleic acid from the cell type or tissue of interest may be immobilized, for example, to a solid support such as a membrane or a plastic surface (for example, on a nylon membrane, a microtiter plate or on polystyrene beads). After incubation, non-annealed, labeled MJP-3α nucleic acid reagents may be easily removed and detection of the remaining, annealed, labeled MIP-3α nucleic acid reagents may be accomplished using standard techniques that are well-known in the art.

Alternative diagnostic methods for the detection of MIP-3α nucleic acids in patient samples or in other cell or tissue sources may involve their amplification, e.g., by PCR (see, for example, the experimental embodiment taught in U.S. Patent No. 4,683,202) followed by detection of the amplified molecules using techniques that are well known to those of skilled in the art. The resulting level of amplified MJP-3α nucleic acid may be compared to those levels that would be expected if the sample being amplified contained only normal levels of MTP-3α nucleic acid, as normal cells or tissues, to determine whether elevated levels of a MJP-3α nucleic acid are expressed.

In one preferred embodiment of such a detection scheme, a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription). A sequence within the cDNA may then be used as a template for a nucleic acid amplification reaction such as PCR. Nucleic acid reagents used as synthesis intitation reagents (e.g., primers) in the reverse transcription and amplification steps of such an assay are preferably chosen from the MTP-3α nucleic acid sequences described herein or are fragments thereof. Preferably, the nucleic acid reagents are at least about 9 to 30 nucleotides in length. The amplification may be performed using, e.g., radioactively labeled or fluorescently labeled nucleotides, for detection. Alternatively, enough amplified product may be made such that the product can be visualized by standard ethidium bromide or other staining methods.

MJP-3α gene expression assays of the invention may also be performed in situ ( .e., directly upon tissue sections of patient tissue, which may be fixed and/or frozen), thereby eliminating the need of nucleic acid purification. MIP-3cc nucleic acid reagents may be used as probes or as primers for such in situ procedures (see, for example, Nuovo, PCR In Situ Hybridization: Protocols And Application, 1992, Raven Press, New York). Alternatively, if a sufficient quantity of the appropriate cells can be obtained, standard Northern analysis can be performed to determine the level of MIP-3α gene expression by detecting levels of MTP-3α mRNA. Detection of MIP-3a gene products. The diagnostic and prognostic methods of the invention also include ones that comprise detecting levels of a MJP-3α protein or other MJP-3α polypeptide and including functionally conserved variants and fragments thereof. For example, antibodies directed against unimpaired, wild-type or mutant MTP-3α gene products or against functionally conserved variants or peptide fragments of a MTP-3α gene product may be used as diagnostic and prognostic reagents. Such reagents may be used, for example, to detect abnormalities in the level of MD?-3α gene product synthesis or expression, or to detect abnormalities in the structure, temporal expression or physical location of a MTP-3α gene product. Antibodies and immunoassay methods such as those described hereinbelow also have important in vitro applications for assessing the efficacy of treatments, e.g., for OA. For example, antibodies, or fragments of antibodies, can be used in screens of potentially therapeutic compounds in vitro to ascertain a compound's effects on MIP-3α gene expression and MTP-3α polypeptide production. Compounds that may have beneficial effects on a disorder associated with abnormal MIP-3α expression can be identified and a therapeutically effective dose for such compounds may be determined using such assays.

As one example, antibodies or fragments of antibodies may be used to detect the presence of a MTP-3α gene product, a variant of a MJ -θα gene product or fragments thereof, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric or fluorimetric detection methods.

In particularly preferred embodiments, antibodies or fragments thereof may also be employed histologically, for example in immunofluorescence or immunoelectron microscopy techniques, for in situ detection of a MIP-3oc gene product. In situ detection may be accomplished by removing a histological specimen (e.g., a tissue sample) from a patient and applying thereto a labeled antibody of the present invention or a fragment of such an antibody. The antibody or antibody fragment is preferably applied by overlaying the labeled antibody or antibody fragment onto a biological sample. Through the use of such a procedure, it is possible to detect, not only the presence of a MIP-3α gene product, but also the gene product's distribution in the examined tissue. A wide variety of histological methods that are well known in the art (for example, staining procedures) can be readily modified by those skilled in the art without undue experimentation to achieve such in situ detection.

Immunoassays for MTP-3α gene products will typically comprise incubating a biological sample (for example, a tissue extract) in the presence of a detectably labeled antibody that is capable of specifically binding a MTP-3oc gene product (including, for example, a functionally conserved variant or a peptide fragment thereof). The bound antibody may then be detected by any of a number of techniques well known in the art.

Therapeutic Methods and Pharmaceutical Preparations

MIP-3α nucleic acids and polypeptides, and MIP-3α specific antibodies may also be used in therapeutic methods and compositions, e.g., to treat diseases and disorders associated with abnormal (preferably elevated) levels of MJP-3α expression. In preferred embodiments such methods are used to treat OA. In particular, Applicants have discovered that MIP-3α polypeptides and nucleic acids are expressed in cartilage cells and tissue at elevated levels in individuals having osteoarthritis (OA) relative to levels of expression in cells and tissue from non-osteoarthritic individuals. Accordingly, in one preferred embodiment the therapeutic methods of the invention comprise administering one or more compounds that modulate (e.g., inhibit) MIP-3α expression or activity; for example, compounds that bind to a MTP-3α nucleic acid or polypeptide of the invention, compounds that modulate expression of a MJJP-3α gene, and/or compounds that interfere with or modulate binding of a MIP-3 nucleic acid or polypeptide with a binding compound.

In another preferred embodiment, the therapeutic methods of the invention may comprise one or more cell-targeted therapies which target compounds (for example, drugs, pro-drugs, toxins or cytotoxins) to cells expressing a MTP-3α nucleic acid or polypeptide.

Inhibitory Approaches. In alternative embodiments, the present invention provides methods and compositions for treating a disease or disorder (for example, OA) associated with abnormal MIP-3α expression or activity by modulating (e.g., increasing or decreasing) the expression or activity of a MIP-3α gene or its gene product. Such methods may simply comprise administering one or more compounds that modulate expression of a MIP-3α gene, synthesis of a MIP-3α gene product or MJP-3α gene product activity so the immune response is modulated (e.g., enhanced or suppressed). Preferably, these one or more compounds are administered until one or more symptoms of the disorder are eliminated or at least ameliorated. The expression of the CCR6 gene may be similarly modulated.

Among the compounds that may exhibit an ability to modulate the activity, expression or synthesis of a MJP-3α or a CCR6 nucleic acid are antisense molecules. Such molecules may be designed to reduce or inhibit wild-type CCR6 or MTP-3α nucleic acids and polypeptides or, alternatively, may target mutant CCR6 or MIP-3α nucleic acids or polypeptides.

Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to target mRNA molecules and preventing protein translation. Antisense approaches involve the design of oligonucleotides that are complementary to a target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.

A sequence that is "complementary" to a portion of a nucleic acid refers to a sequence having sufficient complementarity to be able to hybridize with the nucleic acid and form a stable duplex. The ability of nucleic acids to hybridize will depend both on the degree of sequence complementarity and the length of the antisense nucleic acid. Generally, however, the longer the hybridizing nucleic acid, the more base mismatches it may contain and still form a stable duplex (or triplex in triple helix methods). A tolerable degree of mismatch can be readily ascertained, e.g., by using standard procedures to determine the melting temperature of a hybridized complex.

In one preferred embodiment, oligonucleotides complementary to non-coding regions of a MTP-3α gene may be used in an antisense approach to inhibit translation of endogenous MIP-3α mRNA molecules. Antisense nucleic acids are preferably at least six nucleotides in length, and more preferably range from between about six to about 50 nucleotides in length. In specific embodiments, the oligonucleotides may be at least 10, at least 15, at least 20, at least 25 or at least 50 nucleotides in length.

It is generally preferred that in vitro studies are first performed to quantitate the ability of an antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

While antisense nucleotides complementary to the target gene coding region sequence could be used, those complementary to the transcribed, untranslated region are most preferred.

Antisense molecules are preferably delivered to cells, such as chondrocytes, that express the target gene in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells. For example, antisense molecules can be injected directly into the tissue site (e.g., directly into a tumor), or modified antisense molecules can be designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.

Preferred embodiments achieve intracellular concentrations of antisense nucleic acid molecules which are sufficient to suppress translation of endogenous mRNAs. For example, one preferred approach uses a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol HI or pol II promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation of the target gene mRNA. For example, a vector, as set forth above, can be introduced e.g., such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in the particular cell type (for example in a hemopoietic cell). For example, any of the promoters discussed supra in connection with the expression of recombinant MIP-3α nucleic acids can also be used to express a MTP-3α antisense nucleic acid.

In addition to antisense technology, siRNA (Fire et al., 1998, Nature 391, 806-811; Elbashir et al., 2001, Nature 411, 494-498), RNA aptamers (Good et al., 1997, Gene Therapy 4: 45-54), double stranded RNA (WO 99/32619), ribozymes (Cech. J., 1988, Amer. Med Assn. 260:3030; Cotten et al., 1989, EMBO J. 8:3861-3866; Grassi and Marini, 1996, Annals of Medicine 28: 499-510; Gibson, 1996, Cancer and Metastasis Reviews 15: 287-299) and/or triple helix DNA (Gee, J.E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.) may be used to modulate the activity, expression or synthesis of a target nucleic acid such as MIP-3α or CCR6) according to methods familiar to one of skill in the art.

Pharmaceutical Preparations. Compositions used in the therapeutic methods of this invention may be administered (e.g., in vitro or ex vivo to cell cultures, or, more preferably, in vivo to an individual) at therapeutically effective doses to treat a disease or disorder such as OA that is associated with abnormal MIP-3α expression and/or activity. For example, compounds, including compounds identified in such screening methods as described above, that bind to either an MIP-3α chemokine, a CCR6 receptor, or both, may be administered to the cells or individual so that binding of the chemokine to the CCR6 receptor is inhibited. The invention therefore also provides pharmaceutical preparations for use, e.g., as therapeutic compounds to treat disorders, including OA, that are associated with abnormal MJP-3α expression or activity.

The terms "therapeutically effective dose" and "effective amount" refer to the amount of the compound that is sufficient to result in a therapeutic response. In embodiments where a compound (e.g., a drug or toxin) is administered in a complex (e.g., with a MIP-3α specific antibody), the terms "therapeutically effective dose" and "effective amount" may refer to the amount of the complex that is sufficient to result in a therapeutic response. A therapeutic response may be any response that a user (e.g., a clinician) will recognize as an effective response to the therapy. Thus, a therapeutic response will generally be an amelioration of one or more symptoms of a disease or disorder. In preferred embodiments, where the pharmaceutical preparations are used to treat OA, a therapeutic response may be a reduction in the amount of cartilage degradation observed, e.g., in biopsies from a patient during treatment.

Toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures, for example in cell culture assays or using experimental animals to determine the LD₅₀ and the ED₅₀. The parameters LD₅₀ and EDsn are well known in the art, and refer to the doses of a compound that are lethal to 50% of a population and therapeutically effective in 50% of a population, respectively. The dose ratio between toxic and therapeutic effects is referred to as the therapeutic index and may be expressed as the ratio: LD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used. However, in such instances it is particularly preferable to use delivery systems that specifically target such compounds to the site of affected tissue so as to minimize potential damage to other cells, tissues or organs and to reduce side effects.

Data obtained from cell culture assay or animal studies may be used to formulate a range of dosages for use in humans. The dosage of compounds used in therapeutic methods of the present invention preferably lie within a range of circulating concentrations that includes the ED₅₀ concentration but with little or no toxicity (e.g., below the LD₅₀ concentration). The particular dosage used in any application may vary within this range, depending upon factors such as the particular dosage form employed, the route of administration utilized, the conditions of the individual (e.g., patient), and so forth.

A therapeutically effective dose may be initially estimated from cell culture assays and formulated in animal models to achieve a circulating concentration range that includes the IC₅₀. The IC₅0 concentration of a compound is the concentration that achieves a half-maximal inhibition of symptoms (e.g., as determined from the cell culture assays). Appropriate dosages for use in a particular individual, for example in human patients, may then be more accurately determined using such information.

Measures of compounds in plasma may be routinely measured in an individual such as a patient by techniques such as high performance liquid chromatography (HPLC) or gas chromatography.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

EXAMPLES

The present invention is also described by means of examples, including the particular Examples presented here below. The use of such examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled.

EXAMPLE 1: ANALYSIS OF MIP-3α EXPRESSION IN HUMAN

CHONDROCYTES AND CARTILAGE TISSUE

This example describes experiments in which expression levels of MJP-3α and its receptor, CCR6, were measured in cartilage tissue samples obtained from individuals having osteoarthritis ("OA"). The results show that MIP-3α is expressed at elevated levels in cartilage tissue of osteoarthritis patients compared to levels in cartilage from normal individuals (i.e., from individuals not afflicted with osteoarthritis).

Materials and Methods The experiments described in this example may be readily performed by those skilled in the art, e.g., according to the following protocols.

MIP-3a mRNA expression analysis in human chondrocyte monolayer cultures by RT-PCR. Cartilage samples from OA donors (patients undergoing knee-joint replacement surgery) and normal individuals (cadavers with no history of arthritis) may be rinsed in PBS, minced and digested with protease from Streptomyces griseus (Sigma, St. Louis, MO) and collagenase-2 (Worthington Biochemicals, Lakewood, NJ) as per Kuettner et al. iJ. Cell Biol. 1982, 93:743-50). Chondrocytes from the treated samples can then be seeded at high density (1 X 10⁶ cells/well) in 6 well plates with high glucose DMEM containing 10% heat inactivated bovine serum. The seeded cells are preferably maintained in a CO₂ incubator (5%, 37°C) until cells achieved 80% confluence, and may then be shifted to medium containing 0.2% serum overnight prior to treatment with inducers. Cells are then preferably treated with EL-lβ (lOng/ml, Preprotech, L ondon, UK), TNF-α (lOng/lml, Sigma, St. Louis, MO) or LPS (5ug/ml, Sigma, St. Louis, MO) for 16hrs. RNA may be isolated using standard materials and methods that are known in the art, including but not limited to a RNeasy kit from Qiagen as per manufacturer's instructions. For semi-quantitative RT-PCR, equal amounts of total RNA from uninduced and induced cells is preferably digested with Dnase and reverse transcribed using oligo dT primer and Superscript II (Life Technologies, Rockville, MD) for 1 hour at 42°C. Equal dilutions of this cDNA may then be subjected to 20 cycles of PCR with the following primers, which are designed to amplify a 185 base pair fragment of human MIP-3α (GenBank Accession No. U64197):

5'-AACCATGTTGCTGTACCAAGAGTT-3' (SEQ ID NO.

1)

5'-GCATTGATGTCACAGCCTTCATT-3' (SEQ ID NO.

2) Immunohistochemical Analysis ofMIP-3a and CCR6 Expression in Normal and OA cartilage. Explants of normal or OA human knee cartilage may be cultured in DMEM containing 5% FBS. Cartilage is preferably fixed with 4% paraformaldehyde and embedded with paraffin to cut 5 μm sections. The tissue sections may be placed on slides deparaffinized in toulene, and hydrated in graded series of ethanol, then washed in PBS and in 0.2% peroxidase. The presence of MIP-3α is determined, e.g., using the avidin-peroxidase method with goat antibody to human MTP-3α (R&D Systems, Minneapolis, MN). This antibody does not cross-react with Exodus-2, Exodus-3 (superfamily members) or any other known chemokine. The antibody may be used at a concentration of 5μg/ml, and incubated with the section for lh at RT. A biotinylated goat anti-rabbit may also be used as a secondary antibody. The presence of CCR6 may also determined according to this protocol using, e.g., a mouse antibody (2 μg/ml) to human CCR6 (R&D Systems, Minneapolis, MN). This antibody does not cross-react with other chemokine receptors, particularly CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CXCR1, CXCR2, CXCR3, CXCR4 and STRL33 transfectants. A biotinylated goat anti-mouse may also be used as a secondary antibody. Negative controls are preferably performed, e.g., by replacing the primary antibody with pre-immune serum or immunoglobulin.

Results

MIP-3a expression in primary human chondrocyte cultures. MIP-3α mRNA levels are determined in unstimulated vs. IL-lβ, LPS, or TNF-α stimulated primary human chondrocytes (obtained as described above) by RT-PCR. Control experiments with GAPDH primers reveal equal amounts of cDNA in each reaction. In addition, isolation cloning and DNA sequencing confirm the identity of the PCR product.

Data indicates that MIP-3α is not detectable in un-stimulated monolayers of primary human chondrocytes but is significantly induced by IL-lβ, TNF-α and LPS treatments. This finding is consistent with MIP-3α being a disease related gene that is induced in chondrocytes by inflammatory cytokines associated with OA. Over-expression ofMIP-3a in cartilage from patients with OA. To determine if ML°-3α protein may be over-expressed in human joint disease, immunohistochemistry can be used to examine MIP-3α levels directly on normal and OA cartilage. Analysis of human cartilage by immunohistochemistry using an antibody specific for MIP-3α demonstrates the presence of MlP-α in the superficial and mid zone of OA cartilage but not in normal human cartilage, nor in OA cartilage treated with a non-specific control antibody. Therefore the immunohistochemistry data clearly demonstrate that the ligand MJP-3α is over expressed in the disease state (OA).

CCR6 is not overexpressed in cartilage from patients with OA. In contrast to what is observed for MIP-3α, CCR6 receptor is found to be equally expressed both in normal and OA cartilage tissue. Immunohistochemistry analysis of normal human cartilage using an antibody specific for CCR6 demonstrates abundant staining compared to a control using a non-specific antibody . Similarly, OA cartilage treated with an anti-CCR6 antibody results in specific staining compared with no staining using a control antibody. However, the CCR6 specific antibody stains chondrocytes in the deeper layers of cartilage tissue from OA individuals somewhat more than it stains chondrocytes in cartilage tissue from non-OA individuals.

EXAMPLE 2: ANALYSIS OF MIP-3α BY IN SITU HYBRIDIZATION IN

SYNOVIUM AND REMODELLING BONE

This example describes experiments where MIP-3α expression was measured in synovium and remodeling bone from a patient having rheumatoid arthritis ("RA"), and in identical tissue samples obtained from individuals not having RA. The results show that MIP- 3α, although not expressed in non-arthritic tissue, is expressed in synovium and bone afflicted with rheumatoid arthritis. These results agree with those observed by others (Matsui et al., Clin Exp. Immunol. 2001; 125:155-61). Materials and Methods

The experiments described in this example may be readily performed by those skilled in the art, e.g., according to the following protocols.

In Situ Hybridization. For riboprobe generation, a 381 base pair fragment, which includes the entire coding region of human MP-3α, may amplified using the following primers:

5'-GGTACTCAACACTGAGCAGCAGATCTG-3' (SEQ ID NO. 3)

5'-TCTTTCTGTTCTTGGGCTATGTCC-3' (SEQ ID NO. 4)

The product of this PCR reaction is preferably cloned, e.g., into a pCR-Bluntil vector (available from Invitrogen, Carlsbad CA), which allows the synthesis of both sense and antisense riboprobes. The probes used in these studies are preferably detectably labeled, such as radiolabeled riboprobes, and are transcribed from the T3 (antisense) and SP6 (sense) transcription initiation sites in the presence of ³²P-UTP.

Tissue sections (RA synovium and remodelling bone) from paraffin blocks may be digested with Proteinase K for varying durations and then hybridized with the probes 4xl0⁸ dpm/ml and 52°C for 20 hr, subsequently treated with RNAse A, and washed for 2 hr in 0.1 x SSC. Hybridization signals may then be visualized, e.g., by exposure to Kodak NTB-2 emulsion followed by exposure at 4°C for 2-4 weeks. Such slides are then preferably developed (e.g., using Kodak D-19 Developer and Rapid fixer), stained (e.g., with hemotoxylin and eosin) and then imaged (for example, using a Sony Digital Photo Camera and a Nikon microscope).

Results MIP-3 is expressed in tissue from patients with RA. Expression of MIP-3α mRNA is also examined in RA tissue using in situ hybridization. Sense and antisense probes covering the entire MIP-3α coding region may be prepared as described above. Synovium and bone are isolated from an RA patient and contacted with the antisense probe for MIP-3α as described in the materials and methods section, above. In situ hybridizations are carried out on joint tissue for normal and RA patients. Positive hybridization is indicated by the level of fluorescence observed.

MJP-3α is detected consistently in RA synovium and remodelling bone but not in normal (i.e., non- arthritic) tissues. This finding is of particular significance because, without being limited by any particular theory or mechanism of action, it is believed that MIP- 3α is involved in the regulation of dendritic cell trafficking, and dendritic cells are thought to play a central role in the RA synovium (Thomas et al, J. Leukoc. Biol. 1999; 66:286-92).

EXAMPLE 3: CONSTRUCTION AND PURIFICATION OF

RECOMBINANT HUMAN MIP-3α

This example describes a novel, preferred expression system that produces active, recombinant MIP-3α. The expression system may be used, for example, to produce MIP-3α for screening assays of this invention. Because MIP-3α is a secreted protein, the recombinant protein was expressed and purified in two different expression systems. First, intracellular expression of the mature protein was accomplished in a bacterial expression system. Secretion of the pre-protein was also accomplished in a baculovirus/insect cell expression system.

Materials and Methods

The experiments described in this example may be readily performed by those skilled in the art, e.g., according to the following protocols.

Generation ofHis-Tagged MIP-3a in Bacteria. To generate expression constructs, the human MJP-3α nucleic acid (GenBank Accesion No. U64197; SEQ ID NO. 11) may be sub-cloned, e.g., into the pET23 expression vector (available from Novagen, Madison, WI) by PCR amplification using standard materials and protocols that are readily available to those skilled in the art, including but not limited to the Expand High Fidelity PCR kit (available from Roche Molecular Biochemicals, Berkeley, CA) in 20 cycles according to the manufacturer's recommended protocol.

The following primer pairs are designed and may be used to amplify MIP-3α nucleic acids that could be inserted into Nde 1 and Xho 1 sites of a pET23 vector (available from Novagen, Madison, WI): upstream primer sequence:

5'GAA TTC CAT ATG GCA AGC AAT TTG ACT GCT GTC TTG G-3'

Ndel (SEQ ID NO.5)

downstream primer sequence:

5'-CCG CTC GAG CAT GTG TTC TTG ACT TTT TTA CTG AGG-3'

Xhol (SEQ ID NO. 6)

(SEQ ID NO. 6(SEQ ID NO. 6) Refolding and Purification of Bacterially His-Tagged MIP-3a. To re-fold His-tagged proteins, wet E. coli cell pellets may be suspended in lysis buffer A (phosphate- buffered saline containing 10 mM imidazole, 0.5 M NaCl) and re-centrifuged. Preferably, the resulting pellets are repeatedly suspended in lysis buffer B (50 mM Tris-HCl pH 8.0 containing 5 mM DTT, 5 mM EDTA and 5 mM Benzamidine-HCl) and centrifuged until the supernatants are clear and free from coloration. The pellets are then preferably washed with 50 mM Tris prior to solubilization in buffer C (50 mM Tris containing 6M Guanidine-HCl and 10 mM β-mercaptoethanol). The resulting suspension may be filtered and loaded onto an XK26/10 column (Sepharose FF charged with nickel sulfate). The column is washed (e.g., with phosphate buffered saline containing 8M urea and 5 mM β -mercaptoethanol) and eluted (e.g., with a gradient of 0-500 mM imidazole). Fractions containing the protein may then be pooled, and β-mercaptoethanol is preferably added to a concentration of 10 mM. Refolding may be initiated, e.g., by dialysis against 50 mM Tris, pH 8.0, containing 2 M urea, 3 mM GSH (reduced glutathione), and 0.3 mM GSS. Disulfide bond formation may be determined by standard reverse-phased HPLC, and is preferably followed by subsequent dialysis against 50 mM Tris (pH 8.0) containing 3 mM GSH and 0.3 mM GSSG. After removal of the precipitate (e.g., by centrifugation) 0.1% by volume of trifluoracetic acid is preferably added added, and the correctly re-folded protein may be purified, e.g., by standard reversed-phase HPLC.

Analysis of Bacterial Expressed MIP-3a-6-His. After re-folding, MIP-3α-6- His may be analyzed according to standard protocols, such as by HPLC-MS, to determine if it is a protein of the correct size of (9209.2 Da) (see, Knebel et al, J Chromatogr. B Biomed. Appl. 1995, 673(2):213-22; and Vekey, J. Chromatogr. A. 2001, 921(2):227-36). The actual molecular weight of the expressed MJP-3α is found to be 9090.4 Da, which represents a protein of SEQ ID. NO. 12 that contains 2 disulphide bonds and lacks one methionine residue. The endotoxin level of the refolded MJP-3α -6xHis may be tested, e.g., by using a Limulus Amebocyte Lysate Single Vial Assay (available from Associates of Cape Cod, Inc. Falmouth, MA).

Further analysis of the correctly-folded MIP-3α protein may be demonstrated by PAGE-analysis followed by staining with Coomassie Blue for protein visualization. A standard Western blotting procedure using anti-MJP-3α antibody (available from Qiagen, Valencia CA, diluted 1:2000) may also be performed to demonstrate expression and secretion of recombinant MIP-3α.

Generation of FLAG-Tagged MIP-3a in Insect Cells. To generate a recombinant baculovirus for secretion, the pre-MJP-3α gene may be sub-cloned, e.g., into the BamH 1 and Sac 1 sites of a pBacPAK9 vector (available from Clontech, Palo Alto CA). The following primer sequences may be used to amplify MIP-3α nucleic acids for cloning into that vector according to the manufacturer's protocol:

upstream primer sequence:

5'-CCG GAT CCC ACC ATG TGCTGT ACC AAG AGTTTG-3' (SEQ ID NO.8)

downstream primer sequence:

5 -GGT GAG CTC TCA CTT GTC ATC GTC ATC CTT GTA ATC ACC GGC ATG TTC TTG ACT TTT TTA CTG AGG-3' (SEQ ID NO. 9)

Purification of FLAG-tagged MIP-3a Secreted from Insect Cells. Secreted mature MJP-3α FLAG may be purified by applying 0.2μl filtered culture media (from 6x 15 cm tissue culture dishes, 45 hour post infection) through a Source S15 HR 5/5 column (Amersham Pharmacia, Buckinghamshire, United Kingdom). The bound protein may be eluted, e.g., with a 20mM - 1M NaCl gradient developed in 0.1 x PBS. The fractions containing MIP-3α FLAG are preferably pooled and extensively dialyzed against PBS + 10% glycerol.

The purity of the MJP-3α FLAG is above 90%, as judged by Coomassie stain of the sample (described above). Western blotting may also be performed as described, supra, to confirm expression of soluble mature MIP-3α.

The pre-MJP-3α-FLAG may be expressed upon infection of insect cells (HIGH 5, Invitrogen, Carlsbad, CA), and the mature MIP-3α-FLAG generated during the secretion process. The amino acid sequence of the exemplary secreted MIP-3α-FLAG protein is represented in SEQ ID NO.: 10.

Fluorescent Activated Cell Sorting (FACS). The stably-transfected 293 HEK EBNA cells, described above, may be treated with the His-tagged, bacterially expressed recombinant MJ_P-3α and incubated on ice for 1 hour to allow binding of MJP-3α to the CCR6 receptor. Cells are preferably washed with phosphate buffered saline containing 10% fetal bovine serum and then incubated with an anti-His antibody (Qiagen) for 1 hour on ice. After washing, e.g., with phosphate buffered saline containing 10% fetal bovine serum, cells may be exposed to a fluoroisocyothionate (FITC)-labeled anti-mouse antibody, reactive against the anti-His antibody, and incubated for an additional hour on ice. FACS analysis may be performed according to standard procedures to confirm binding of the His-tagged MIP-3α to the CCR6-expressing 293 HEK-EBNA cells.

Calcium-Flux Fluormetric Imaging Plate Reader (FLIPR) Assay. FLJPR technology (Molecular Devices, Sunnyvale, CA) may be used to quantitate changes in intracellular calcium following stimulation of 293 HEK EBNA cells, stably transfected with the CCR6 receptor (e.g., in the pCEP4 565 vector, Novartis Pharmaceuticals, New Jersey), with the recombinant-tagged MIP-3α described above. The protocol used preferably follows the manufacturer ' s suggestion.

Results Bacterial Expression of His-tagged MIP-3o A bacterially-expressed MJP- 3α-6-His fusion protein is generated as described in the Materials and Methods section, above. Expression of the protein is confirmed by SDS-PAGE analysis, followed by staining with Coomassie, or by Western blot as described, supra, in the Materials and Methods section. The secreted protein, denoted MJP-3α -6xHis (in BL21[DE3]), is found to be insoluble. Therefore, the protein is re-folded from inclusion bodies as described above. After re-folding, analysis of MIP-3α -6xHis by HPLC-MS indicates a protein of correct size of 9090.4 Daltons. Furthermore, the initial Met is removed by the host bacteria during expression.

The bacterially expressed recombinant protein is the mature MJP-3α containing an extra methionine (Met) at the protein's N-terminus and six Histidine (His) residues at the protein ' s C-terminus. The amino acid sequence of the His-tagged MD?-3α is represented in SEQ ID NO. 7. This sequence lacks initial 25 amino acid secretion sequence of the native protein.

The bacterial host cells used in this expression system produces large amounts of endotoxin. Because such endotoxin can interfere with mammalian cell-based assays even when present in only modest amounts, the levels of endotoxin contamination are carefully monitored. The endotoxin level of the re-folded MJP-3α -6xHis is only about 0.015 ng endotoxin/μg MIP-3α. This level is significantly below the levels of 0.1 ng/μg found in commercial preparations of MJP-3α (e.g., available from R & D Systems Inc., Minneapolis, MN) and is safe for use in mammalian cell-based assays.

Baculovirus expression of FLAG-tagged MIP-3a. In addition to the bacterial expression system described above, another improved expression system is developed using a recombinant baculovirus carrying the entire pre-MJP-3α gene and with FLAG tag at its 3' end. Upon infection, the HIGH 5 host insect cell is able to process the pre-protein into its mature form as demonstrated by SDS-PAGE followed by protein visualization with Coomassie stain and immunoblotting.

Expression and secretion of the protein is shown by both Coomassie staining and by Western blotting (described above) with an anti-FLAG antibody (M2; available from Sigma-Aldrich, Corp., Saint Louis MO). The HIGH 5 insect cells infected with baculovirus- containing FLAG-tagged MIP-3α secrete into the medium a recombinant protein having the expected molecular weight of 9090.4 Daltons. In contrast, no detectable MJP-3α is secreted into the medium by non-infected HIGH 5 cells. The amino acid sequence of the secreted MIP-3α-FLAG protein is represented in SEQ ID NO. 10.

Recombinant MIP-3a is Biologically Active. Both the soluble mature MIP-3α FLAG and MTP-3α -6xHis fusion proteins are tested for bioactivity using calcium flux and FACS binding assays. FACS analysis using an antibody specific for the His-tag demonstrates the ability of His-tagged MIP-3α to bind the CCR6 receptor that is expressed on HEK 293-EBNA cells. This confirms that the re-folding of the MJP-3α described above results in a conformationally-active protein.

To confirm that the recombinant MIP-3α is active, intracellular calcium is measured in response to treatment of CCR6-expressing 293 HEK-EBNA cells with recombinant MIP-3α. An increase in cellular calcium is observed after treatment, compared with treatment of untransfected 293 HEK-EBNA cells, as assayed using the FLIPR technology described above. This increase in intracellular calcium is indicative of receptor activity, and therefore, demonstrates that the recombinant MIP-3α proteins are at least as active as the MJP- 3α protein that is currently available in the art (e.g., from R&D Systems Inc., Minneapolis MN).

Claims

WHAT IS CLAIMED IS:

1. A method for identifying compounds that may be used to treat osteoarthritis, which method comprises:

(a) contacting a reaction mixture comprising (i) an MIP-3α polypeptide, and (ii) a CCR6 receptor, wherein the reaction mixture conditions permit binding of the MIP-3α polypeptide to the CCR6 receptor thereby permitting formation of a binding complex, with or without a test compound;

(b) detecting levels of formation of said binding complex in the reaction mixtures; and

(c) comparing the level of said binding complex formed in the presence of said test compound to the level of said binding complex formed in the absence of said test compound, wherein a decrease in the level of said binding complex formed in the presence of said test compound indicates that the test compound may be used to treat osteoarthritis.

2. A method according to claim 1 in which the MIP-3α polypeptide comprises the amino acid sequence set forth in:

(a) SEQ ID NO:12;

(b) SEQ ID NO:7; or

(c) SEQ ID NO O.

3. A method according to claim 1 in which the CCR6 receptor comprises the amino acid sequence set forth in SEQ ID NO:14.

4. A method for identifying an individual having osteoarthritis, which method comprises detecting an MIP-3α nucleic acid in cartilage or chondrocytes derived from the individual, wherein detection of the MIP-3α nucleic acid indicates that the individual has osteoarthritis.

5. A method according to claim 4 wherein the MIP-3α nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO:11.